Two different types of nonthrombogenic surfaces: PEG suppresses platelet adhesion ATP-independently but HEMA-St block copolymer requires ATP consumption of platelets to prevent adhesion

Poly(ethylene glycol) (PEG) and a hydrophobic-hydrophilic microdomain structured block copolymer comprising poly(2-hydroxyethyl methacrylate) and polystyrene (HEMA-St) have been reported to show good blood compatibility owing to inhibition of platelet activation. By using a computer-assisted novel technique to analyze platelet behavior on the surfaces, we found two different mechanisms to prevent platelet adhesion. Platelets were prevented from adhesion and spreading on the microdomain surface and retained cell movement for a long time. The platelet movement velocity was not significantly different between PEG-grafted surfaces and HEMA-St block copolymer-cast surfaces. However, platelet motion was qualitatively different. Platelets on HEMA-St block copolymer-cast surfaces moved with rolling, spinning, and vibrating, whereas platelet movement was limited to oscillatory vibration on PEG-grafted surfaces. When platelets were treated with NaN(3), an adenosine triphosphate (ATP) synthesis inhibitor, before contacting the surfaces, platelets movement velocity was decreased only on HEMA-St block copolymer-cast surfaces. Such an inhibitory effect was hardly observed with platelets on PEG-grafted surfaces. We propose two different mechanisms to prevent platelet adhesion onto surfaces. One is ATP-independent as observed with PEG, and the other is ATP-dependent for HEMA-St block copolymer, where platelets consume ATP to prevent adhesion.     

Synthesis of a new antithrombogenic block copolymer containing fluorinated segments: poly(nonafluorohexyl acrylate-b-styrene)

Two types of block copolymers, 3a and 3b , were synthesized from α-hydro-ω-(2-hydroxyethylthio)poly[1-(3,3,4,4,5,5,6,6,6-nonafluorohexyloxycarbonyl)ethylene] ( 1a ) or α-hydro-ω-(2-aminoethylthio)poly[1-(hexyloxycarbonyl)ethylene] ( 1b ) and α,ω-bis(4-cyanatophenylthio)-poly(1-phenylethylene) ( 2 ), respectively, and the adhesion of blood platelets to these polymer surfaces was evaluated. Adhesion and activation of platelets were found to be effectively suppressed at the lamellar-microdomain surface of block copolymer 3a , having a surface free energy gap between microdomains of about 20 dyn/cm, whereas no such a suppression was observed for the microdomain structured surface of block copolymer 3b with a small surface free energy gap between the microdomains. Further, a random copolymer from nonafluorohexyl acrylate and styrene without microdomain structure does not suppress the adhesion and activation of platelets. From these results, it was concluded that the microdomain morphology and the surface free energy gap between microdomain is important to produce antithrombogenicity.     

Hydrophilic-hydrophobic microdomain surfaces having an ability to suppress platelet aggregation and their in vitro antithrombogenicity

Block copolymers were synthesized by a coupling reaction of hydrophilic chains of poly(2-hydroxyethyl methacrylate) (PHEMA) with hydrophobic chains of polystyrene (PSt), or poly(dimethyl siloxane) (PDMS). Microstructures of films of the block copolymers exhibited a hydrophilic-hydrophobic microphase separated structure. For evaluation of in vivo antithrombogenicity, small diameter tubes (1.5 mm I.D. and 20 cm length) coated by the copolymers on their internal surfaces were implanted in rabbits as arteriovenous shunts. Occlusion times of the tubes, measured by formation of thrombus, were three days for PHEMA, two days for PSt, and three days for PDMS. The block copolymers showed excellent antithrombogenic properties: occlusion times were 20 days for HEMA-St block copolymer and 12 days for HEMA-DMS block copolymers. In vitro examination of polymer-platelet interaction in terms of platelet adhesion and aggregation, which are important initial processes of blood coagulation, demonstrated suppressed adhesion and aggregation on microdomain surfaces constructed of hydrophilic and hydrophobic block copolymers. From both in vivo and in vitro examination, it was concluded that HEMA-St and HEMA-DMS block copolymers showed promising antithrombogenic activities by suppressing activation and aggregation of platelets.     

Adhesion behavior of rat lymphocyte subpopulations (B cell and T cell) on the surface of polystyrene/polypeptide graft copolymer

Polyvinyl/polypeptide graft copolymers having microdomain structure on their surfaces were newly synthesized for the development of a specific cell separator, and the adhesion behavior of rat lymphnode lymphocytes with these materials was examined by column method. Morphologic change of lymphocytes adherent to the graft copolymers was found to be less than that of cells adherent to corresponding homopolymers, i.e., polystyrene and poly (gamma-benzyl L-glutamate). Separation of lymphocyte subpopulations (B cell and T cell) was examined in the absence of serum proteins. The adhesion selectivity for B cell was found to depend on the microdomain structure, since the highest value, 2,2, was observed for the graft copolymer with the polypeptide content of 50 wt%. Synthetic polypeptide derivatives may be promising materials which substitute for a conventional system with nylon fiber and fetal calf serum.     

Effect of microphase separated structure of polystyrene/polyamine graft copolymer on adhering rat platelets in vitro

Rat platelet adhesion on microphase separated surface of polystyrene/polyamine graft copolymers (SA copolymers) was investigated. Shapes of adhering platelet were very much changeable in response to the mode of microphase separation of SA copolymer surfaces. We assume that the microphase separated structure of SA copolymer may regulate the shape change of platelet through its effect on a redistribution of proteins and/or lipids present at the plasma membrane of platelets.     

Platelet adherence and detachment: a flow study with a series of hydroxyethyl methacrylate-ethyl methacrylate copolymers using video microscopy

The adhesion and detachment of platelets were studied on glass coatings of a series of copolymers of hydroxyethyl methacrylate (HEMA) and ethyl methacrylate (EMA). Observations of the interactions of mepacrine labelled washed platelets with these surfaces from a flowing (500 s-1 wall shear rate) suspension in Tyrode's solution containing albumin and red cells were made with epifluorescent video microscopy (EVM). Total platelet adhesion, including platelets which adhere on first contact and platelets which attach temporarily before adhesion, and the number of detaching platelets were minimal for the 0 and 20% EMA copolymers, reached a maximum for the 50% EMA copolymer and showed reduced values for the 80% and 100% EMA copolymers. For the 50, 80, and 100% EMA copolymers, the adhesion values expressed, as a percentage of total contacting platelets, were not different. Albumin adsorption to these copolymers shows a continuous increase from the 0% to the 100% EMA copolymer. It is likely that the peak in platelet adhesion at the 50% EMA composition is related to: low protein adsorption on the 0 and 20% EMA copolymers, too little albumin adsorption to block adhesion on the 50% EMA copolymer, and full-scale blocking on the 80 and 100% EMA copolymers due to greater albumin adsorption.     

Effect of microstructure of poly(propylene-oxide)-segmented polyamides on platelet adhesion

The relationship between microstructure and platelet adhesivity of six types of poly(propylene oxide) (PPO)-segmented polyamides based on the polyamide segments nylon 210, 310, 410, 510, 610, and 710 were investigated. These multiblock PPO-segmented copolymers were prepared by interfacial polycondensation. Physical characterization of these copolymers was by means of thermal analysis, transmission electron microscope, wide-angle X-ray diffraction (WAXD), and small-angle X-ray scattering (SAXS). The WAXD and SAXS measurements showed that the copolymers had microstructures containing crystalline and amorphous phases and that these microstructures, represented by means of crystallite thickness and long period, varied with incorporation of PPO segments. Blood compatibility of these copolymers was evaluated by estimating the amount of adhering platelets on the copolymer surfaces. The amount of adhering platelets was minimum for the surfaces of the copolymers having a crystallite thickness of 6.0-6.5 nm and a long period of 12-13 nm. This result suggests that the particular size and distribution of the crystalline and amorphous phases in the copolymer could be determining factors for suppressing platelet adhesion on the copolymer surface, and that the control of these factors could lead to ideal antithrombogenic polymers.     

Platelet adherence and detachment with adsorbed fibrinogen: a flow study with a series of hydroxyethyl methacrylate-ethyl methacrylate copolymers using video microscopy.

The adhesion and detachment of platelets were studied on glass coatings of a series of copolymers of hydroxyethyl methacrylate (HEMA) and ethyl methacrylate (EMA) with preadsorbed fibrinogen. Observations of the interactions of acridine-orange-labeled washed platelets with these surfaces from a flowing (500 s-1 wall shear rate) suspension in Tyrode's solution containing albumin and red cells were made with epifluorescent video microscopy (EVM). In some cases preadsorbed materials were incubated for 24 h, during which little or no loss of protein occurred. Protein surface concentration, by itself, was a poor indicator of expected cell adhesion and morphology. Surface chemistry was a second important factor which must be considered. A third observation is that for the 100% EMA copolymer, 24 h of incubation led to a large reduction in platelet adhesion when compared to the 100% EMA material without incubation. For the 0% and 100% EMA polymers, the percentage of contacting platelets which adhere and detach is greater for the 24-h incubation cases than for those not incubated. These results led to the conclusion that our most hydrophilic surface favors adhesion with detachment, transient cell contact, over long-term adhesion, as does incubation of adsorbed protein. A brief discussion is presented of a possible connection between this behavior and platelet consumption in vivo for hydrogels.     

Interaction of polystyrene/poly(gamma-benzyl L-glutamate) and poly(methyl methacrylate)/poly(gamma-benzyl L-glutamate) block copolymers with plasma proteins and platelets

A-B-type block copolymers, consisting of polystyrene (PST) or poly(methyl methacrylate) (PMMA) forming segment (A) and poly(gamma-benzyl L-glutamate) (P[Glu(OBzl)]) segment (B), were synthesized and the thrombus formation on these block copolymer films was investigated in relation to the adsorption of plasma proteins and the activation of platelets. The relative amount of thrombus formation was higher on homopolymers than on block copolymers. The amount of thrombus formation became less, with decreasing content of P[Glu(OBzl)] in the PST block copolymers and with increasing content of P[Glu(OBzl)] in the PMMA block copolymers. Adsorption of bovine serum albumin(BSA), bovine gamma-globulin (B gamma G) and bovine plasma fibrinogen(BPF) onto polymer films was also investigated. More proteins were adsorbed and denatured when adsorbed onto PST and PMMA than onto block copolymers. With increasing content of P[Glu(OBzl)] in the PST block copolymers, the degree of denaturation of adsorbed proteins increased, while the amount of protein adsorption was unaffected. Conversely, with increasing content of P[Glu(OBzl)] in the PMMA block copolymers, the degree of denaturation of adsorbed proteins decreased, while similarly the amount of protein adsorption was unaffected. Adhesion of platelets from platelet suspension (WP) to polymer films coated with one of the plasma proteins showed that the activation of adhered platelets was suppressed when there was a lower degree of denaturation of coated proteins. In the same experiments using platelet-rich plasma(PRP), neither the number of platelets adhered nor the degree of activation of the adhered platelets was correlated with the composition of the polymer films.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antithrombogenicity and oxygen permeability of block and graft copolymers of polydimethylsiloxane and poly(alpha-amino acid)

A-B-A-type block copolymers consisting of poly(alpha-amino acid) as A component and polydimethylsiloxane as B component and graft copolymers consisting of polydimethylsiloxane as trunk polymer and poly(alpha-amino acid) as branch polymer were synthesized. gamma-Benzyl-L- or DL-glutamate, epsilon-benzyloxycarbonyl L-lysine, and sarcosine were used as alpha-amino acid. Different microphase-separated structures were found on the film surface according to the copolymer composition and the casting conditions. In vitro antithrombogenicity test showed higher antithrombogenicity of block or graft copolymers than homopolymers. The best antithrombogenicity was independent of the kind of alpha-amino acid and the degree of polymerization of copolymers. The best ratio was 65-75% in block copolymer and 40-50% in the case of graft copolymer. The oxygen permeability of block and graft copolymer film was intermediate between those of homopolymers and varied with changing the composition of the copolymer. These experiments showed that the microphase-separated structure on the film surface was most important both for the antithrombogenicity and oxygen permeability of these copolymer films.     

In vitro and ex vivo platelet interactions with hydrophilic-hydrophobic poly(ethylene oxide)-polystyrene multiblock copolymers

Hydrophilic-hydrophobic multiblock copolymers synthesized from telechelic oligomers of poly(ethylene oxide) (PEO) and polystyrene (PS) have been used to study the influence of hydrophilic and hydrophobic balance on interfacial interactions of these surfaces with blood components. In vitro coagulation assays show no inherent ability of these amphiphilic surfaces to affect contact activation or coagulation factors. In vitro platelet adhesion and release reactions from rabbit platelet-rich plasma were shown to be greatest on Biomer and PS homopolymer surfaces and least on cross-linked PEO surfaces, with the PEO-PS block copolymers demonstrating intermediate responses. These same substrates were tested in a new low-flow, low-shear arterio-artery shunt system in rabbits. Whole blood occlusion times were not a direct function of hydrophilic content as both PEO and PS homopolymers and Biomer showed short occlusion times, while PEO-PS block copolymers prolonged occlusion times considerably, depending on composition. Overall, results suggest that PEO-PS block copolymers promote unique whole blood responses in contrast to homopolymer and Biomer controls which are more complex than direct correlations to bulk hydrophilic and hydrophobic contents.     

Biocompatibility of poly(epsilon-caprolactone)/poly(ethylene glycol) diblock copolymers with nanophase separation

In this study, we prepared diblock copolymers of poly(epsilon-caprolactone) (PCL) and poly(ethylene glycol) (PEG) by aluminum alkoxide catalysts. The biological responses to the spin cast surface of different PCL/PEG diblock copolymers were investigated in vitro. Our results showed that surface hydrophilicity improved with the increased PEG segments in diblock copolymers and that bacteria adhesion was inhibited by increased PEG contents. PCL-PEG 23:77 showed nanotopography on the surface. The number of adhered endothelial cells, platelets and monocytes on diblock copolymer surfaces was inhibited in PCL-PEG 77:23 and enhanced in PCL-PEG 23:77. Nevertheless, the platelet and monocyte activation on PCL-PEG 23:77 was reduced. PCL-PEG 23:77 had better cellular response as well as lower degree of platelet and monocyte activation. The current study was the first one to demonstrate that surface nanotopography could influence not only cell adhesion and growth but also platelet and monocyte activation.     

Platelet adhesion on a bioresorbable poly(propylene fumarate-co-ethylene glycol) copolymer

Platelet adhesion and aggregation on poly(propylene fumarate-co-ethylene glycol), P(PF-co-EG), hydrogels was examined under both static and flow conditions. Adherent platelets were quantified under static conditions using both 111Indium oxine-labeled platelets as well as a lactate dehydrogenase, LDH, assay. The radiolabeling assay showed a significant decrease in platelet attachment on the copolymer hydrogel films relative to the poly(propylene fumarate), PPF, homopolymer. In addition, there were reductions in adhesion resulting from the increase in poly(ethylene glycol), PEG, weight percent or molecular weight. There was good agreement between both assays under static conditions for the copolymer films. Platelet surface coverage was quantified under flow conditions in a parallel plate flow chamber using the LDH assay. There was a dramatic decrease in the number of adherent platelets on the copolymers relative to glass and silicone rubber controls. All of the copolymer surfaces showed minimal aggregation with no thrombus formation or platelet spreading as assessed qualitatively using scanning electron microscopy. These results suggest that P(PF-co-EG) is a good candidate for development as a cardiovascular implant.     

Microstructure of poly[polytetrahydrofuran-block-poly(sebacoylchloride-alt-hexamethylenediamine)] and its role in blood compatibility

The microstructure of poly[polytetrahydrofuran-block-poly(sebacoyl chloride-alt-hexamethylenediamine)]s 1–4 , containing polytetrahydrofuran (PTHF) blocks of various molecular weights, and their blood compatibility were studied. These multiblock copolymers were prepared by interfacial polycondensation. The characterization of these copolymers was carried out by means of transmission electron microscopy (TEM), differential scanning calorimetry (DSC), dynamic mechanical measurements, wide-angle X-ray diffraction (WAXD), small-angle X-ray scattering (SAXS), and electron spectroscopy for chemical analysis (ESCA). The TEM observation revealed the formation of a spherulitic structure at the copolymer surfaces, which is closely related to the homopolymer, polyamide (PA) 610. The DSC and dynamic mechanical measurements indicate the presence of distinct phase separation between PTHF and PA 610 blocks, and of the PTHF block in the copolymer being partially crystallized. The WAXD and SAXS indicate the formation of microstructures composed of crystalline and amorphous phases in the copolymer. Moreover, ESCA measurements verify that the surface chemical composition of the copolymer is identical to their bulk composition. Blood compatibility of these copolymers was evaluated by estimating platelet adhesion on the copolymer surfaces. Platelet adhesion was found to be affected by the PA 610 crystallinity, including the size and distribution of the crystalline phase in the case of the copolymers in which the PTHF blocks are completely amorphous (M?_n = 980). On the contrary, platelet adhesion at the copolymers in which the PTHF blocks are partially crystallized (M?_n ≥ 1560) depends upon the crystallinity of both PA 610 and PTHF, including the balance of crystalline (PA 610 and PTHF) and amorphous (mainly PTHF) phases. This result suggests that the balance of the crystalline and amorphous phase distribution in the copolymer is the most determinative factor for suppressing platelet adhesion at the copolymer surface.     

Fibroblast adhesion to micro- and nano-heterogeneous topography using diblock copolymers and homopolymers

Polymeric substrates of different surface chemistry and length scales were found to have profound influence on cell adhesion. The adhesion of fibroblasts on surfaces of oxidized polystyrene (PS), on surfaces modified with random copolymers of PS and poly(methyl methacrylate) [P(S-r-MMA)] with topographic features, and chemically patterned surfaces that varied in lateral length scales from nanometers to microns were studied. Surfaces with heterogeneous topographies were generated from thin film mixtures of a block copolymer, PS-b-MMA, with homopolymers of PS and PMMA. The two homopolymers macroscopically phase separated and, with the addition of diblock copolymer, the size scales of the phases decreased to nanometer dimensions. Cell spreading area analysis showed that a thin film of oxidized PS surface promoted adhesion whereas a thin film of P(S-r-MMA) surface did not. Fibroblast adhesion was examined on surfaces in which the lateral length scale varied from 60 nm to 6 microm. It was found that, as the lateral length scale between the oxidized PS surfaces decreased, cell spreading area and degree of actin stress fiber formation increased. In addition, scanning electron microscopy was used to evaluate the location of filopodia and lamellipodia. It was found that most of the filopodia and lamellipodia interacted with the oxidized PS surfaces. This can be attributed to both chemical and topographic surface interactions that prevent cells from interacting with the P(S-r-MMA) at the base of the topographic features.     

MPC-BMA共聚物的合成及其血小板粘附行为研究

本研究首先合成了含有磷酸胆碱基团的单体2-甲基丙烯酰氧乙基-2′-三甲胺乙基磷酸酯.内盐(MPC)和甲基丙烯酸正丁酯(BMA)的共聚物,采用红外光谱对其主要基团进行了表征分析,利用血小板粘附实验研究了磷脂聚合物膜的血小板粘附性,通过扫描电镜对血小板在聚合物膜上的形态和粘附量进行观察。结果表明:MPC含量越高,血小板的粘附量和变形程度越小;与其它亲水性单体如HEMA、HPOEM360、HPOEM526相比,等量MPC更能有效的降低其聚合物膜的血小板粘附性。     

Cell adhesion on poly(propylene fumarate-co-ethylene glycol) hydrogels

We synthesized poly(propylene fumarate-co-ethylene glycol) block copolymers [P(PF-co-EG)] that were crosslinked to form hydrogels and investigated the effect of copolymer composition on cell adhesion to the hydrogels. These copolymers were water soluble when the molar ratio of ethylene glycol repeating unit to propylene fumarate repeating unit was higher than 4.4. The water content of swollen hydrogels increased from 29 to 63% and the water contact angle decreased from 38 to 21 degrees as the molar ratio increased from 0.6 to 4.4. No significant change in either property was observed for ratios higher than 4.4. In a cell adhesion assay under serum-free conditions, the number of adherent platelets and smooth muscle cells decreased from 21 to 2% and from 78 to 20% of the initial seeding density, respectively, as the molar ratio increased from 0.6 to 7.8. Adherent smooth muscle cells did not spread on the hydrogels of the compositions tested. Adherent platelets did not show any filopodia. These results suggest that the hydrophilicity of P(PF-co-EG) hydrogels is one of the factors affecting cell adhesion, and that copolymer modification may be required for enhancing cell adhesion for an application involving the copolymers as in situ crosslinkable cell carriers.     

Surface modification with PEO-containing triblock copolymer for improved biocompatibility: in vitro and ex vivo studies

Poly(ethylene oxide) (PEO) has been frequently used to modify biomaterial surfaces for improved biocompatibility. We have used PEO-polybutadiene-PEO triblock copolymer to graft PEO to biomaterials by gamma-irradiation for a total radiation dose of 1 Mrad. The molecular weight of PEO in the block copolymer was 5000. In vitro study showed that fibrinogen adsorption to Silastic, polyethylene, and glass was reduced by 70 to approximately 95% by PEO grafting. On the other hand, the reduction of fibrinogen adsorption was only 30% on expanded polytetrafluoroethylene (e-PTFE). In vitro platelet adhesion study showed that almost no platelets could adhere to PEO-coated Silastic, polyethylene, and glass, while numerous platelet aggregates were found on the ePTFE. The platelet adhesion in vitro corresponded to the fibrinogen adsorption. When the PEO-grafted surfaces were tested ex vivo using a series shunt in a canine model, the effect of the grafted PEO was not noticeable. Platelet deposition on ePTFE was reduced by PEO grafting from 8170 +/- 1030 to 5100 +/- 460 platelets 10(-3) microm2, but numerous thrombi were still present on the PEO-grafted surface. The numbers of platelets cumulated on Silastic, polyethylene, and glass were 100 +/- 80, 169 +/- 35, and 24 +/- 22 platelets 10(-3) microm2, respectively. This is about 35% reduction in platelet deposition by PEO grafting. While the numbers of deposited platelets were small, the decreases were not as large as those expected from the in vitro study. This may be due to a number of reasons which have to be clarified in future studies, but it appears that in vitro platelet adhesion and fibrinogen adsorption studies may not be a valuable predictor for the in vivo or ex vivo behavior of the PEO-grafted surfaces.     

Reversibility of granulocyte adhesion using polyamine-grafted nylon-6 as a new column substrate for granulocyte separation

Reversibility of leucocytes adhered onto the surface of polyamine-grafted nylon-6 was investigated to estimate its feasibility as a new column substrate for granulocyte separation from whole blood. Polyamine-grafted nylon-6 was synthesized by a radical polymerization of 2-diethylaminoethyl methacrylate monomer onto nylon-6. The surfaces of these graft copolymers were found to show a microphase-separated structure composed of island-like phases of cationic polyamine and continuous phases of nonionic nylon-6. Interaction between leucocytes and these copolymer surfaces was studied by passing rabbit heparinized blood through a column packed with glass beads precoated with these copolymers. Columns of these copolymers showed a selective adhesion of granulocytes among leucocyte populations. Also, the adhering granulocytes were able to be recovered from the column fay a gentle elution procedure. From these results, it was concluded that polyamine-grafted nylon-6 having a microphase-separated structure is suitable for use as a column substrate in granulocyte separation from whole blood.     

Non-fouling biomaterials based on blends of polyethylene oxide copolymers and polyurethane: simultaneous measurement of platelet adhesion and fibrinogen adsorption from flowing whole blood

Measurements of platelet adhesion and fibrinogen adsorption from flowing whole blood to a series of polyethylene oxide (PEO)-based materials were carried out. A unique experimental design was used in which both quantities were measured in the same experiment. The materials consisted of a polyurethane (PU) as a matrix into which various triblock copolymers of general structure PEO-PU-PEO were blended; the PU block was the same in all materials but the PEO blocks ranged in molecular weight from 550 to 5000. Platelets were isolated from fresh human blood and labeled with ⁵¹Cr; purified fibrinogen was labeled with ¹²⁵I. A whole blood preparation containing these labeled species was used for the adhesion/adsorption studies. The surfaces were exposed to the flowing blood in a cone and plate device at a wall shear rate of 300?s^?1. It was found that both platelet adhesion and fibrinogen adsorption decreased with increasing copolymer content in the blends and with decreasing PEO block size for a given copolymer content. The block size effect was due probably to higher PEO surface coverage for the lower molecular weight blocks. Fibrinogen adsorption and platelet adhesion were linearly and strongly correlated. The best performing materials showed very low fibrinogen adsorption of the order of 25?ng/cm², and correspondingly low platelet densities around 10,000 per cm², i.e. fractional platelet coverage in the vicinity of 0.2%.