Plasma-deposited tetraglyme surfaces greatly reduce total blood protein adsorption, contact activation, platelet adhesion, platelet procoagulant activity, and in vitro thrombus deposition

The ability of tetraethylene glycol dimethyl ether (tetraglyme) plasma deposited coatings exhibiting ultralow fibrinogen adsorption to reduce blood activation was studied with six in vitro methods, namely fibrinogen and von Willebrand's factor adsorption, total protein adsorption, clotting time in recalcified plasma, platelet adhesion and procoagulant activity, and whole blood thrombosis in a disturbed flow catheter model. Surface plasmon resonance results showed that tetraglyme surfaces strongly resisted the adsorption of all proteins from human plasma. The clotting time in the presence of tetraglyme surfaces was lengthened compared with controls, indicating a lower activation of the intrinsic coagulation cascade. Platelet adhesion and thrombin generation by adherent platelets were greatly reduced on tetraglyme-coated materials, compared with uncoated and Biospan-coated glass slides. In the in vitro disturbed blood flow model, tetraglyme plasma coated catheters had 50% less thrombus than did the uncoated catheters. Tetraglyme-coated materials thus had greatly reduced blood interactions as measured with all six methods. The improved blood compatibility of plasma-deposited tetraglyme is thus not only due to their reduced platelet adhesion and activation, but also to a generalized reduction in blood interactions.     

In vitro studies of platelet adhesion on laser-treated polyethylene terephthalate surface.

In order to improve blood compatibility, the surfaces of polyethylene terephthalate (PET) were treated using CO2 pulsed and KrF excimer lasers. The physico-chemical characterization of the laser-treated PET surfaces was carried out through attenuated total reflectance infrared spectroscopy and contact-angle measurements. The hemocompatibility of the laser-irradiated PET films was examined in vitro to evaluate their capability of inducing platelet adhesion in comparison with unmodified PET. The number of adhered platelets was determined by lactate dehydrogenase (LDH) activity measurement. Platelet adhesion on the untreated PET was relatively high compared to the laser-treated samples. Laser irradiation of PET surface reduced the number of adherent platelets and prevented platelet spreading on the surface. Reduction of platelet adhesion was attributed to the change in morphology, chemical structure, and crystallinity of the PET surface due to laser irradiation with various numbers of pulses. The morphology of adhered platelets on the PET surfaces was investigated by scanning electron microscopy (SEM). The SEM observations were consistent with the results obtained from LDH activity measurement.     

Serum protein adsorption and platelet adhesion on aspartic-acid-immobilized polysulfone membranes

Polysulfone (PSf) membranes that covalently conjugated with aspartic acid (ASP-PSf) were prepared and analyzed for hemocompatability. Compared to PSf or other types of surface-modified PSf membranes, the ASP-PSf membranes had a reduced ability to adsorb protein from either a plasma solution or a mixed solution of albumin, globulin and fibrinogen. This appears to be due to the creation of a hydrophilic surface by the aspartic acid zwitterion immobilized on the ASP-PSf membranes. Furthermore, the analyses of membrane protein adsorption showed that a mixed protein solution recapitulates the cooperative adsorption of proteins that occurs in plasma. We also found that the number of adhering platelets was the lowest on the ASP-PSf membranes and, in general, that platelet adhesion decreased in parallel with fibrinogen adsorption. In summary, aspartic acid immobilized on the ASP-PSf membranes, which have zwitterions with a net zero charge, effectively contributes to the hydrophilic and hemocompatible sites on the surface of the hydrophobic PSf membranes.     

Serum protein adsorption and platelet adhesion on aspartic-acid-immobilized polysulfone membranes

Polysulfone (PSf) membranes that covalently conjugated with aspartic acid (ASP-PSf) were prepared and analyzed for hemocompatability. Compared to PSf or other types of surface-modified PSf membranes, the ASP-PSf membranes had a reduced ability to adsorb protein from either a plasma solution or a mixed solution of albumin, globulin and fibrinogen. This appears to be due to the creation of a hydrophilic surface by the aspartic acid zwitterion immobilized on the ASP-PSf membranes. Furthermore, the analyses of membrane protein adsorption showed that a mixed protein solution recapitulates the cooperative adsorption of proteins that occurs in plasma. We also found that the number of adhering platelets was the lowest on the ASP-PSf membranes and, in general, that platelet adhesion decreased in parallel with fibrinogen adsorption. In summary, aspartic acid immobilized on the ASP-PSf membranes, which have zwitterions with a net zero charge, effectively contributes to the hydrophilic and hemocompatible sites on the surface of the hydrophobic PSf membranes.     

Serum protein adsorption and platelet adhesion on pluronic-adsorbed polysulfone membranes

We examined plasma protein adsorption and platelet adhesion to polysulfone (PSf) flat membranes coated with Pluronic with varying polyethylene oxide (PEO) block length. Adsorption of albumin, globulin and fibrinogen to Pluronic-coated PSf membranes was independent of plasma dilution when concentrations of human blood plasma above 20% were applied. Increasing coating concentrations of aqueous Pluronic solution resulted in decreased protein adsorption by the PSf membranes. Pluronic F68, which was more hydrophilic than Pluronic L62 or L64 and had 80% of PEO content, was the most effective at suppressing the adsorption of plasma proteins and platelet adhesion to PSf membranes. We developed a mixed protein solution containing human albumin, gamma-globulin and fibrinogen to attempt to mimic the competitive and cooperative binding effects found in plasma. Fibrinogen adsorption from plasma could be recapitulated by the mixed protein solution. The number of platelets adhering to the PSf membranes decreased as the coating concentration of Pluronic solution was increased, and platelet adhesion decreased in parallel with fibrinogen adsorption. These results suggest that the bioinert property of PEO segments in the Pluronic, which is ascribed to their high flexibility in aqueous media, suppresses the adsorption of plasma proteins and platelets to the Pluronic-coated PSf membranes.     

In vitro short-term platelet adhesion on various metals

The in vitro short-term platelet adhesion on various metals, as accelerated by the addition of Ca²⁺, was evaluated in this study. Metals used for medical devices [an austenitic stainless steel, a cobalt (Co)-chromium (Cr)-molybdenum (Mo) alloy, a titanium (Ti)-6 aluminum (Al)-4 vanadium (V) alloy, a Ti-6Al-7 niobium (Nb) alloy, a Tinickel (Ni) alloy, and commercially pure Ti] were immersed into a platelet-rich plasma solution for 5 or 20 min, and platelet adhesion and aggregation on the surfaces were observed using a scanning electron microscope. The platelet adhesion level on each metal after 5 min of immersion in a platelet-rich plasma solution was the smallest in this order: stainless steel ≤ Co-Cr-Mo alloy < Ti-6Al-4V alloy < Ti-6Al-7Nb alloy < Ti-Ni alloy = Ti. The levels after 5 min of immersion were almost the same as those after 20 min of immersion. Platelet adhesion was minimal on stainless steel and Co-Cr-Mo alloy, which have a Cr₂O₃-containing passive surface oxide film, but was accelerated on Ti and Ti alloys having a TiO₂-contanining film. A Cr₂O₃-containing oxide film has a lower relative permittivity than a TiO₂-contanining film; it thus supports a larger electrostatic force than the latter, adsorbs more albumins, which work as inhibitory proteins, and inhibits platelet aggregation. Therefore, platelet adhesion and aggregation are controlled by the composition of the surface oxide film on a metal due to the relative permittivity of the metal, which influences the amount of adsorbed proteins.     

Fibrinogen adsorption, platelet adhesion and activation on mixed hydroxyl-/methyl-terminated self-assembled monolayers

The effect of surface wettability on fibrinogen adsorption, platelet adhesion and platelet activation was investigated using self-assembled monolayers (SAMs) containing different ratios of longer chain methyl- and shorter chain hydroxyl-terminated alkanethiols (C15CH3 vs. C11OH) on gold. Protein adsorption studies were performed using radiolabeled human fibrinogen (HFG). Platelet adhesion and activation studies with and without pre-adsorbed fibrinogen, albumin and plasma were assessed using scanning electron microscopy (SEM) and a glutaraldehyde-induced fluorescence technique (GIFT). Results demonstrated a linear decrease of HFG adsorption with the increase of OH groups on the monolayer (increase of the hydrophilicity). Platelet adhesion and activation also decrease with increase of hydrophilicity of surface. Concerning SAMs pre-immersed in proteins, fibrinogen adsorption was related with high platelet adhesion and activation. The passivant effect of albumin on platelet adhesion and activation was only demonstrated on SAMs contained C11OH. When all the blood proteins are present (plasma) platelet adhesion was almost absent on SAMs with 65% and 100% C11OH. This could be explained by the higher albumin affinity of the SAMs with 65% C11OH and the lower total protein adsorption associated with SAMs with 100% C11OH.     

Biocompatible and biodegradable phosphorylcholine ionomers with reduced protein adsorption and cell adhesion

In this paper a recently developed biodegradable phosphorylcholine ionomer (PC ionomer) was evaluated in different biological environments with a focus on the adsorption of proteins (fibrinogen) and the adhesion of cells. Our results have shown that the polar phosphoryl choline (PC) group may be enriched at the surface of cast films with an added hydrophilic environment. X-ray photoelectron spectroscopy confirmed the surface depletion of PC groups in dry conditions, as nitrogen and phosphorous atoms were found in the bulk of the material but not at the outermost surface layer. The surface enrichment leads to a strongly hydrophilic surface that prevents the adsorption of proteins and reduces the adhesion of cells. The non-functional and hydrophobic reference poly(trimethylene carbonate) (PTMC) adsorbs both proteins and cells, thus the wetting and low adhesion behavior of the PC ionomer can be attributed to the introduced PC functionality. Since the in vivo acceptance of biomaterials is determined by their ability to withstand protein adsorption the PC ionomer described in this paper is highly interesting for a number of in vivo applications in which the adsorption of proteins may be critical, for example, blood contact events.     

Biocompatible and biodegradable phosphorylcholine ionomers with reduced protein adsorption and cell adhesion

In this paper a recently developed biodegradable phosphorylcholine ionomer (PC ionomer) was evaluated in different biological environments with a focus on the adsorption of proteins (fibrinogen) and the adhesion of cells. Our results have shown that the polar phosphoryl choline (PC) group may be enriched at the surface of cast films with an added hydrophilic environment. X-ray photoelectron spectroscopy confirmed the surface depletion of PC groups in dry conditions, as nitrogen and phosphorous atoms were found in the bulk of the material but not at the outermost surface layer. The surface enrichment leads to a strongly hydrophilic surface that prevents the adsorption of proteins and reduces the adhesion of cells. The non-functional and hydrophobic reference poly(trimethylene carbonate) (PTMC) adsorbs both proteins and cells, thus the wetting and low adhesion behavior of the PC ionomer can be attributed to the introduced PC functionality. Since the in vivo acceptance of biomaterials is determined by their ability to withstand protein adsorption the PC ionomer described in this paper is highly interesting for a number of in vivo applications in which the adsorption of proteins may be critical, for example, blood contact events.     

Molecular responses of vascular smooth muscle cells and phagocytes to curcumin-eluting bioresorbable stent materials

A major complication of coronary stenting is restenosis, often accompanied by inflammatory reactions and smooth muscle cell proliferation. Curcumin has been shown to possess anti-inflammatory and anti-proliferative properties, thus we hypothesize that locally released curcumin by coronary stent would diminish in-stent restenosis. As a first test of this hypothesis, curcumin-eluting PLLA films (C-PLLA) were produced and the anti-inflammatory and anti-proliferative properties were then tested using peritoneal phagocytes and human coronary artery smooth muscle cell (hCASMCs) culture systems. We find that the addition of curcumin reduced phagocyte accumulation and activation on C-PLLA films. On the other hand, C-PLLA significantly reduced the proliferation, but not the adhesion, of hCASMCs. The molecular responses of hCASMCs to C-PLLA were further assessed by cDNA microarray analysis. Curcumin up-regulated genes related to apoptosis and enhanced the expression of anti-proliferative and anti-inflammatory factors, and of antioxidants. Equally important, C-PLLA inhibited the cell cycle progression of adherent hCASMCs. The results suggest that curcumin regulates gene expression and cell function through the protein kinase (PK) and mitogen-activated protein kinase (MAPK) pathways. These results support the use of curcumin to inhibit in-stent restenosis.     

In vitro adhesion and platelet aggregation properties of bacteremia-associated lactobacilli

Eight bacteremia-associated Lactobacillus strains were evaluated in vitro for the ability to adhere to human intestinal mucosa and to aggregate platelets. Adherence varied significantly among the strains, and platelet aggregation was induced by three strains. In conclusion, strong binding ability does not appear to be a prerequisite for the involvement of lactobacilli in bacteremia or to their ability to aggregate platelets.     

Photochemically immobilized polymer coatings: effects on protein adsorption,cell adhesion,and leukocyte activation

Amphiphilic chains of 4-benzoylbenzoic acid moieties and polymer were photochemically immobilized onto silicone rubber to ask whether the covalently coupled polymers would passivate the silicone rubber by inhibiting protein adsorption and subsequent cell adhesion and activation. Three groups of polymers were utilized: the hydrophilic synthetic polymers of polyacrylamide, polyethylene glycol, and polyvinylpyrrolidone; the glycosaminoglycan, hyaluronic acid; and poly(glycine-valine-glycine-valine-proline), a polypeptide derived from the sequence of clastin. Each coating variant decreased the adsorption of fibrinogen and immunoglobulin G compared to uncoated silicone rubber. All except the methoxy-polyethylene glycol coating nearly abolished fibroblast growth, but none of the coating variants inhibited monocyte or polymorphonuclear leukocyte adhesion. Interleukin-1β, interleukin-1 receptor antagonist, and tumor necrosis factor-α secretion by leukocytes were not statistically different between any of the coating variants and uncoated silicone rubber. However, the methoxy-polyethylene glycol and elastin-based polypeptide coatings, which supported the highest numbers of adherent monocytes, also elicited the lowest levels of pro-inflammatory cytokine secretion. When these in vitro data were collectively evaluated, the coating that most effectively passivated silicone rubber was the polypeptide derived from elastin.     

Photochemically immobilized polymer coatings: effects on protein adsorption, cell adhesion, and leukocyte activation

Amphiphilic chains of 4-benzoylbenzoic acid moieties and polymer were photochemically immobilized onto silicone rubber to ask whether the covalently coupled polymers would passivate the silicone rubber by inhibiting protein adsorption and subsequent cell adhesion and activation. Three groups of polymers were utilized: the hydrophilic synthetic polymers of polyacrylamide, polyethylene glycol, and polyvinylpyrrolidone; the glycosaminoglycan, hyaluronic acid; and poly(glycine-valine-glycine-valine-proline), a polypeptide derived from the sequence of elastin. Each coating variant decreased the adsorption of fibrinogen and immunoglobulin G compared to uncoated silicone rubber. All except the methoxy-polyethylene glycol coating nearly abolished fibroblast growth, but none of the coating variants inhibited monocyte or polymorphonuclear leukocyte adhesion. Interleukin-1beta, interleukin-1 receptor antagonist, and tumor necrosis factor-alpha secretion by leukocytes were not statistically different between any of the coating variants and uncoated silicone rubber. However, the methoxy-polyethylene glycol and elastin-based polypeptide coatings, which supported the highest numbers of adherent monocytes, also elicited the lowest levels of proinflammatory cytokine secretion. When these in vitro data were collectively evaluated, the coating that most effectively passivated silicone rubber was the polypeptide derived from elastin.     

Effect of reduced protein adsorption on platelet adhesion at the phospholipid polymer surfaces

We prepared polymers having a phospholipid polar group, poly[ω-methacryloyloxyalkyl phosphorylcholine (MAPC)-co-n-butyl methacrylate(BMA)], as new biomedical materials and evaluated their blood compatibility with attention to protein adsorption and platelet adhesion. The total amount of proteins adsorbed on the polymer surface from human plasma was determined, and the distribution of adsorbed proteins on the plasma-contacting surface was analyzed. The amount of proteins adsorbed on every poly(MAPC-co-BMA) was small compared with that observed on polymers without the phospholipid polar group. However, there was no significant difference in the amount of adsorbed proteins on the poly(MAPC-co-BMA) even when the methylene chain length between the phospholipid polar group and the backbone in the MAPC moiety was altered. Platelet adhesion on the polymer surface from a platelet suspension in a buffered solution was evaluated with and without plasma treatment on the surface. When a rabbit platelet suspension was brought into contact with the poly(BMA) surface after treatment with plasma, many platelets adhered and aggregated. However, a reduced amount of platelet adhered on the poly(BMA) was found in the case of direct contact with the platelet suspension. On the other hand, the poly(MAPC-co-BMA)s could inhibit platelet adhesion under both conditions. Based on these results, it can be concluded that the proteins adsorbed on the surface play an important role in determining the platelet adhesion and suppression of the protein adsorption on the surface, which is one of the most significant ways of inhibiting platelet adhesion.     

In vitro platelet adhesion and in vivo antithrombogenicity of heparinized polyetherurethaneureas

We investigated in-vitro platelet adhesion to polyetherurethaneureas, to which heparin was bound covalently or by ionic bonding. When heparin was bound to polymers, platelet adhesion and platelet activation upon adhesion were suppressed with the increasing content of bound heparin in the polymer. Platelets were activated upon adsorption to different degrees according to the method of heparinization. The platelet adhesion and the platelet activation upon adhesion appeared to be regulated by the electrostatic repulsion between platelet and anionic surface of covalently or ionically heparinized polymer, rather than by the physiological action of bound heparin. The effect of the method of heparinization seemed to be related to the molecular heterogeneity of heparin. Heparinized polyurethanes which interacted very weakly with platelets in vitro were tested for in-vivo antithrombogenicity. The test was carried out by the implantation of a suture of the heparinized polyurethane into canine veins. Ionically heparinized polyurethane did not form a thrombus and maintained a smooth surface over a long period. On the other hand, covalently heparinized polyurethane formed a small amount of thrombus and grew endothelial cells from the insertion point.     

Resisting protein adsorption on biodegradable polyester brushes

The protein adsorption and degradation behaviors of poly(lactic acid), poly(glycolic acid) (PGA) and poly(ε-caprolactone) (PCL) brushes and their co-polymer brushes with oligo(ethylene glycol) (OEG) were studied. Both brush structure and relative amount of OEG and polyester were found to be important to the protein resistance of the brushes. A protein-resisting surface can be fabricated either by using OEG as the top layer of a copolymer brush or by increasing the amount of OEG relative to polyester when using a hydroxyl terminated OEG (OEG-OH) and a methoxy terminated OEG (OEG-OMe) mixture as the substrate layer. The degradation of single polyester brushes and their co-polymer brushes using OEG-OH as a substrate layer or using OEG as a top layer was hindered. This phenomenon was rationalized by the inhibition of the proposed back-biting process as the hydroxy end groups of polyester were blocked by OEG molecules. Among these brushes tested, PGA co-polymer brushes using the methoxy/hydroxyl OEG mixture as the substrate layer proved to be both protein-resistant and degradable due to the relatively large amount of OEG moieties and the good biodegradability of PGA.     

In vitro and in vivo studies on biodegradable CaMgZnSrYb high-entropy bulk metallic glass

In order to enhance the corrosion resistance of the Ca₆₅Mg₁₅Zn₂₀ bulk metallic glass, which has too fast a degradation rate for biomedical applications, we fabricated the Ca₂₀Mg₂₀Zn₂₀Sr₂₀Yb₂₀ high-entropy bulk metallic glass because of the unique properties of high-entropy alloys. Our results showed that the mechanical properties and corrosion behavior were enhanced. The in vitro tests showed that the Ca₂₀Mg₂₀Zn₂₀Sr₂₀Yb₂₀ high-entropy bulk metallic glass could stimulate the proliferation and differentiation of cultured osteoblasts. The in vivo animal tests showed that the Ca₂₀Mg₂₀Zn₂₀Sr₂₀Yb₂₀ high-entropy bulk metallic glass did not show any obvious degradation after 4 weeks of implantation, and they can promote osteogenesis and new bone formation after 2 weeks of implantation. The improved mechanical properties and corrosion behavior can be attributed to the different chemical composition as well as the formation of a unique high-entropy atomic structure with a maximum degree of disorder.     

The role of adsorbed fibrinogen in platelet adhesion to polyurethane surfaces: a comparison of surface hydrophobicity, protein adsorption, monoclonal antibody binding, and platelet adhesion

Ten specially synthesized polyurethanes (PUs) were used to investigate the effects of surface properties on platelet adhesion. Surface composition and hydrophilicity, fibrinogen (Fg) and von Willebrand's factor (vWf) adsorption, monoclonal anti-Fg binding, and platelet adhesion were measured. PUs preadsorbed with afibrinogenemic plasma or serum exhibited very low platelet adhesion, while adhesion after preadsorption with vWf deficient plasma was not reduced, showing that Fg is the key plasma protein mediating platelet adhesion under static conditions. Platelet adhesion to the ten PUs after plasma preadsorption varied greatly, but was only partially consistent with Fg adsorption. Thus, while very hydrophilic PU copolymers containing PEG that had ultralow Fg adsorption also had very low platelet adhesion, some of the more hydrophobic PUs had relatively high Fg adsorption but still exhibited lower platelet adhesion. To examine why some PUs with high Fg adsorption had lower platelet adhesion, three monoclonal antibodies (mAbs) that bind to sites in Fg thought to mediate platelet adhesion were used. The antibodies were: M1, specific to gamma-chain C-terminal; and R1 and R2, specific to RGD containing regions in the alpha-chain N- and C-terminal, respectively. Platelet adhesion was well correlated with M1 binding, but not with R1 or R2 binding. When these mAbs were incubated with plasma preadsorbed surfaces, they blocked adhesion to variable degrees. The ability of the R1 and R2 mAbs to partially block adhesion to adsorbed Fg suggests that RGD sites in the alpha chain may also be involved in mediating platelet adhesion and act synergistically with the C-terminal of the gamma-chain.     

Fluorine doping into diamond-like carbon coatings inhibits protein adsorption and platelet activation

The first major event when a medical device comes in contact with blood is the adsorption of plasma proteins. Protein adsorption on the material surface leads to the activation of the blood coagulation cascade and the inflammatory process, which impair the lifetime of the material. Various efforts have been made to minimize protein adsorption and platelet adhesion. Recently, diamond-like carbon (DLC) has received much attention because of their antithrombogenicity. We recently reported that coating silicon substrates with fluorine-doped diamond-like carbon (F-DLC) drastically suppresses platelet adhesion and activation. Here, we evaluated the protein adsorption on the material surfaces and clarified the relationship between protein adsorption and platelet behaviors, using polycarbonate and DLC- or F-DLC-coated polycarbonate. The adsorption of albumin and fibrinogen were assessed using a colorimetric protein assay, and platelet adhesion and activation were examined using a differential interference contrast microscope. A higher ratio of albumin to fibrinogen adsorption was observed on F-DLC than on DLC and polycarbonate films, indicating that the F-DLC film should prevent thrombus formation. Platelet adhesion and activation on the F-DLC films were more strongly suppressed as the amount of fluorine doping was increased. These results show that the F-DLC coating may be useful for blood-contacting devices.