Immobilization Of Heparin Research Articles

Copolymerization of 2-Methylene-1,3-dioxepane and Glycidyl Methacrylate, a Well-Defined and Efficient Process for Achieving Functionalized Polyesters for Covalent Binding of Bioactive Molecules

The understanding of cell-material interactions is important for creating personalized implants for tissue engineering. This has resulted in an interest in developing polymers with functional groups with the possibility of controlling the macromolecular surface. We have in a one-pot reaction synthesized a series of amorphous and degradable polyester-based copolymers with active functional groups by copolymerization of 2-methylene-1,3-dioxepane and glycidyl methacrylate. The properties of the final polymers were varied by varying the feed ratios of the monomers, and it was seen that it was possible to control the amount of active functional groups. The resulting epoxy-functionalized polyester was further modified by covalent immobilization of heparin. The heparinization was done in order, in a future aspect, to enhance the osteogenic differentiation of mesenchymal stem cells. Heparin binds directly with the growth factor bone morphogenetic protein-2 and helps to retain its activity. The molecular structure of the copolymers was characterized by nuclear magnetic resonance, size exclusion chromatography, and Fourier transform infrared spectroscopy. Differential scanning calorimetry and tensile testing showed that the monomer feed ratio had a great influence on the properties of the final polymer and that it thus was possible to control the mechanical properties to suit an intended application. The presence of heparin was verified by toluidine blue staining, and all of the films tested showed positive signals for heparin.

Hemocompatible and antibacterial porous membranes with heparinized copper hydroxide nanofibers as separation layer

Here we report the fabrication of a novel heparinized copper hydroxide (Cu(OH)2) nanofiberous membrane with satisfying hemocompatibility and antibacterial properties. The positively charged Cu(OH)2 nanofibers were prepared in a weakly alkaline copper nitrate solution in the presence of 2-aminoethanol. A heparin (Hep) solution was then added dropwise into the solution of nanofibers to immobilize negatively charged heparin onto the Cu(OH)2 nanofibers by electrostatic interaction. A composite Hep@Cu(OH)2 nanofiberous membrane was prepared by filtration and deposition of the heparinized nanofibers onto a polysulfone (PSF) porous membrane. Chemical composition analysis of membrane surface using X-ray photoelectron spectroscopy (XPS) confirmed the successful immobilization of heparin on Cu(OH)2 nanofibers. The amount of immobilized heparin on nanofiberous membrane was determined by a colorimetric assay of toluidine blue dye and the results showed that the amount of immobilized heparin was strongly dependent on the heparin dosage in reaction solution. The results of contact angle measurement indicated that the hydrophilicity of Cu(OH)2 nanofiberous membranes was enhanced by the immobilization of heparin. The adhesion, activation and transmutation of platelets on Hep@Cu(OH)2 membrane were suppressed remarkably due to the introduction of heparin, which suggested that the Hep@Cu(OH)2 membranes had good hemocompatibility. In addition, Cu(OH)2 and Hep@Cu(OH)2 nanofiberous membranes exhibited very good antibacterial activities against Escherichia coli and Staphyloccocus aureus.

Immobilization of Heparan Sulfate on Electrospun Meshes to Support Embryonic Stem Cell Culture and Differentiation *

As our understanding of what guides the behavior of multi- and pluripotent stem cells deepens, so too does our ability to utilize certain cues to manipulate their behavior and maximize their therapeutic potential. Engineered, biologically functionalized materials have the capacity to influence stem cell behavior through a powerful combination of biological, mechanical, and topographical cues. Here, we present the development of a novel electrospun scaffold, functionalized with glycosaminoglycans (GAGs) ionically immobilized onto the fiber surface. Bound GAGs retained the ability to interact with GAG-binding molecules and, crucially, presented GAG sulfation motifs fundamental to mediating stem cell behavior. Bound GAG proved to be biologically active, rescuing the neural differentiation capacity of heparan sulfate-deficient mouse embryonic stem cells and functioning in concert with FGF4 to facilitate the formation of extensive neural processes across the scaffold surface. The combination of GAGs with electrospun scaffolds creates a biomaterial with potent applicability for the propagation and effective differentiation of pluripotent stem cells.

Simultaneous antibacterial and anticoagulant properties of polypropylene non-woven textiles

The aim of this work was to prepare a non-woven Polypropylene (PP) textile functionalized with bioactive molecules to improve simultaneously its anticoagulation and antibacterial properties. The immobilization of either heparin (anticoagulation agent) or gentamicin (aminoglycoside antibiotic) was already proven to be effective on non-woven PP textiles. This work details how we managed to immobilize both gentamicin and heparin on the textile [1]. The immobilization times were studied in order to determine the best compromise between cytocompatibility, anticoagulant effect and antimicrobial activity.

Surface modification of polysulfone based hemodialysis membranes with layer by layer self assembly of polyethyleneimine/alginate-heparin: a simple polyelectrolyte blend approach for heparin immobilization

This study intends to improve blood compatibility of polysulfone (PSF) membranes by generating a nonthrombogenic surface through heparin immobilization. To achieve this task, the support membrane prepared from a blend of PSF and sulfonated polysulfone (SPSF) was modified with layer by layer (LBL) deposition of polyethyleneimine (PEI) and alginate (ALG) and heparin blended with ALG was immobilized only on the outermost surface of the LBL assembly. The results have shown that the adsorption of human plasma proteins and platelet activation on the LBL modified membranes decreased significantly compared with the unmodified PSF and PSF-SPSF blend membranes. Furthermore, blending ALG with a small amount of heparin remarkably prolonged the APTT values of heparin free PEI/ALG coated membranes. It is envisaged that the use of a blend of HEP and ALG only in the terminating layer of the LBL assembly can be an economical and alternative modification technique to create nonthrombogenic surfaces.

Polysaccharides immobilized in polypyrrole matrices are able to induce osteogenic differentiation in mouse mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) have attracted considerable interest due to their ability to differentiate and contribute to the regeneration of mesenchymal tissues. The present study illustrates that the proper immobilization of heparin (Hep) and hyaluronic acid (HA) into a polypyrrole (PPy) matrix by electropolymerization results in an optimal interface for MSC differentiation towards osteoblast lineage. The obtained thin films showed good thermal stability, hydrophilicity and slow controlled polysaccharide release. The in vitro tests showed the main role of the interface chemical composition. Indeed, PPyHep and PPyHA thin films were able to induce osteogenic differentiation as determined by levels of specific early osteogenic markers (Runx2 and osterix) even in the absence of differentiating medium. Increased levels of ALP and Alizarin red staining, both indicating mineralization processes, confirmed the presence of mature osteoblasts.

Anti-platelet and tissue engineering approaches to biomaterial blood compatibilization: how well have these been translated into the clinic?

In this article, we provide an update on the various approaches to "blood compatibilization", and include both passive and active approaches to compatibilizing biomaterials in contact with blood. Broadly speaking, the surface modification approaches involved either repel platelets or attract endothelial cells. For platelet-repelling surfaces, heparin immobilization seems to be the most successful approach. At least two such surfaces have been approved by the health authorities in various countries for applications involving short-term contact with blood. For active endothelialization, ex vivo seeding with autologous cells has been translated into the clinic, while selective endothelial cell capture is a promising approach. In spite of over 30years of research in this area, a truly intrinsically non-clotting surface has not been developed yet; certain promising avenues have been indicated by the research, which we will critically assess here.

Polycarbonate urethane films modified by heparin to enhance hemocompatibility and endothelialization

AbstractPolyurethane with pendant carboxyl groups was synthesized by a two‐step reaction using methylene diphenyl diisocyanate as hard segments, polycarbonate diols as soft segments and 2,2‐bis(hydroxymethyl) propionic acid as chain extenders. The surfaces of the casting polymer films were further covalently conjugated with heparin as confirmed by Fourier transform infrared spectroscopy. The associated surface‐exposed heparin content was measured to be 1.92 µg cm−2 and was shown to be active. Meanwhile, surface hydrophobicity decreased after heparin modification. Heparin immobilization greatly improved the hemocompatibility of polyurethane films both in vitro and in vivo. The in vitro cell responses revealed that the expression of proinflammatory markers CD106 and CD62E of human vein endothelial cells was significantly suppressed on the surfaces of the heparinized polyurethane films, while the affinity and proliferation of the cells were improved. The in vivo performance of this material before and after heparin modification was evaluated as a stent coating material in a porcine coronary artery injury model. Histological analysis indicated that heparinized polyurethane induced mild foreign body reactions and inflammation, which were much lower than those induced by unmodified polyurethane. Heparin immobilization also accelerated the endothelialization process on the surface of the polymer‐coated stents when implanted in porcine coronary arteries. More detailed long‐term studies are needed to confirm the biostability of the heparin and the endothelialized surfaces. Copyright © 2012 Society of Chemical Industry

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

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.

Haemocompatibility improvement of metallic surfaces by covalent immobilization of heparin–liposomes

Stainless steel surfaces were processed by means of plasma enhanced chemical vapor deposition (PE-CVD) fed with acrylic acid vapors in order to functionalize them with carboxyl groups, which were subsequently activated for covalent immobilization of heparin-loaded (HEP) NH2 group-functionalized (Fun) nanoliposomes (NLs). Empty Fun or HEP non-functionalized (control) NLs were used as controls. NLs were characterized for mean diameter, surface charge and heparin encapsulation/release. Different lipid compositions were used for NL construction; PC/Chol (2:1mol/mol) or PC/Chol (4:1mol/mol) (fluid type vesicles) [which allow gradual release of heparin] and DSPC/Chol (2:1mol/mol) (rigid type vesicles). Surface haemocompatibility was tested by measuring blood clotting time. Platelet adhesion on surfaces was evaluated morphologically by SEM and CLSM. The haemocompatibility of plasma-processed surfaces was improved (compared to untreated surfaces); Fun-HEP NL-coated surfaces demonstrated highest coagulation times. For short surface/blood incubation periods, surfaces coated with Fun-HEP NLs consisting of PC/Chol (2:1) had higher coagulation times (compared to DSPC/Chol NLs) due to faster release of heparin. Heparin release rate from the various NL types and surface platelet adhesion results were in agreement with the corresponding blood coagulation times. Concluding, covalent immobilization of drug entrapping NLs on plasma processed surfaces is a potential method for preparation of controlled-rate drug-eluting metallic stents or devices.

Fabrication of PP-g-PEGMA-g-heparin and its hemocompatibility: From protein adsorption to anticoagulant tendency

We described a two-step process to fabricate the heparinized polypropylene (PP) film using cyanuric chloride (CC) as a trifunctional reagent and poly (ethylene glycol) methacrylate (PEGMA) as a spacer. The modified PP films were characterized by attenuated total reflectance FT-IR and X-ray photoelectron spectroscopy; the content of PEGMA and heparin were determined by gravimetric method and a toluidine blue assay, respectively. For the PP-g-PEGMA films, it was found that small size protein BSA tended to adsorb on the surface of low molecular weight monomer grafted PP, whereas big spindle-shaped fibrinogen tended to adsorb on the surface of high molecular weight monomer grafted PP. We gave a definition of anti-protein adsorptive factor r with two model proteins, albumin and fibrinogen. The results by platelet adhesion and plasma recalcification time (PRT) experiments indicated that the factor r could be used to quantitatively evaluate the anticoagulant tendency of PP-g-PEGMA modified films. For the PP-g-PEGMA-g-heparin modified films, the surface was proved to have a high bioactivity by the adsorption of AT III assay and very low platelet adhesion. It indicated that immobilization of heparin on the PP film with PEGMA as a spacer was an effective way to improve the hemocompatibility of PP.

THREE-DIMENSIONAL NANOCOMPOSITE SCAFFOLDS FOR BONE TISSUE ENGINEERING: FROM DESIGN TO APPLICATION

Selective laser sintering (SLS), a rapid prototyping technology, was investigated for producing bone tissue engineering scaffolds. Completely biodegradable osteoconductive calcium phosphate (Ca-P)/poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) scaffolds were successfully fabricated via SLS using Ca-P/PHBV nanocomposite microspheres. In the SLS manufacturing route, the architecture of tissue engineering scaffolds (pore shape, size, interconnectivity, etc.) can be designed and the sintering process can be optimized for obtaining scaffolds with desirable porous structures and mechanical properties. SLS was also shown to be very effective in producing highly complex porous structures using nanocomposite microspheres. To render SLS-formed Ca-P/PHBV scaffolds osteoinductive, recombinant human bone morphogenetic protein-2 (rhBMP-2) could be loaded onto the scaffolds. For achieving a controlled release of rhBMP-2 from scaffolds, surface modification of Ca-P/PHBV scaffolds by gelatin entrapment and heparin immobilization was needed. The immobilized heparin provided binding affinity for rhBMP-2. Surface modified Ca-P/PHBV nanocomposite scaffolds loaded with rhBMP-2 enhanced the proliferation of human umbilical cord derived mesenchymal stem cells (hUCMSCs) and also their alkaline phosphatase activity. In in vivo experiments using a rabbit model, surface modified Ca-P/PHBV nanocomposite scaffolds loaded with rhBMP-2 promoted ectopic bone formation, exhibiting their osteoinductivity. The strategy of combining advanced scaffold fabrication, nanocomposite material, and controlled growth factor delivery is promising for bone tissue regeneration.

Anticoagulant and antimicrobial finishing of non-woven polypropylene textiles

The aim of this work is to prepare non-woven polypropylene (PP) textile functionalized with bioactive molecules in order to improve its anticoagulation and antibacterial properties. This paper describes the optimization of the grafting process of acrylic acid (AA) on low-pressure cold-plasma pre-activated PP, the characterization of the modified substrates and the effect of these modifications on the in vitro biological response towards cells. Then, the immobilization of gentamicin (aminoglycoside antibiotic) and heparin (anticoagulation agent) has been carried out on the grafted samples by either ionic interactions or covalent linkages. Their bioactivity has been investigated and related to the nature of their interactions with the substrate. For gentamicin-immobilized AA-grafted samples, an inhibition radius and a reduction of 99% of the adhesion of Escherichia coli have been observed when gentamicin was linked by ionic interactions, allowing the release of the antibiotic. By contrast, for heparin-immobilized AA-grafted PP samples, a strong increase of the anticoagulant effect up to 35 min has been highlighted when heparin was covalently bonded on the substrate, by contact with the blood drop.

Plasma graft polymerization of Acrylic Acid and immobilization of Heparin to improve blood compatibility of Polyethylene terephthalate (PET).

ABSTRACTPolymeric materials have been attracted the attention of researchers in various R&D applications. In this research, attempts were made to evaluate the changes in blood compatibility of polyethylene terephthalate (PET) by grafting acrylic acid (AAc) and immobilizing heparin by employing two-step plasma treatment. The PET surface was modified by using a novel method named “two-step plasma treatments” (TSPT). While first plasma is creating active sites for grafting, the second plasma polymerizing pre-adsorbed reactive monomer onto the surface of films. Finally, heparin immobilization was performed in the presence of 1-ethyl-3-(dimethylaminopropyl) carbodiimide. All films were characterized by attenuated total reflection Fourier transformed infrared (ATR-FTIR) spectroscopy and scanning electron microscopy (SEM). The surface hydrophilicity of films was studied by water contact angle test and blood compatibility evaluated by Lactate dehydrogenase method (LDH Test). In vitro studies based on LDH results demonstrared that platelet adhesion onto the modified surfaces with heparin was drastically reduced in comparison with unmodified PET.

Blood Compatibility Improvement of Titanium via Self-polymerization of Dopamine and Covalent Graft of Heparin

elf-polymerization of dopamine and immobilization of heparin were applied for surface-modification of titanium in order to improve its blood compatibility.Surface modification was developed via self-polymerization of dopamine and covalent graft of heparin.Surface elemental composition was analyzed qualitatively by X-ray photoelectron spectroscopy(XPS).Surface heparin content was detected by toluidine blue O(TBO).Surface hydrophilicity and hydrophobicity were characterized by water contact angle measurement.Hemolysis rate test,which was applied for detection ofhemolytic ability,was combined with activated partial thromboplastin time test(APTT) and platelet adhesion test to evaluate blood compatibility.The results showed that self-polymerization of dopamine was achieved on titanium surface.Heparin was successfully immobilized onto polydopamine layer by covalent graft.The heparin-modified surface could result in a significantly improved hydrophilicity,low hemolysis rate,prolonged APTT and a remarkablely decrease both in adhesion amount and activation degree of platelet.The blood compatibility of titanium could be significantly improved by the method of self-polymerization of dopamine and covalent graft of heparin.This study provides potential application for cardiovascular implants materials with anticoagulant properties.

Nanocoating of Diazoresin/Polysaccharides Multilayer Boosts Biocompatibility of Nitinol Biomedical Devices

The surface of a biomaterial and a biological environment are key factors determining the biocompatibility of the material. Determining biocompatibility of medical implants is a critical step toward approval and clinical application. In this study, the biocompatibility of a covalently attached nanocoating of alginate(Alg)/diazoresin(DR)/heparin(Hep) multilayer has been evaluated by thrombin inactivation, protein adsorption, leukocyte adhesion, cytotoxicity in vitro and subcutaneous implantation in vivo. It was found that the outermost layer of the coating played a key role on anticoagulant activity. No significant difference was seen in albumin adsorption between uncoated and coated one. However, the fibrinogen adsorption on DR/polysaccharides coatings was lower than the uncoated Nitinol. Compared with the uncoated Nitinol surface, DR/polysaccharides multilayer coating presented a drastically reduced adhesion in vitro of polymorphonuclear neutrophil leukocytes (PMN) and peripheral blood mononuclear cells (PBMC). Cell biocompatibility tests indicated that the nanocoating of DR/polysaccharides multilayer did not lead to an increase in the cytotoxicity of the Nitinol and the adherent cells maintained good viability. Subcutaneous implantation in C57BL/6 mice suggested that the coated Nitinol had good tissue biocompatibility. The use of alginate interlayer for heparin immobilization instead of proteins such as albumin, collagen, and gelatin may avoid the risk of contamination with pathogenic agents. Hence, the nanocoating of covalently attached DR/polysaccharide multilayer boosts the biocompatibility of Nitinol biomedical devices. Keywords: heparin, thrombin, Nitinol, alginate, biocompatibility, Layer-by-layer, Nitinol Biomedical Devices, medical implants, Diazoresin

Enhancement of Blood Compatibility of Poly(urethane) Substrates by Mussel-Inspired Adhesive Heparin Coating

Heparin immobilization on surfaces has drawn a great deal of attention because of its potential application in enhancing blood compatibility of various biomedical devices such as catheters, grafts, and stents. Existing methods for the heparin immobilization are based on covalent linkage formation and electrostatic interaction between substrates and heparin molecules. However, complicated multistep procedures and uncontrolled desorption of heparin are limitations of these methods. In this work, we report a new heparin derivative that exhibits robust adhesion on surfaces. The derivative, called hepamine, was prepared via conjugation of dopamine, a mussel-inspired adhesive moiety, onto a heparin backbone. Immersion of poly(urethane) substrates into an aqueous solution of hepamine resulted in robust heparin coating of the poly(urethane), the most widely used polymeric material for blood-contacting medical devices. The hepamine-coated poly(urethane) substrate showed significant inhibition of blood coagulation and platelet adhesion. The use of hepamine for surface modification is advantageous for several reasons: for example, no chemical pretreatment of the substrates is necessary, and surface functionalization is a simple, one-step procedure. Thus, the heparin immobilization method described herein is an excellent alternative approach for the introduction of heparin molecules onto surfaces.

Improved Hemocompatibility Guided by Pulsed Plasma Tailoring the Surface Amino Functionalities of TiO2 Coating for Covalent Immobilization of Heparin

New biomedical metals are constantly developed to fulfillthe clinical needs for better mechanical properties,biocompatibility, and resistance to corrosion. The 316Lstainlesssteel(SS),Co Cralloysandnickel–titaniumalloyshave gained wide applications of fabricating vascularinterventionaldevices,suchasstentsandvenacavafilters,due to their excellent mechanical properties.

Low Temperature Plasma Treatment for Immobilization of Biomaterials on Polymer Surface

In some medical use, surface property such as biocompatibility and blood compatibility is very important. To improve the surface property of polymers, ammonia plasma was used to introduce amino group onto polymer surface in order to bind with heparin. The amount and the selectivity of amino group were improved by plasma treatment. The ratio of sulfur contained in heparin to carbon also increased from 0.5% to 10% by plasma process. The positive correlation can be found between heparin immobilization and amino group introduced by plasma. The immobilized heparin is strongly connected and it enhances the anticoagulant property on the surface.

Heparinized polyvinyl alcohol to specifically adsorb low-density lipoprotein from plasma

Introduction A medical adsorbent for blood purification was developed to specifically adsorb low-density lipoprotein (LDL) from hypercholesterolemia patient’s plasma by covalently immobilizing heparin onto the surface of polyvinyl alcohol (PVA) with the couplant toluence-2,4-diisocyanate (TDI). Methods We used IR to demonstrate the success of covalently immobilizing heparin onto the surface, and investigated its adsorption of LDL, and primarily evaluated its hemo-compatibility using tests for platelet adhesion, the degree of platelet activation and a hemolysis test. Results (1) Heparin was successfully covalently immobilized onto the surface, the maximum amount of heparin immobilized on the surface of 1 g PVA–1799 granules was about 5 μg; (2) one optimal condition for adsorption of LDL from hyperlipidemia plasma was a pH within the range of 7.2∼9.5, accordingly the adsorptive ratio (adsorbent/g: plasma/L = 1:2) for LDL was about 70%; (3) it exhibited good hemo-compatibility. Conclusion The adsorbent results in satisfactory adsorption of LDL with good hemo-compatibility; it could potentially be used as a blood purification material, and immobilization of heparin onto medical materials may be a way to develop an LDL-specific adsorbent for blood purification.