Glycocalyx-mimetic dextran-modified poly(vinyl amine) surfactant coating reduces platelet adhesion on medical-grade polycarbonate surface.

A dextran-modified poly(vinyl amine) comb-like surfactant polymer, poly(N-vinyl dextran aldonamide-co-N-vinyl hexanamide), that can surface-adsorb on hydrophobic polymeric substrates, was designed to improve the interfacial blood-compatibility of polymeric biomaterials. Medical-grade polycarbonate was selected as a model substrate because of its extensive use in blood-contacting biomedical devices like hemodialyzers, blood pumps and oxygenators. The surfactant polymer was physisorbed from aqueous solution onto the polycarbonate substrate. The surfactant coating was stable under dynamic shear conditions in whole blood, as confirmed by fluorescence microscopy and total internal reflection fluorescence (TIRF) experiments with fluorescein-labeled surfactant polymer. The coated disks and uncoated control disks were exposed to platelet-rich plasma (PRP) and whole human blood in a rotating disk system (RDS) to study platelet-adhesion under dynamic shear stress environments. Adhered platelets were stained with fluorescein isothiocyante (FITC)-tagged anti-CD41a monoclonal antibody and imaged by epifluorescence microscopy. Complimentary images were obtained by phase-contrast microscopy. Platelet adhesion on the surfactant-coated disks was reduced by approximately 90%, compared with uncoated disks. The images also showed a concomitant reduction in platelet-derived microparticles on surfactant-coated disks, compared with uncoated disks. The results suggest potential application of carbohydrate-modified surfactant polymers as a glycocalyx-mimetic non-thrombogenic interfacial coating for blood-contacting biomaterials.

Molecular views and measurements of hemostatic processes using atomic force microscopy.

Hemostasis and thrombosis are highly complex and coordinated interfacial responses to vascular injury. In recent years, atomic force microscopy (AFM) has proven to be a very useful approach for studying hemostatic processes under near physiologic conditions. In this report, we review recent progress in the use of AFM for studying hemostatic processes, including molecular level visualization of plasma proteins, protein aggregation and multimer assembly, and structural and morphological details of vascular cells under aqueous conditions. AFM offers opportunities for visualizing surface-dependent molecular and cellular interactions in three dimensions on a nanoscale and for sensitive, picoNewton level, measurements of intermolecular forces. AFM has been used to obtain molecular and sub-molecular, resolution of many biological molecules and assemblies, including coagulation proteins and cell surfaces. Surface-dependent molecular processes including protein adsorption, conformational changes, and subsequent interactions with cellular components have been described. This review outlines the basic principles and utility of AFM for imaging and force measurements, and offers objective perspectives on both the advantages and disadvantages. We focus primarily on molecular level events related to hemostasis and thrombosis, particularly coagulation proteins, and blood platelets, but also explore the use of AFM in force measurements and surface property mapping.

Reduced protein adsorption and platelet adhesion by controlled variation of oligomaltose surfactant polymer coatings.

A series of oligomaltose surfactant polymers were prepared by the simultaneous coupling of hydrophilic maltolactone [of 2(M2), 7(M7), or 15(M15) glucose units] and hydrophobic N-(hexanoyloxy)succinimide (Hex) groups to the amino groups of a poly(vinyl amine) backbone. The surfactants were characterized by FTIR and 1H-NMR spectroscopies for purity and composition. Contact-angle and AFM measurements confirmed full monolayer adsorption for all surfactants on a model surface, highly oriented pyrolitic graphite, while full coverage was observed on polyethylene only for PVAm (M7:Hex) due to the optimal M7:Hex ratio and Hex chain density. On graphite, protein resistance increased with increasing coating thickness from 81.4 to 85.8 to 95.8% for the M2, M7, and M15 surfactants, respectively. Additionally, static platelet adhesion on all three surfactants dropped substantially to 15% (M2), 17% (M7), and 16% (M15)compared to glass (adhesion normalized to 100%) and a polyurethane (24%) surface. Protein- and platelet-resistant properties of the controlled oligomaltose layers are discussed by analysis of molecular modeling, oligomaltose and hexanoyl chain densities, and surfactant stability.

Shear-induced platelet activation and adhesion on human pulmonary artery endothelial cells seeded onto hydrophilic polymers.

We evaluated platelet activation and adhesion on two plasma polymerized surfaces, N-vinyl pyrrolidone (NVP) and gamma-butyro lactone (GBL), which have been shown previously to promote endothelial cell growth and adhesion as well as fibronectin-coated glass (1 microg/cm(2)) coverslips. Human pulmonary artery endothelial cells were seeded onto coverslips at a low density ( approximately 20,000 cells/cm(2)) and grown to confluence (3-5 days). The materials, both with and without ECs, were then exposed to a shear rate of 400 s(-1) in a closed loop recirculating flow system containing human platelet-rich plasma. Plasma samples were taken at 0, 5, 15, 30, and 60 min and analyzed for platelet and coagulation activation. The coverslips were examined for EC coverage and platelet adherence. EC retention over a 1-h period was approximately 75% for all three materials. All three materials without ECs were highly platelet activating having similar P-selectin expression, platelet factor 4 (PF4) release, mepacrine uptake, and microparticle production. Both microparticle production and platelet adhesion were significantly lower in EC-seeded materials. Dense granule and PF4 release were both slightly diminished in all three materials seeded with ECs. P-selectin expression was reduced slightly for GBL, but remained the same for the other two materials. The EC-seeded materials displayed favorable characteristics with respect to platelet activation and adhesion; however, they still demonstrated some thrombogenic tendencies due to EC loss and exposure of the underlying substrate. Therefore, both EC coverage and EC hemostatic function are important factors in determining the thromboresistance of an EC-seeded surface.

Individual plasma proteins detected on rough biomaterials by phase imaging AFM.

In the past several years, atomic force microscopy (AFM) has provided topographic images of adsorbed plasma proteins in situ at unprecedented resolution. Imaging has been limited to adsorbed protein on relatively smooth model substrates such as mica, graphite, or self-assembled monolayers on which the small height of the protein can be observed from the background. The inherent roughness of biomaterial surfaces has prevented observation of adsorbed proteins in topographic images. We report imaging isolated fibrinogen molecules adsorbed on National Heart Lung and Blood Institute (NHLBI) reference materials polydimethylsiloxane and low-density polyethylene in situ using phase imaging AFM. Fibrinogen, a plasma protein important for blood coagulation and platelet aggregation, was adsorbed from dilute solution onto reference biomaterial surfaces at sub-monolayer coverage. Tapping mode AFM was used to image the samples. For polydimethylsiloxane, the lateral size of the surface features is much greater than the dimensions of proteins. This allowed adsorbed proteins to be observed in topographic images. The phase imaging signal of tapping mode AFM provides information on differences in material properties of the surface, and was used to distinguish individual protein molecules from the underlying polymer surface. On the low-density polyethylene surface, characteristic topographical features are of the same magnitude as the protein molecules, so that protein cannot be distinguished from the surface using topographic images. However, phase images were used to successfully locate and characterize the distribution of the protein. Phase imaging was not able to distinguish fibrinogen adsorbed onto expanded polytetrafluoroethylene. The utility and limitations of the phase imaging technique for characterizing protein adsorption to rough surfaces is discussed.

Determining intramolecular binding sites on surface-bound von Willebrand factor under aqueous conditions.

The results described in this report demonstrate the feasibility of using AFM in combination with immuno-gold labeling to investigate the accessibility of various binding sites on vWF and to localize the binding site within a vWF multimer. With the aid of monoclonal antibodies [5, 11 and 23] it should be possible to use this approach to perform a quantitative assessment of the differential accessibility of various binding sites on vWF. This should allow localization and quantification of binding sites within the observed tertiary structure of the vWF, providing a measure of the accessibility, a point of reference with which the tertiary structure of vWF could be correlated to the primary sequence, and a means to determine the structural features of the antibody binding regions under physiologic buffer conditions. There are a number of obvious questions that are not addressed here: The role of different biologic and artificial surfaces; time-dependent effects; vWF orientation with respect to different thrombogenic surfaces; and the location of critical binding sites, such as for platelet GPIalpha and GPIIb-IIIa binding regions in the hydrated tertiary structure of vWF. Nevertheless, the work described in this report provides essential groundwork that should provide a novel basis on which to explore the molecular steps, both structural and functional, of vWF in thrombus development. In a wider sense, this experimental approach is applicable to structure-function studies on a wide variety of proteins in physiologic environments.

Use of Dacron as an alternative carrier for evaluating oxidizing sterilants in the AOAC sporicidal test.

The AOAC sporicidal method (966.04) recommends the use of porcelain penicylinders and black waxed silk sutures as carriers for demonstrating the sporicidal activity of sterilants. However, the silk carriers are not suitable for evaluating the sporicidal efficacy of oxidizing agents, and an inert polyester material (Dacron) is recommended as an alternative. Dacron provides an equivalent microbial and physical challenge to silk. Microbiologically, both materials demonstrated similar HCI resistance, which is required by the AOAC test, as well as equivalent spore loading and spore wash-off. Electron microscopy showed that both materials present the same braided microstructure, providing an equivalent physical challenge to the test sterilant. Dacron was more consistent than silk, and did not require extraction prior to spore loading. The extraction method for black waxed silk was variable and incomplete, which may compromise the activity of oxidizing sterilants and add to method variability. Silk was also structurally altered in the presence of oxidizing sterilants and increased sterilant degradation. Dacron did not affect the sterilant and was inert in the presence of oxidizing agents. Dacron sutures are proposed as inert alternatives to silk for evaluating the sporicidal efficacy of oxidizing agents.

Adsorption of plasma proteins on polyethylene oxide-modified lipid bilayers studied by total internal reflection fluorescence.

Distearoylphophatidylcholine (DSPC) mixed with various mole percentages of polyethylene oxide (number average molecular weight 2000)-grafted distearoylphosphatidylethanolamine (PEO2000-DSPE) were deposited on DSPE-coated quartz surfaces by the Langmuir-Blodgett deposition. Structural transitions in PEO2000 from pancake to mushroom, and from mushroom to brush conformations were revealed from film balance experiments. Adsorption kinetics of proteins from 1% platelet-poor plasma (PPP) on the supported lipid bilayers were studied using intrinsic total internal reflection fluorescence. All the supported lipid bilayers exhibited over a magnitude reduction in adsorbed plasma proteins, compared with the quartz substrate. The increase of PEO2000-DSPE density in the mixed bilayers slightly increases the amount of adsorbed proteins on the bilayers.

Surfactant polymers designed to suppress bacterial (Staphylococcus epidermidis) adhesion on biomaterials.

We describe a series of surfactant polymers designed as surface-modifying agents for the suppression of bacterial adhesion on biomaterials. The surfactant polymers consist of a poly(vinyl amine) backbone with hydrophilic poly(ethylene oxide) (PEO) and hydrophobic hexanal (Hex) side chains (PVAm/PEO:Hex). Surface modification is accomplished by simple dip coating from aqueous solution, from which surfactant polymers undergo spontaneous surface-induced assembly on hydrophobic biomaterials. The stability of PVAm/PEO:Hex on pyrolytic graphite (HOPG) and polyethylene (PE) was demonstrated by the absence of detectable desorption under flow conditions of pure water over a 24-h period. PEO surfactant polymers with four different PEO:Hex ratios (1:1.4, 1:2.5, 1:4.6, and 1:10.7) and a dextran surfactant polymer were compared with respect to S. epidermidis adhesion under dynamic flow conditions. Suppression of S. epidermidis adhesion was achieved for all modified surfaces over the shear range 0-15 dyn/cm(2). The effectiveness depended on the surfactant polymer composition such that S. epidermidis adhesion to modified surfaces decreased significantly with increasing PEO packing density. Modified HOPG was more effective in reducing bacterial adhesion compared with the corresponding modification on PE, which we attribute to the presence of defects in surfactant polymer assembly on PE. Our results are discussed from the perspective of critical factors, such as optimal PEO packing density and hydration thickness, that contribute to the effectiveness of surfactant polymers to shield a biomaterial from adhesive bacterial interactions.

Endothelial cell expression of monocyte chemotactic protein-1, tissue factor, and thrombomodulin on hydrophilic plasma polymers.

Endothelial cells (EC) from human aortas, microvessels, and pulmonary arteries were examined for their expression and activity of monocyte chemotactic protein-1 (MCP-1), tissue factor, and thrombomodulin in response to tumor necrosis factor-alpha (TNFalpha) on the hydrophilic plasma polymers gamma-butyrolactone (GBL) and N-vinyl-2-pyrrolidone (NVP), along with a fibronectin (FN) control. RNAs isolated from EC grown on these substrates were subjected to reverse transcription-polymerase chain reaction (RT-PCR) and dot-blot analysis. EC expression of MCP-1 and tissue factor was very low in the absence of TNFalpha but high for constitutively expressed thrombomodulin. TNFalpha induced EC expression and activity of MCP-1 and tissue factor and suppressed that of thrombomodulin on all substrates. Greater differences were seen with regard to cell origin, but little difference was seen among substrates. Basal secretion of MCP-1 was very low in aortic and pulmonary artery EC and even less in microvascular EC. TNFalpha increased MCP-1 secretion significantly in aortic and pulmonary artery EC but to a lesser extent in microvascular EC. In contrast, tissue factor expression was greater in pulmonary artery EC compared to microvascular and aortic EC. Basal expression of thrombomodulin was largely comparable for all three cell types grown on different surfaces, but TNFalpha suppressed thrombomodulin to different extents depending on the origin of the EC. The activity of tissue factor and thrombomodulin and the secretion of MCP-1 by EC were largely correlated with the expression of these genes. We conclude that EC origin may be an important determinant of cellular function on hydrophilic plasma polymer substrates. However, the differences in cellular function due to variations in substrate surface hydrophilicity could have been masked by the extracellular matrix remodeling that presumably occurred during EC growth to confluence.

Surface-dependent conformations of human fibrinogen observed by atomic force microscopy under aqueous conditions.

Conformational differences in human fibrinogen under aqueous conditions on hydrophobic, positively charged and negatively charged surfaces, were examined by atomic force microscopy (AFM). Hydrophobic and positively charged surfaces were prepared by depositing octadecyltrichlorosilane (OTS) and 3-aminopropyltriethoxysilane (APTES) respectively on cleaned glass coverslips forming self-assembled monolayers. The negatively charged surface was prepared by freshly cleaving muscovite mica. AFM operated in fluid tapping mode with an ultrasharp carbon spike probe was used to obtain the molecular scale images. Fibrinogen displayed a characteristic trinodular structure on all three surfaces. although additional U-shaped conformations were observed on mica. In its native hydrated state, fibrinogen is well represented by three connected ellipsoids in close proximity. Quantitative dimensional analysis, which yielded structural information in three dimensions, indicates that surface-dependent structural deformation or spreading of fibrinogen increases according to the order: mica < APTES < OTS. Molecular length, and D and E domain widths of fibrinogen are increased, while the corresponding heights are decreased. The results provide direct evidence that material surface properties affect the conformational state of interacting fibrinogen.

Platelet-derived microparticles on synthetic surfaces observed by atomic force microscopy and fluorescence microscopy.

Platelet activation on a thrombogenic surface includes the release of membrane-derived microparticles that provide catalytic sites for blood coagulation factors. Here, we describe a quantitative investigation on the production and dimensions of platelet-derived microparticles observed on glass and polyethylene under aqueous conditions, using atomic force microscopy (AFM) and complementary fluorescence microscopy. The results show that contact-activated platelet microparticles are not evenly distributed over a thrombogenic surface, but in clusters in close proximity to adherent platelets. The microparticles are localized near the platelet periphery, and in some cases appear to emanate from platelet pseudopodia, suggesting that formation may result from vesiculation of the pseudopodia. The microparticles measured 125 +/- 21 nm (n = 73) in the x-y dimensions and 5.2 +/- 3.6 nm in height. The results compared closely with 125 +/- 22 nm width and 4.1 +/- 1.6 nm height obtained for control preparations of thrombin activated microparticles, that were filtered and deposited on glass. Large differences between the measured widths and heights of adsorbed microparticles suggest that platelet microparticles may undergo spreading after attachment to a surface. The adsorbed microparticles expressed platelet membrane receptor GPIIb/IIIa, and many expressed the platelet activation marker P-selectin as determined by fluorescence microscopy. The high number distribution of procoagulant microparticles per unit area of surface compared with platelets suggests that platelet-derived microparticles provide a mechanistic route for amplifying thrombus formation on a thrombogenic surface.

Intermolecular force mapping of platelet surfaces on collagen substrata.

The interactions between plasma proteins and platelets are responsible for surface adsorption and activation of platelets, which leads to initiation of platelet-mediated thrombotic events at biomaterial surfaces. We are seeking to gain a fundamental understanding of these interactions. The atomic force microscope (AFM) has been used to create force maps across platelets adsorbed onto collagen substrata using peptide-modified probes. Combining the imaging and force-measuring capabilities of AFM, the force-mapping mode has been used to measure interactions of peptide-modified AFM probes with the surface. Observed differences in the force of adhesion are clearly evident in the platelet samples fixed in air, proving the ability of the AFM system to map adhesion. When this system is changed to a fluid environment we are no longer able to see such evident adhesion because of the membrane flexibility; instead the deformability of the surface is mapped. The specific interaction between the peptide sequence RGD and platelets was measured in a non-mapping mode of the AFM. Although this does not provide a force map, we can see significant differences between the forces measured on the substrate and those measured with a control hexapeptide.

Bacterial surface properties of clinically isolated Staphylococcus epidermidis strains determine adhesion on polyethylene.

The role of surface physiochemical properties of Staphylococcus epidermidis strains in adhesion to polyethylene (PE) was investigated under physiological flow conditions in phosphate buffered saline (PBS) and 1% platelet poor plasma (PPP). Four clinically isolated strains were divided into two groups: low and high relative hydrophobicity, and the F1198 and RP62A strains showing significantly greater hydrophobicity than the F21 and F1018 strains. In PBS, adhesion of all S. epidermidis strains was shear dependent from 0 to 15 dyn/cm2, after which adhesion becomes shear independent. Strains with higher surface hydrophobicity showed higher adhesion to PE, demonstrating the influence of bacterial surface hydrophobicity in nonspecific adhesion. Bacterial adhesion correlated well with bacterial surface hydrophobicity at low shear stresses (0-8 dyn/cm2). In 1% PPP, adhesion of all strains dramatically decreased and we found no correlation between bacterial surface hydrophobicity and adhesion. The presence of plasma proteins reduced nonspecific adhesion. S. epidermidis surface charge did not correlate with bacterial adhesion in either test media. The results suggested that S. epidermidis surface hydrophobicity may mediate nonspecific adhesion to PE at low shear stresses in protein-free media.

Atomic force microscopy for characterization of the biomaterial interface.

The molecular processes that occur at the interface of an implanted biomaterial determines the host response, including phenomena such as protein adsorption, conformational changes, and subsequent interactions with cellular components. Until recently, such processes could not be observed directly. Over the past decade, atomic force microscopy (AFM) has provided mechanistic insights into the molecular level interactions that occur at the biomaterial interface. Several unique operational modes have been developed which utilize intermittent contact with the sample and decrease applied shear forces. These dynamic modes also can be used to study the role of different structural components on biomaterial micromechanical properties. Force detection techniques allow molecular level studies of individual receptor-ligand binding events, and force mapping for determining structure/function relationships. Advancements in tip manufacturing, image processing techniques, the use of model surfaces and labeling all have contributed to the advancement of the AFM as a state-of-the-art research instrument. In this report, we examine the applicability of the AFM to the study of biomaterials and cell/molecular interactions.

Biomimetic engineering of non-adhesive glycocalyx-like surfaces using oligosaccharide surfactant polymers.

The external region of a cell membrane, known as the glycocalyx, is dominated by glycosylated molecules, which direct specific interactions such as cell-cell recognition and contribute to the steric repulsion that prevents undesirable non-specific adhesion of other molecules and cells. Mimicking the non-adhesive properties of a glycocalyx provides a potential solution to the clinical problems, such as thrombosis, that are associated with implantable devices owing to non-specific adsorption of plasma proteins. Here we describe a biomimetic surface modification of graphite using oligosaccharide surfactant polymers, which, like a glycocalyx, provides a dense and confluent layer of oligosaccharides. The surfactant polymers consist of a flexible poly(vinyl amine) with dextran and alkanoyl side chains. We show that alkanoyl side chains assemble on graphite through hydrophobic interaction and epitaxial adsorption. This constrains the polymer backbone to lie parallel to the substrate, with solvated dextran side chains protruding into the aqueous phase, creating a glycocalyx-like coating. The resulting biomimetic surface is effective in suppressing protein adsorption from human plasma protein solution.

Adhesion of Staphylococcus epidermidis and transposon mutant strains to hydrophobic polyethylene.

Staphylococcus epidermidis capsular polysaccharide adhesin (PS/A) and slime were studied as possible mediators of bacterial adhesion to NHLBI polyethylene (PE) under dynamic flow. This putative interaction was examined by quantifying the adhesion of M187 (PS/A+, slime+) parent strain and isogenic transposon mutant strain sn3 (PS/A-, slime-) to polyethylene (PE) under a range of physiologic shear stress conditions in both phosphate-buffered saline (PBS) and 1% platelet poor plasma (PPP). No significant differences in adhesion were noted between the M187 and sn3 strains in either test medium. However, adhesion of both strains in 1% PPP was decreased 75-95% compared to adhesion in PBS. In PBS, adhesion was shear stress dependent from 0-15 dyne/cm2, after which adhesion was comparatively shear stress independent. Adhesion in 1% PPP was independent of shear stress. Epifluorescent imaging of both strains labeled for slime confirmed the presence of slime on the surface of M187 and suggested that PS/A and slime promote the formation of large aggregates, as aggregates were totally absent in the images of the sn3 strain. The results suggest that PS/A and slime do not mediate S. epidermidis adhesion to bare PE or PE with adsorbed plasma proteins, but may be necessary for intercellular adhesion, which is important for biofilm formation.

A novel fiber-optic pH sensor incorporating carboxy SNAFL-2 and fluorescent wavelength-ratiometric detection.

A fiber-optic pH sensor was prepared using the self-referencing fluorescent pH probe carboxy seminaphtho-fluorescein (SNAFL-2). The ratio of the emission from the base form of this dye and the emission near the isoemissive point is insensitive to variations in the excitation intensity and photobleaching. The carboxy SNAFL-2 and a photoaffinity crosslinker, 4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester, were attached onto poly(acrylamide-co-vinylamine) to form a hydrophilic functional membrane for the fiber-optic sensor. Photo crosslinking was used to create a crosslinked pH-sensing membrane and covalently bind the membrane onto the surface of the PMMA optical fiber. The fluorescent properties of the membrane-fiber conjugate have been determined. The membrane is stable, and the pH sensor shows a fast response time and excellent resolution in a wide pH range of 3 to 11.

Three dimensional structure of human fibrinogen under aqueous conditions visualized by atomic force microscopy.

Fibrinogen plays a central role in surface-induced thrombosis. However, the interactions of fibrinogen with different substrata remain poorly understood because of the difficulties involved in imaging globular proteins under aqueous conditions. We present detailed three dimensional molecular scale images of fibrinogen molecules on a hydrophobic surface under aqueous conditions obtained by atomic force microscopy. Hydrated fibrinogen monomers are visualized as overlapping ellipsoids; dimers and trimers have linear conformations predominantly, and increased affinity for the hydrophobic surface compared with monomeric fibrinogen. The results demonstrate the importance of hydration on protein structure and properties that affect surface-dependent interactions.

Shear-dependent changes in the three-dimensional structure of human von Willebrand factor.

The three-dimensional tertiary structure of human von Willebrand Factor (vWF) on a hydrophobic surface under aqueous conditions and different shear stress regimes was studied by atomic force microscopy (AFM). vWF was imaged by AFM at molecular level resolution under negligible shear stress, under a local applied shear force (7.4 to 19 nN) using the AFM probe in contact mode scanning, and after subjecting vWF to a range of shear stress (0 to 42.4 dyn/cm2) using a rotating disk system. The results demonstrate that vWF undergoes a shear stress-induced conformational transition from a globular state to an extended chain conformation with exposure of intra-molecular globular domains at a critical shear stress of 35 +/- 3.5 dyn/cm2. The globular vWF conformation (149 nm by 77 nm and height 3.8 nm) is representative of native vWF after simple diffusion to the hydrophobic surface, followed by adhesion and some spreading. In a shear stress field above the critical value, protein unfolding occurs and vWF is observed in extended chain conformations oriented in the direction of the shear stress field with molecular lengths ranging from 146 to 774 nm and 3.4 nm mean height. The shear stress-induced structural changes to vWF suggest a close conformation-function relationship in vWF properties for thrombogenesis in regions of high shear stress.