In vitro and in vivo studies of heparinized-collageno-elastic tubes

Heparin was covalently coupled to collageno-elastic grafts (CET) derived from lamb carotid arteries, by using the crosslinking agent 1-ethyl-3 (3-dimethyl-aminopropyl) carbodiimide (EDC). The collagenous grafts were pretreated with various aminating agents in order to enhance the number of available binding sites on the collagen surface. By varying the EDC/heparin weight ratio, the pH of the immobilization media, and the pretreatment agent, a global search pattern maximized heparin loading at 3.90 +/- 0.36 USP heparin/cm2 collagenous graft surface when the EDC/heparin ratio was 2:1 at a pH of 1.5 with 1 M hydroxylamine sulfate as the pretreatment agent. Heparinized CETs were superior to nonheparinized CETs by exhibiting both enhanced antiplatelet activity in using an in vitro differential recirculation reactor with chromium-51 tagged platelets and enhanced patency when interposed in canine carotid arteries. Both antiplatelet activity and patency duration for heparinized CETs were independent of heparin loading.     

Effects of modified collagen matrices on human umbilical vein endothelial cells

The most commonly used biomaterials fail to ensure sufficient angiogenesis for fast in vivo incorporation. This results in central necrosis and consequent infection. One way of obtaining a high angiogenic response is the application of vascular endothelial growth factor (VEGF). To obtain a sustained release of these cytokines, heparin was incorporated into collagen matrices using 1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinmide (NHS). The functionality of the heparinized collagen matrices was then enhanced by immobilization of VEGF via its heparin-binding domain. This procedure changed in vitro degradation behavior and water-binding capacity. Accelerated endothelial cell proliferation was also achieved. A range of different heparin and EDC/NHS concentrations in combination with VEGF induced variation in endothelial cell growth and tubulogenic formation. Polymerized collagen scaffolds presented biointeractive systems with integrated angiogenic activity. This may become a useful tool in the clinical therapy of disorders connected with wound healing.     

Preparation and surface characterization of HMDI-activated 316L stainless steel for coronary artery stents

Poor compatibility between blood and metallic coronary artery stents is one reason for arterial restenosis. Immobilization of anticoagulant agents on the stent's surface is feasible for improving compatibility. We examined possible surface-coupling agents for anticoagulant agent immobilization. Hexamethylene diisocyanate (HMDI) and 3-aminopropyl-triethoxysilane (APTS) were examined as surface-coupling agents to activate 316L stainless steel (e.g., stent material). The activated surface was characterized using Fourier transformation infrared spectroscopy (FTIR), atomic force microscope (AFM), surface plasmon resonance (SPR), and trinitrobenzene sulfonic acid (TNBS) assay. In FTIR analysis, HMDI and APTS were both covalently linked to 316L stainless steel. In AFM analysis, it was found that the HMDI-activated surface was smoother than the APTS-activated one. In SPR test, the shift of SPR angle for the APTS-activated surface was much higher than that for the HMDI-activated surface after being challenged with acidic solution. TNBS assay was used to determine the amount of immobilized primary amine groups. The HMDI-activated surface was found to consist of about 1.32 micromol/cm(2) amine group, whereas the APTS-activated surface consisted of only 0.89 micromol/cm(2) amine group. We conclude that the HMDI-activated surface has more desirable surface characteristics than the APTS-activated surface has, such as chemical stability and the amount of active amine groups.     

Carboxyl groups and tryptophan residues in the active site of Rhizopus niveus glucoamylase

Functionally important carboxyl groups in the glucoamylase, Gluc1, from Rhizopus niveus were investigated by site-specific modification using water-soluble carbodiimide, 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide, and nucleophiles. A total of 7.5 carboxyl groups of the 37 present in Gluc1 were substituted in the presence of the substrate maltose, and there was a slight loss of enzymatic activity. After removal of maltose, re-treatment of the deprotected enzyme reduced its activity to 3% with the modification of 1.2 carboxyl groups. It is conceivable that there is only one carboxyl group located in the active site. Fluorescence spectra of the enzyme suggested an interaction of tryptophan residues and carboxyl groups at the locus of the enzyme active site. Distinctive disruption of the protein structure in the modified enzyme was excluded by CD spectroscopy and immunological investigation.     

Optimization of the methods for introduction of amine groups onto the silica nanoparticle surface

The luminescent silica nanoparticle has attracted the researchers' concentration in bioanalysis recently. Its extensive application is based on the immobilization of various biomolecules such as deoxyribonucleic acid, antibody, and so forth onto the surface. By comparing different introduction methods of amine groups, it was confirmed that the "two-step" route is more preferable by adding tetraethyl orthosilicate and 3-aminopropyl-(triethoxyl)silane in sequence, to attain ideal amine-modified silica nanoparticles. On this basis, carboxyl groups were derived from amine groups on the nanoparticle surface and then were activated by 1-ethyl-3-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy-succinimide. Finally, mouse monoclonal antihuman CD71 antibody (McAb CD71) and transferrin were effectively linked with the carboxyl groups and successfully labeled the receptors in the membrane of fibroblast cells, respectively.     

Surface modification of polycaprolactone with poly(methacrylic acid) and gelatin covalent immobilization for promoting its cytocompatibility

Polycaprolactone (PCL) membrane was modified by grafting copolymerization of methacrylic acid (MAA) initiated under UV light. The covalent immobilization of gelatin on PCL-g-PMAA surface was consequently performed by using condensing agent, 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride. The occurrence of grafting copolymerization of PMAA and further immobilization of gelatin was confirmed by ATR-FTIR and X-ray photoelectron spectroscopy characterizations. The existence of carboxyl groups grafted on PCL surface was verified quantitatively by absorbance spectroscopy where rhodamine 6G was employed to react with carboxyl groups to generate an absorbance at 512 nm. The endothelial cell culture proved that the PCL membrane slightly modified with suitable amount of PMAA or gelatin had better cytocompatibility than control PCL or PCL membrane heavily modified with PMAA or gelatin.     

Properties of carbodiimide treated heparin

Heparin, a well-known anticoagulant, has been frequently used to coat surfaces for attaining blood compatibility of polymeric materials. Since carbodiimides are often used for immobilization of heparin on these biomaterials, the present study intended to evaluate some properties of carbodiimide-treated heparin. It was observed that the properties of heparin were altered after treatment with carbodiimide, mainly in the molar excess of this reagent. Thus, dye fixation and electrophoretic behaviour of heparin were modified, as well as its degradability by specific enzymes. Also, these modifications resulted in loss of anticoagulant activity. Infrared spectra of this carbodiimide-treated heparin presented evidence that can confirm its modification.     

Some characteristics of collagen-heparin complex

In various forms of purified collagen (powder of insoluble collagen from bovine skin, fibers from rat tail tendons, membrane from bovine gut), carboxyl groups were activated by carbodiimide to allow covalent binding of heparin. Collagen powder and collagen fibers from rat tail tendons were also incubated in a heparin solution under the same reaction conditions but without carbodiimide present to account for other forms of collagen-heparin interaction. It was found that the linkage of heparin to collagen formed in the presence of carbodiimide is stable, as heparin was minimally extractable by 0.2M buffers with a pH ranging from 5 to 9. Collagen powder incubated with heparin in the absence of carbodiimide released heparin almost completely into Tris buffer of pH 9.0. As a consequence of covalent binding of heparin to collagen, the collagen fibers became more stable as shown by their significantly reduced swelling capacity and significantly increased shrinkage temperature. Collagen fibers interacted with heparin in the absence of carbodiimide also showed some stabilization of their structure, which was, however, significantly less than with carbodiimide reaction. By two independent methods it was shown that heparin linked to collagen by a stable bond retains its anticoagulant activity. It is concluded that, in the presence of carbodiimide, heparin covalently binds to collagen thus forming an antithrombogenic surface. At the same time, collagen is crosslinked. Incubation of collagen in the solution of heparin without carbodiimide also stabilizes collagen structure, but to a significantly lesser degree. Such a linkage is unstable as heparin dissociates and is readily extractable into 0.2M Tris buffers with pH 7-9.     

Immobilization of Heparin on the Surface of Polypropylene Non-Woven Fabric for Improvement of the Hydrophilicity and Blood Compatibility

Abstract

A polypropylene non-woven fabric (PPNWF) was exposed to oxygen plasma to produce peroxides on its surface. These peroxides were used to initiate graft polymerization of acrylic acid (AA) on the surface of PPNWF. Direct heparinization was accomplished via a reaction between heparin and PP-PAA (AA grafted PPNWF) which was activated by EDC (N-ethyl-N’-[3-(dimethylamino)propyl] carbodiimide). Indirect heparinized PPNWF was prepared by grafting poly(ethylene oxide) (PEO) on a PP-PAA surface to form PP-PAA-PEO, followed by reaction with heparin which was activated by EDC before use. The surface modified PPNWFs were characterized by attenuated total reflection Fourier transform infrared (ATR–FT-IR) spectroscopy, electron spectroscopy for chemical analysis (ESCA) and contact angle goniometry. It was found that hydrophilicity was greatly improved, as indicated by the decrease of the water contact angle from 142 to 33°. In vitro blood compatibility evaluation of modified PPNWFs, including hemolysis rate, platelet adhesion, plasma protein adsorption and activated partial thromboplastin time (APTT) was investigated. The results suggested that both heparinized PPNWFs showed lower hemolysis rates and better platelet anti-adhesion than non-heparinized controls. Furthermore, PPNWF obtained via indirect immobilization of heparin showed better hydrophilicity and blood compatibility than direct heparinization of PPNWF.     

Immobilization of the direct thrombin inhibitor-bivalirudin on 316L stainless steel via polydopamine and the resulting effects on hemocompatibility in vitro

Bivalirudin (BV), a peptidic direct thrombin inhibitor, derived from hirudin, has gained increasing interest in clinical anticoagulant therapy in the recent years. In this work, a hemocompatible surface was prepared by immobilization of BV on 316L stainless steel (SS) using a bonding layer of polydopamine (DA). X-ray photoelectron spectroscopy (XPS) was used to determine the chemical composition of the surfaces to characterize polydopamine intermediate layer and the immobilized BV. The quantity of bound BV was measured by quartz crystal microbalance (QCM). The hemocompatibility in vitro was evaluated by coagulating time of activated partial thromboplastin time (aPTT) and prothrombin time (PT) assay, platelet adhesion and activation, fibrinogen adsorption, and activation and whole blood test. The effect of sterilizing method on the bioactivity of immobilized BV was also evaluated. The results showed that BVs were successfully immobilized on SS surface with the DA interlayer at a density of 98 ng/cm(2) . BV coating surface prolonged aPTT and PT, inhibited the activation of platelet and fibrinogen significantly. Sterilization by ultraviolet radiation was possible with only marginal loss of activity. Thus, the approach described here may provide a basis for the preparation of 316L SS surface modification for use in cardiovascular implants.     

Synthesis and characterization of heparinized polyurethanes using plasma glow discharge

Polyurethanes (PU) were synthesized from 4,4'-diphenylmethane diisocyanate and polytetramethylene glycol, and subsequently with ethylene diamine as a chain extender. The PU film was exposed to oxygen plasma glow discharge to produce peroxides on the surfaces. These peroxides were then used as catalysts for the copolymerization of acrylic acid (AA) and methyl acrylate (MA) in order to prepare carboxyl group-introduced PU (PU-C). Heparin-immobilized PU was prepared using the coupling reaction of PU-C with polyethylene oxide (PEO) followed by the reaction of grafted PEO with heparin. The surface-modified PUs were then characterized by attenuated total reflection Fourier transform infrared spectroscopy, electron spectroscopy for chemical analysis (ESCA), and a contact angle goniometer. The concentration of carboxylic acid groups on the PU surfaces could be controlled within the range of 0.47-1.68 micromol cm(-2) by the copolymerization of AA and MA. The amounts of heparin coupled to terminus amino groups on PU-6 and PU-33 were 1.30 and 1.16 microg cm(-2), respectively. The water contact angle of the PU was decreased by AA grafting, and further decreased by PEO grafting and heparin immobilization, showing an increased hydrophilicity of the modified PUs. A 3% loss from the originally bound heparin appeared within several hours and thereafter almost no heparin was released when heparin-immobilized PUs were immersed in a physiological solution for 100 h, indicating the covalent immobilization of heparin on the surfaces.     

Immobilization of heparin to EDC/NHS-crosslinked collagen. Characterization and in vitro evaluation

In the present study, heparin immobilization to a non-cytotoxic crosslinked collagen substrate for endothelial cell seeding was investigated. Crosslinking of collagen using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) resulted in a material containing 14 free primary amino groups per 1000 amino acid residues (E/N14C). At a fixed molar ratio NHS:EDC of 0.6, the amount of heparin covalently immobilized to E/N14C increased with increasing molar ratios of EDC to heparin carboxylic acid groups (Hep-COOH), to a maximum of approximately 5-5.5 wt% at a ratio of 2. Upon incubation in cell culture medium of endothelial cells, 4 to 7% of the immobilized heparin was released during 11 days. Immobilization of increasing amounts of heparin to E/N14C progressively reduced activation of contact activation proteases. Optimal anticoagulant activity, as measured by thrombin inhibition, was obtained after heparin immobilization using a ratio of EDC to Hep-COOH of 0.2-0.4 (14-20 mg heparin immobilized per gram of collagen). Platelets deposited to (heparinized) E/N14C showed only minor spreading and aggregation, although heparin immobilization slightly increased the number of adherent platelets. The results of this study suggest that heparin immobilization to EDC/NHS-crosslinked collagen may improve the in vivo blood compatibility of this material.     

Grafting of dermatan sulfate on polyethylene terephtalate to enhance biointegration

The aim of the present study was to achieve the immobilization of dermatan sulfate (DS) on polyethylene terephthalate (PET) surfaces and to evaluate its biocompatibility. DS obtained from the skin of Scyliorhinus canicula shark was immobilized via carbodiimide on knitted PET fabrics, modified with carboxyl groups. PET-DS characterization was performed by SEM, ATR-FTIR and contact angle measurements. Biocompatibility was evaluated by investigating plasma protein adsorption and endothelial cell proliferation, as well as by subcutaneous implantations in rats. The results indicated that DS immobilization on PET was achieved at ~8 μg/cm2. ATR-FTIR evidenced the presence of sulfate groups on the PET surface. In turn, contact angle measurements indicated an increase in the surface wettability. DS immobilization increased albumin adsorption on the PET surface, whereas it decreased that of fibrinogen. In vitro cell culture revealed that endothelial cell proliferation was also enhanced on PET-DS. Histological results after 15 days of subcutaneous implantation showed a better integration of PET-DS samples in comparison to those of nonmodified PET. In summary, DS was successfully grafted onto the surface of PET, providing it new physicochemical characteristics and biological properties for PET, thus enhancing its biointegration.     

Free electron laser induces specific immobilization of heparin on polysulfone films

Covalent immobilization of heparin has been developed to reduce the amount of heparin administered systematically during long-term dialysis. Recently, it was doubted partially because of the complexion during immobilization process. In this study, we investigated a novel method for specific immobilization of heparin on polysulfone (PSF) via free electron laser (FEL) irradiation. Laser wavelengths of 6.18 or 6.31 μm, the typical absorption bands of carboxyl groups of heparin and aromatic rings in PSF, respectively, were chosen to irradiate the thin heparin membrane formed on PSF surfaces. The amount of heparin immobilized on PSF was measured by the toluidine blue method. The binding of heparin on PSF was analyzed by X-ray photoelectron spectroscopy (XPS). The immobilization of heparin resulted in a hydrophilic surface on which decreased platelet adhesion was observed. The efficiency differences, depending on laser wavelengths, were discussed from the point of view of structural and environmental differences of light-absorbing groups.     

Investigation on clotting and hemolysis characteristics of heparin-immobilized polyether sulfones biomembrane

In this study, a novel heparin-immobilized polyethersulfone (PES) was synthesized. PES was initially sulfonated with chlorosulfonic acid and then 1,6-hexanediamine was grafted to the -SO(3)H groups of sulfonated PES, which subsequently reacted with heparin through a covalent bond by using (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) as catalyst. The hydrophobic/hydrophilic property was characterized by measuring the water contact angle. The data shows decline from 62.29 degrees +/- 1.2 degrees to 47.86 degrees +/- 0.3 degrees for water and 86.79 degrees +/- 0.8 degrees to 68.34 degrees +/- 1.0 degrees for glycerol, which indicates an enhancement of hydrophilicity. Plasma hemolysis assay shows a comparatively low hemolysis ratio of 1.04%, which is below permissible limit of 5%. A higher content of dissociated blood cells and Ca(2+) concentration was found in red blood cell counting and coagulation factor IV test in heparinized PES. Plasma recalcification time of 360 s also offers positive evidence that heparinized PES seems to have a good anticoagulation property. This new heparin-immobilized PES biomaterials may have the potential for biomedical applications.     

Fabrication and characterization of poly(γ-glutamic acid)-graft-chondroitin sulfate/polycaprolactone porous scaffolds for cartilage tissue engineering

The development of blended biomacromolecule and polyester scaffolds can potentially be used in many tissue engineering applications. This study was to develop a poly(γ-glutamic acid)-graft-chondroitin sulfate-blend-poly(ε-caprolactone) (γ-PGA-g-CS/PCL) composite biomaterial as a scaffold for cartilage tissue engineering. Chondroitin sulfate (CS) was grafted to γ-PGA, forming a γ-PGA-g-CS copolymer with 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC) system. The γ-PGA-g-CS copolymers were then blended with PCL to yield a porous γ-PGA-g-CS/PCL scaffold by salt leaching. These blended scaffolds were characterized by ¹H NMR, ESCA, water-binding capacity, mechanical test, degradation rate and CS assay. The results showed that with γ-PGA-g-CS as a component, the water-binding capacity and the degradation rate of the scaffolds would substantially increase. During a 4 week period of culture, the mechanical stability of γ-PGA-g-CS/PCL scaffolds was raised gradually and chondrocytes were induced to function normally in vitro. Furthermore, a larger amount of secreted GAGs was present in the γ-PGA-g-CS/PCL matrices than in the control (PCL), as revealed by Alcian blue staining of the histochemical sections. Thus, γ-PGA-g-CS/PCL matrices exhibit excellent biodegradation and biocompatibility for chondrocytes and have potential in tissue engineering as temporary substitutes for articular cartilage regeneration.     

Free electron laser induces specific immobilization of heparin on polysulfone films

Covalent immobilization of heparin has been developed to reduce the amount of heparin administered systematically during long-term dialysis. Recently, it was doubted partially because of the complexion during immobilization process. In this study, we investigated a novel method for specific immobilization of heparin on polysulfone (PSF) via free electron laser (FEL) irradiation. Laser wavelengths of 6.18 or 6.31 microm, the typical absorption bands of carboxyl groups of heparin and aromatic rings in PSF, respectively, were chosen to irradiate the thin heparin membrane formed on PSF surfaces. The amount of heparin immobilized on PSF was measured by the toluidine blue method. The binding of heparin on PSF was analyzed by X-ray photoelectron spectroscopy (XPS). The immobilization of heparin resulted in a hydrophilic surface on which decreased platelet adhesion was observed. The efficiency differences, depending on laser wavelengths, were discussed from the point of view of structural and environmental differences of light-absorbing groups.     

Anticoagulant activity of immobilized heparin on the polypropylene nonwoven fabric surface depending upon the pH of processing environment

Antenna coupling microwave plasma enables a highly oxidative treatment of the outmost surface of polypropylene (PP) nonwoven fabric within a short time period. Subsequently, grafting copolymerization with acrylic acid (AAc) makes the plasma-treated fabric durably hydrophilic and excellent in water absorbency. With high grafting density and strong water affinity, the pAAc-grafted support greatly becomes feasible as an intensive absorbent and as a support to promote heparin immobilization through amide bonds. For heparin immobilized in acidic condition, the carbonate groups of the molecule tend to dissolve and passive encapsulation of the molecule prevents its functional groups from bonding with the carboxylic acid of pAAc. This effect leads to inhibit the immobilization process and consequently reduces the quantity as well as the bioactivity of the immobilized heparin. In alkaline processing environment, the oxidized uronic acid residues in heparin-related glycans are presumably cleaved and the removal of some oxidized residuals before immobilization process is likely to reduce the chain length of heparin. In the latter case, anticoagulant Factors X and XII, but not thrombin, are unaffected. Anticoagulant activity test using activated partial thromboplastin time (aPTT) is more sensitive in assessing heparin-immobilized surfaces, since it corresponds to Factor X and initiates the inhibition of Factor XII and thrombin. Likewise, platelets adhesion on the surfaces decreases as the process shifted from acidic to alkaline condition, whereas the hydrophilic character of the grafted pAAc markedly contributes to extend physical insertion of platelets. The immobilized heparin has a great part of original bioactivity, depending on the pH of the processing environment and the immobilized quantity. Relative bioactivity based upon aPTT tests is partially held longer than 90 days for the sample prepared in the alkaline or neutral environment.     

Direct thrombin inhibitor-bivalirudin functionalized plasma polymerized allylamine coating for improved biocompatibility of vascular devices

The direct thrombin inhibitor of bivalirudin (BVLD), a short peptide derived from hirudin, has drawn an increasing attention in clinical application because it is safer and more effective than heparin for diabetic patients with moderate- or high-risk for acute coronary syndromes (ACS). In this study, BVLD was covalently conjugated on plasma polymerized allylamine (PPAam) coated 316L stainless steel (SS) to develop an anticoagulant surface. QCM-D real time monitoring result shows that 565?±?20?ng/cm(2) of BVLD was bound to the PPAam surface. Infrared spectroscopy (IR) and X-ray photoelectron spectroscopy (XPS) confirmed the immobilization of BVLD. The conjugation of BVLD onto the PPAam coating led to enhanced binding of thrombin, and the activity of the thrombin adsorbed on its surface was effectively inhibited. As a result, the BVLD immobilized PPAam (BVLD-PPAam) substrate prolonged the clotting times, and exhibited inhibition in adhesion and activation of platelets and fibrinogen. We also found that the BVLD-PPAam coating significantly enhanced endothelial cell adhesion, proliferation, migration and release of nitric oxide (NO) and secretion of prostaglandin I(2) (PGI(2)). In?vivo results indicate that the BVLD-PPAam surface restrained thrombus formation by rapidly growing a homogeneous and intact endothelium on its surface. These data suggest the potential of this multifunctional BVLD-PPAam coating for the application not only in general vascular devices such as catheters, tubes, oxygenator, hemodialysis membranes but also vascular grafts and stents.     

Covalent immobilization of biomolecules onto polystyrene MicroWells for use in biospecific assays

Modification of polystyrene for higher binding capacity and/or for specific covalent immobilization of biomolecules is discussed. The benefit of covalent coupling of biomolecules onto a new commercially available surface type for covalent immobilization, CovaLink NH, is illustrated. The CovaLink NH solid phase has spacer arms covalently grafted onto the polystyrene solid phase, approximately 10(14) groups/cm2. Coupling procedures for covalent immobilization of biotin and peptides are demonstrated, and the advantage of using carbodiimide for coupling of carboxylic acid containing compounds is shown.