In vitro study of blood-contacting properties and effect on bacterial adhesion of a polymeric surface with immobilized heparin and sulphated hyaluronic acid

The blood-contacting properties and the effect on bacterial adhesion of a material based on polyurethane and poly(amido-amine) (PUPA), both in its native form and with the anticoagulant molecules heparin or sulphated hyaluronic acid (HyalS3.5) electrostatically bonded to its surface, were evaluated and compared in vitro. The presence of the biological molecules on the surface was revealed by a dye test and ATR/FTIR analysis. Bound heparin was found to maintain its physiological action, in terms of thrombin inactivation, as well as did free heparin. Moreover, it reduced the degree of platelet adhesion. On the contrary, bound HyalS3.5 lost its anticoagulant activity, though it reduced platelet adhesion. The number of platelets on both modified surfaces was low. Their shape distribution, as determined by SEM, did not differ significantly on the two modified surfaces or with respect to the bare PUPA surface. HyalS3.5 and heparin also inhibited adhesion of Staphylococcus epidermidis to the material. A possible relationship between the platelet and bacterial adhesion is ascribed to the mediating role of plasma proteins.     

Platelet adhesion to commercial and modified polymer materials in animals under psychological stress and in a no-stress condition

It is well known that stressful stimuli change blood functions and that protein and platelet parameters are altered in humans and animals subjected to stress. We have examined the influence of psychological stress on the morphological responses of platelets on commercially available materials [polyester (VP), fluoropassivated polyester (VPF), non-woven benzylic ester of hyaluronic acid (Hyaff11)] and on materials synthesised (PUPA) and/or surface modified by sulphation (Hyaff11S) or by immobilisation of the anticoagulant molecules heparin and sulphated hyaluronic acid (PUPA-Heparin, PUPA-HyalS, HyalS-PET). Moreover, the anticoagulant activity (i.e. thrombin inactivation) of the materials was analysed. In the no-stress condition, the surfaces with a low degree of platelet adhesion were Hyaff11S, HyalS-PET, PUPA-Heparin and PUPA-HyalS. Hyaff11, PET and PUPA had the highest number of adherent platelets within the series. VP and VPF exhibited an intermediate behaviour. The exposure of animals to stress induced a dramatic change in platelet number and morphology on PET, HyalS-PET, PUPA, PUPA-HyalS and Hyaff11: there was a higher degree of platelet adhesion, increased platelet spreading and the appearance of pseudopodia. In VP, VPF, Hyaff11S and PUPA-Heparin, there were no changes in platelet adhesion in stress conditions with respect to the no-stress condition; the latter two materials, the only ones able to prolong thrombin time, had a very low number of adherent platelets.     

Immobilization of an antithrombin-heparin complex on gold: anticoagulant properties and platelet interactions

The anticoagulant properties and platelet interactions of gold surfaces modified with an antithrombin-heparin (ATH) complex are reported. ATH was attached to gold through either a short disulfide (linker) or a thiol-terminated polyethylene oxide (PEO) (linker, spacer). Analogous surfaces were prepared with uncomplexed heparin. Antithrombin (AT) uptake was measured before and after selectively destroying the active pentasaccharide sequence of the heparin moiety, and was found to be predominantly through the active sequence on all of the surfaces. AT binding was higher on the ATH surfaces than on the corresponding heparin surfaces. Heparin activity was assessed by an anti-factor Xa assay. The ratio of active heparin density (from the anti-FXa assay) to total heparin density was taken as a measure of heparin bioactivity. The ratio was greater on the ATH- than on the heparin-modified surfaces, i.e. the PEO-ATH surfaces showed the greater proportion of active heparin. Platelet adhesion from flowing whole blood was found to be reduced on PEO- and ATH-modified surfaces compared to bare gold. The PEO-ATH modified surfaces, but not the heparinized surfaces, were shown to prolong the clotting time of recalcified plasma.     

Effect of surface modification of siliconeon Staphylococcus epidermidis adhesion and colonization

Cerebrospinal fluid (CSF) shunts for the treatment of hydrocephalus are generally made of silicone rubber. The growth of bacterial colonies on the silicone surface leads to frequent CSF shunt complications. A systematic study of the effect of the surface modification of silicone on Staphylococcus epidermidis adhesion and colonization was performed for different incubation times by means of colony counting and scanning electron microscopy (SEM). Silicone was modified with different biopolymers and silanes, including heparin, hyaluronan, octadecyltrichlorosilane (OTS), and fluoroalkylsilane (FAS) to provide a stable and biocompatible surface with different surface functional groups and degrees of hydrophobicity. The modified silicone surfaces were studied by using contact angle measurements, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). After 4 and 8 h of incubation, the FAS- and OTS-coated silicone and the hyaluronan coated OTS/silicone surfaces showed significantly reduced bacterial adhesion and colonization compared to blank silicone by both quantification methods. However, the heparin coated OTS/silicone showed significantly increased bacterial adhesion. These results indicate that the nature of the surface functional group and surface roughness determine the extent of bacterial adhesion and colonization. However, the degree of hydrophobicity of the surface did not appear to play a determining role in bacterial adhesion and colonization.     

Construction and hemocompatibility study of highly bioactive heparin-functionalized surface

A simple method is developed to construct anticoagulant surfaces via passive adsorption of heparin onto the protonated plasma-polymerized allylamine (PPAam) films from phosphate-buffered saline (PBS). These protonated PPAam surfaces are found to have high affinity to heparin. Importantly, the heparin-functionalized PPAam (Hep-PPAam) surfaces show good retention of heparin after long-term immersion in PBS. The Hep-PPAam surface prolongs the activated partial thromboplastin time for about 20 s as compared to 316L stainless steel even though the adsorption amount of heparin is only about 300 ng/cm(2) . This indicates that the heparin bound to the protonated PPAam surfaces in this way maintains a high bioactivity. Blood platelet adhesion and activation on this surface is remarkably reduced and adsorption and activation of fibrinogen is inhibited. Thus, Hep-PPAam surface modification leads to a significant improve of the hemocompatibility. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3124-3133, 2012.     

利用等离子体表面接枝技术提高医用聚氨酯血液相容性的研究

目的：聚氨酯作为与血液接触的植入物和组织器官替代材料在心血管系统有重要而广泛的应用前景,本研究采用等离子体表面接枝技术,通过“空间桥梁”在聚氨酯材料表面引入具有抗凝血功能的肝素分子,对材料表面的微观化学组成、表面接触角等理化性能进行了测定分析,并通过测定血小板在材料表面的粘附数量,对改性表面的抗凝血性能做了评价。结果：聚氨酯表面接枝肝素分子后,表面的氧／氮元素比提高,水接触角减小。对血小板的吸附和活化性下降,抗凝血性能得到提高。     

Use of plasma glow for surface-engineering biomolecules to enhance bloodcompatibility of Dacron and PTFE vascular prosthesis

The search for a nonthrombogenic material having patency to be used for small diameter vascular graft applications continues to be a field of extensive investigation. The purpose of the present study was to examine whether surface modification of polytetra fluoroethylene (PTFE, Teflon) and polyethylene-terephthalate (Dacron) vascular grafts might extend graft biocompatibility without modifying the graft structure. A series of surface coatings were prepared by modifying the argon plasma-treated PTFE and Dacron grafts with collagen IV and laminin and subsequently immobilizing bioactive molecules like PGE1, heparin or phosphatidyl choline via the carbodiimide functionalities. Surface analysis by Fourier transform infrared spectroscopy-attenuated total reflectance revealed the presence of new functional groups on the modified graft surfaces. In vitro studies showed that fibrinogen adsorption and platelet adhesion on modified grafts were significantly reduced. This study proposes that surface grafting of matrix components (collagen-type IV and laminin) and subsequent immobilization of bioactive molecules (PGE1, heparin or phosphatidyl choline) changed the surface conditioning of vascular grafts and subsequently improved their biocompatibility. However, more detailed in vivo studies are needed to confirm these observations.     

In vitro hemocompatibility of albumin-heparin multilayer coatings on polyethersulfone prepared by the layer-by-layer technique

Polyethersulfone foils (PES)--a unique material for blood purification membranes--were coated with a multilayer assembly of heparin (unfractionated or high anticoagulant activity fraction heparin) and albumin (albumin-heparin coatings), or with a multilayer of albumin (albumin coating), using the layer-by-layer technique. The coatings combine advantages of albumin (reduction of nonspecific interactions) and heparin (specific interactions with blood coagulation proteins). The differences between the two heparins, while significant for their biological activity, had only a minor effect on the multilayer assembly with albumin monitored in situ by reflection infrared spectroscopy (FTIR MIRS). Uncoated as well as modified PES surfaces were evaluated using an in vitro assay with freshly drawn, slightly heparinized (1.5 IU heparin/mL) human whole blood. The blood was circulated with a roller pump over the sample surfaces in shear flow across rectangular slit channels ( app. 6 mL/min and 120 s(-1)) for 1.5 h at 37 degrees C. All coatings effectively reduced platelet adhesion and activation according to the PF4 release. The activation of coagulation evaluated as TAT generation was significantly lowered for the coating composed of albumin and high activity heparin. A further beneficial effect of the heparin containing coatings was reduced complement activation as determined by different complement fragments.     

Platelet adhesion to heparin coated oxygenator fibers under in vitro static conditions: impact of temperature

Heparin coating of cardiopulmonary bypass (CPB) circuitry may attenuate the platelet consumption associated with CPB. We investigated the effect of temperature on the interaction between platelet and heparin coated surfaces under in vitro static conditions. Heparin coated and non coated oxygenator fibers were incubated with heparinized whole blood at 37 degrees C and 22 degrees C. The incubation time was set at 30, 60, 180, and 300 minutes. The number of platelets adhering to each fiber was assessed with enzyme immunoassay using monoclonal antibody against platelet receptor protein CD 61(GPIIbIIIa). As an index of platelet activation, plasma soluble(s) P-selectin levels were measured by enzyme-linked immunosorbent assay. Under normothermia, the number of adherent platelets on the non coated surface increased significantly after 300 min of incubation. Platelet adhesion was reduced significantly by heparin coating of the surface and was kept constant after 300 min. Under hypothermia, heparin coating was also associated with significant reduction of platelet adhesion. The levels of sP-selectin did not correlate with the extent of platelet adhesion. Our results suggest that heparin coating is effective in decreasing platelet adhesion to the synthetic surface tested regardless of the temperature under static conditions. Inhibition of platelet activation on the heparin coated surface may be masked by standard dose heparinization.     

Surface modification of chitosan membranes by complexation-interpenetration of anionic polysaccharides for improved blood compatibility in hemodialysis

Chitosan membrane surface was modified by cornplexation and interpenetration of anionic polysaccharides - heparin and dextran sulfate - for improved blood compatibility in hemodialysis. Electron spectroscopy for chemical analysis results showed a characteristic sulfur (S) and sodium (Na) peaks after modification with dextran sulfate. The sulfur/carbon (S/C) atomic composition ratio increased from 0.03 to 0.08 when the bulk dextran sulfate concentration used for modification was increased from 2.5 to 10 mg ml^-1 . The permeability of urea and creatinine did not change significantly upon modification with heparin or dextran sulfate. Surface modification, however, did decrease the permeability coefficients of glucose, vitamin B-2, and vitamin B-12. Unlike Cuprophan, chitosan and surface-modified chitosan membranes did not significantly activate the complement system as measured by the serum iC3b concentration. Compared to forty and sixty fully-activated platelets present on control surfaces, surface modification with heparin and dextran sulfate significantly reduced the number of adherent platelets per 25 000 μm² area and the extent of platelet activation. Surface modification with anionic polysaccharides, however, did significantly shorten the plasma recalcification time leading to fibrin clot formation. The results of this study show that chitosan membrane surface can be modified by complexation-interpenetration of anionic modifying agents. The modified membranes do resist complement activation and platelet adhesion and activation.     

Fibrinogen adsorption and platelet lysis characterization of fluorinated surface-modified polyetherurethanes

A polyetherurethane (PU) was modified using fluorinated surface-modifying macromolecules (SMMs). A double radiolabel method was used simultaneously to measure the number of adhered platelets ((51)Cr) and the quantity of adsorbed Fg ((125)I), in a cone-and-plate instrument. The objectives were to determine if adsorbed Fg levels correlated to platelet adhesion on the surfaces, and to assess if any reductions in platelet adhesion for the SMM-treated surfaces resulted from surface-induced platelet lysis, rather than changes directly related to lower platelet activation and attachment on the novel surfaces. Platelet lysis was determined from lactate dehydrogenase (LDH) and unbound (51)Cr released into plasma isolated from whole blood exposed to test materials. The corresponding Fg adsorption, evaluated under the same platelet adhesion conditions, did not account for the reduced platelet adhesion on the treated surfaces. LDH and (51)Cr platelet release were very low and indicated no statistically significant differences between the materials. It was therefore concluded that platelet lysis did not contribute to the reduction in platelet adhesion characteristic observed on the SMM-treated surfaces. More importantly, the work emphasizes that the platelet activation cannot be inferred to by assessing the quantity of fibrinogen as is commonly done in the literature. The finding suggests a much more complex mechanism of action for the SMM surface modifiers. On-going work is investigating other Fg parameters such as protein binding affinity and protein conformational state in order to establish the mechanism by which the fluorinated surface modifiers may be reducing platelet adhesion via intermediary changes in initial protein adsorption.     

The effect of protein adsorption on the anticoagulant activity of surface immobilized heparin

The anticoagulant activity of albumin-heparin conjugates covalently immobilized on carboxylated polystyrene beads was determined before and after exposure to different plasma/PBS dilutions using a thrombin inhibition assay, a FXa inhibition assay, and a modified aPTT assay. Exposure of albumin-heparin modified surfaces (alb-hep surfaces) to plasma dilutions resulted in surfaces with a lower anticoagulant activity than surfaces which were not exposed to plasma dilutions. The reduction of the activity increased up to ±80% when the surfaces were exposed to solutions containing more than 70% plasma. Alb-hep surfaces incubated in plasma which was preexposed to heparin-Sepharose retained 30% of their initial activity. These observations were attributed to non-specific adsorption of plasma proteins onto the surface and to interaction of heparin binding proteins with the immobilized heparin. Both processes result in a decreased accessibility of the immobilized heparin and thus in a reduced anticoagulant activity displayed by the heparinized surface. Identification of adsorbed proteins with SDS gel electrophoresis and immunoblotting revealed that many different proteins were present at the heparinized surface. Only small differences were observed between the gel electrophoresis pattern of adsorbed proteins obtained from heparinized surfaces and from a surface containing immobilized albumin.     

Poly(dimethylsiloxane)-poly(ethylene oxide)-heparin block copolymers. II: Surface characterization and in vitro assessments

Amphiphilic block copolymers containing poly(dimethylsiloxane), poly(ethylene oxide), as well as heparin-coated glass beads and tubes were evaluated for the amounts and activities of surface-immobilized heparin. Because the amphiphilic copolymer system is thermodynamically predicted to demonstrate low-energy phase enrichment on the surfaces of air-cast films, studies were also undertaken to understand the in vitro results. Solvent-cast copolymer films have a heterogeneous microphase-separated structure according to transmission electron micrographs. Wilhelmy plate contact angle analysis indicates significant surface restructuring occurs upon hydration. Attenuated total reflectance infrared spectroscopy studies of the desiccated and hydrated films at two different sampling depths show compositional heterogeneity as a function of depth, as well as near surface restructuring allowing surface enrichment of the high-energy segments following contact with water. Significant concentrations of heparin are detected on the surface of these coatings by toluidine blue assays. In addition, a portion of the surface-bound heparin maintains its original bioactivity as determined by recalcification times, thrombin times, and Factor Xa assays. These substrates were also tested for platelet adhesion and activation reactions in vitro using polymer-coated beads in rabbit platelet-rich plasma. Heparinized polymers promoted low levels of platelet adhesion and serotonin release. Surface concentrations of heparin from bioactivity assays were then correlated with platelet adhesion and the extent of platelet release to assess the efficacy of this heparin-immobilized copolymer as a blood-compatible material or coating.     

Poly(DL-lactic acid) film surface modification with heparin for improving hemocompatibility of blood-contacting bioresorbable devices

This work describes a simple method to immobilize heparin by covalent bonding to the surface of poly(lactic acid) film with the aim of showing improved hemocompatibility. Carboxyl groups present in heparin molecules were activated by reaction with N-hydroxy-succinimide and allowed to react with free amino groups created at the surface of poly(DL-lactic acid) films by controlled aminolysis. Contact angle measurements and XPS analysis confirmed the binding. Quantification was determined by radioactivity using heparin labeled with tritium. The surface exhibited anti factor Xa activity, thus confirming the presence of bounded heparin that kept some biological activity. Finally platelets adhesion showed less platelet adhesion on heparin modified films as well as preserved morphology.     

Immobilization of NaIO4-treated heparin on PEG-modified 316L SS surface for high anti-thrombin-III binding

Poor compatibility between blood and metallic coronary artery stents is one reason for arterial restenosis; however, the immobilization of anticoagulant agents on the surface of the stent is a feasible method of improving stent compatibility. Heparin, a well-known anticoagulant, has been frequently used to coat the surfaces of certain biomaterials to attain blood compatibility. The compound 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide has often been utilized for the immobilization of heparin, but the critical carboxyl groups of heparin (with regards to heparin's anticoagulant activity) will be reduced by this method. This study examined possible methods of heparin immobilization without consuming these carboxyl groups. The 316L stainless steel surface was first activated with hexamethylene diisocyanate and then coupled with bis-amine-terminated poly (ethylene glycol) (BA-PEG) so as to create active amine groups. Sodium periodate (NaIO(4); SP) was then used to oxidize heparin to form aldehyde groups. The treated heparin could then be grafted onto the activated surface of the test material without losing its carboxyl groups. Effective surface modification of the hexamethylene diisocyanate-activated and BA-PEG-grafted 316L SS surface was confirmed using Fourier Transform Infrared Spectroscopy, electron spectroscopy for chemical analysis and a water contact angle test. After the heparin was immobilized on the BA-PEG-grafted 316L SS surface by SP, the surface showed an improvement in antithrombrin III (AT III) binding ability, its anticoagulant property, and hemocompatibility in comparison with heparin grafted by 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide.     

Analysis of cellular morphology, adhesion, and proliferation on uncoated and differently coated PVC tubes used in extracorporeal circulation (ECC)

The biggest challenge to improve extracorporeal circulation (ECC) circuits lays on avoiding platelet adhesion to their surfaces, because this contributes to thrombus formation, resulting in the activation of blood coagulation. One approach to minimize this effect is to improve the biocompatibility of ECC circuits by modifying their surfaces. This can be achieved by coating them with heparin or phospholipids. The present study investigated the adhesion and morphology characteristics of fibroblastic and blood cells cultured on uncoated poly (vinyl) chloride PVC tubes as well as on heparin, phosphatidylcholine (DMPC), and phosphatidylethanolamine (DMPE) -coated tubing. The results showed the importance of uniform coating regardless of the substance used, because the coatings cover the grooves on PVC surfaces, which favor cell adhesion. The comparison among the three different coatings showed the best biocompatibility results for the PVC tubes coated with heparin, followed by the coating with DMPE and with DMPC. For all coated tubes, cells did not spread on the PVC surfaces and, consequently, did not adhere to their surfaces, increasing the overall biocompatibility of PVC tubes. However, possible DMPE's alkylation, caused by sterilization, resulted in increased material hydrophobicity, which explains the decrease in fibroblastic adhesion. Furthermore, sterilization of DMPC-PVC improves its hydrophilic character, also decreasing adhesion. Based on these results, coating PVC with the phospholipids DMPC and DMPE seems to be a promising technique to improve the biocompatibility of PVC tubes, and is worthy of further investigation.     

<Emphasis Type="Italic">N</Emphasis>-desulfated non-anticoagulant heparin inhibits leukocyte adhesion and transmigration in vitro and attenuates acute peritonitis and ischemia and reperfusion injury in vivo

OBJECTIVE: Heparin, a highly sulfated proteoglycan, is known to have strong anticoagulant and anti-inflammatory activities. Here we sought to generate a heparin derivative, which had a significantly lower anticoagulant activity while retaining its strong anti-inflammatory activity. MATERIALS AND METHODS: Heparin was chemically modified and this discrete set of the heparin derivatives was tested for their anticoagulant activities, such as activated partial thromboplastin time (APTT), and anti-inflammatory activities, such as leukocyte adhesion and transmigration in vitro and acute peritonitis and ischemia and reperfusion injury in vivo. RESULTS: We found that an N-desulfated heparin had 188-fold (compared to heparin) and 32-fold (compared to low molecular weight heparin; LMWH) reductions of APTT. The N-desulfated heparin inhibited adhesion of human promyeloid HL-60 cells to the stimulated human umbilical vein endothelial cells (HUVECs) under a physiological shear stress. It also prevented the transmigration of human neutrophils through the monolayers of the stimulated HUVECs. Further, intravenous administration of this compound attenuated the peritoneal infiltration of neutrophils in a mouse model of acute peritonitis, and reduced tissue edema and leukocyte deposition in a rabbit ear model of ischemia and reperfusion injury. CONCLUSION: It is to our best knowledge that the N-desulfated heparin has the lowest anticoagulant activity among LMWH and chemically modified heparin derivatives, while preserving a potent anti-inflammatory activity. These combined properties appear to suggest it as a safer medicine for treatment of inflammation.     

Synthesis and physicochemical characterization of a new material (PUPA) based on polyurethane and poly(amido-amine) components capable of strongly adsorbing quantities of heparin

The synthesis of new materials (PUPAs) based on a commercial polyurethane and a heparin-complexing polymer, poly(amido-amine), was studied. PUPAs are capable of adsorbing heparin because the basic nitrogens of poly(amido-amine), once protonated, interact with the negative charges carried by the heparin molecule. Six different samples of PUPA were synthesized having a varied ratio of the components. The quantity of basic nitrogen on the surface and the bound heparin for each sample was determined. Two different kinds of heparin are present on a PUPA surface: one is strongly bound but can be detached by 0.1 M NaOH solution, the other is physically adsorbed and is slowly released by a stream of saline solution. A relationship between the quantity of strongly bound heparin and basic nitrogen was found. SEM and FTIR-ATR analysis were performed on all the PUPA samples. The mechanical characteristics change according to chemical composition.     

Heparin immobilization onto segmented polyurethane-urea surfaces--effect of hydrophilic spacers

Heparin was immobilized onto segmented polyurethane-urea surfaces (Biomer) using hydrophilic poly(ethylene oxide) spacers of different chain lengths. The use of the hydrophilic spacer, poly(ethylene oxide), reduces protein adsorption and subsequent platelet adhesion on the surface. In addition, the bioactivity of the immobilized heparin is enhanced by the incorporation of these spacers. Immobilized heparin bioactivity is shown to be a function of PEO spacer length. Use of hydrophilic PEO spacers demonstrates that immobilized heparin's bioactivity is consistently higher than that of the C6 alkyl spacer, but heparin-immobilized surfaces demonstrate no chain length effect on platelet adhesion, even though they show less platelet adhesion compared to Biomer controls. In the case of PEO-grafted surfaces, platelet adhesion is decreased compared to Biomer controls, and C6 alkyl spacer-grafted surfaces, and exhibits a minimum at PEO 1000. In ex vivo A-A shunt experiments under low flow and low shear conditions, all heparinized surfaces exhibit significant prolongation of occlusion times compared to Biomer controls, indicating an ability of immobilized heparin to inhibit thrombosis in whole blood.     

Physiochemical characterization and coating of polyurethane with a new heparin-adsorbing material

The coating of polyurethane devices was obtained by using a solution of a new heparin-adsorbing material, PUPA. The structure of the coated material was investigated at different depths by FTIR techniques. The bond with heparin is electrostatic, as revealed by subtracting the spectrum of the native PUPA from that of the heparinized sample.