Surface Heparinization Research Articles

Surface modification of PE porous membranes based on the strong adhesion of polydopamine and covalent immobilization of heparin

Based on the self-polymerization and strong adhesion characteristic of dopamine in wet conditions, the hydrophobic polyethylene (PE) porous membranes were surface-modified via simply immersing them into dopamine aqueous solution for 24 h. Subsequently, heparin was immobilized covalently onto the resultant membrane by the coupling between heparin and reactive polydopamine layer. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) were utilized to determine the chemical compositions of membrane surface, which confirmed the successful introduction of polydopamine and immobilization of heparin molecules. Scanning electronic microscopy (SEM) and atomic force microscopy (AFM) were employed to investigate the changes in surface morphologies after surface modification. The data of water contact angle measurements indicated that the hydrophilicity of PE membranes was remarkably improved after polydopamine coating and heparin immobilization. The results of in vitro hemocompatibility test proved that surface heparinization significantly suppressed the adhesion of platelet and enhanced the anticoagulation ability of PE membranes. This work offered a convenient approach to improve the permeability and biocompatibility of inert PE porous membranes for their biomedical and blood-contacting applications.

Cytokines in Blood from Septic Patients Interact With Surface-Immobilized Heparin

When passing blood from septic patients through a column packed with surface heparinized beads, we were able to significantly reduce concentrations of the proinflammatory cytokine tumor necrosis factor (TNF)-alpha from initially very high levels. Passage of blood over nonheparinized beads did not affect the TNF levels. Meanwhile, concentrations of the regulated on activation, normal T-cells expressed, and secreted leukocyte activating cytokine (RANTES) remained unchanged following passage through the heparinized column, but rose significantly after passage through a column packed with the nonheparinized control beads. We conclude that surface heparinization may be a useful technique for selectively regulating the levels of heparin-binding cytokines from whole blood. This may have potential implications for the treatment of hyper-inflammatory conditions such as severe sepsis. Our data also suggests that surface activation and its associated inflammatory response may be avoided by using heparinization of the extracorporeal circuit.

Functionalization of chitosan/poly(lactic acid‐glycolic acid) sintered microsphere scaffolds via surface heparinization for bone tissue engineering

Scaffolds exhibiting biological recognition and specificity play an important role in tissue engineering and regenerative medicine. The bioactivity of scaffolds in turn influences, directs, or manipulates cellular responses. In this study, chitosan/poly(lactic acid-co-glycolic acid) (chitosan/PLAGA) sintered microsphere scaffolds were functionalized via heparin immobilization. Heparin was successfully immobilized on chitosan/PLAGA scaffolds with controllable loading efficiency. Mechanical testing showed that heparinization of chitosan/PLAGA scaffolds did not significantly alter the mechanical properties and porous structures. In addition, the heparinized chitosan/PLAGA scaffolds possessed a compressive modulus of 403.98 +/- 19.53 MPa and a compressive strength of 9.83 +/- 0.94 MPa, which are in the range of human trabecular bone. Furthermore, the heparinized chitosan/PLAGA scaffolds had an interconnected porous structure with a total pore volume of 30.93 +/- 0.90% and a median pore size of 172.33 +/- 5.89 mum. The effect of immobilized heparin on osteoblast-like MC3T3-E1 cell growth was investigated. MC3T3-E1 cells proliferated three dimensionally throughout the porous structure of the scaffolds. Heparinized chitosan/PLAGA scaffolds with low heparin loading (1.7 microg/scaffold) were shown to be capable of stimulating MC3T3-E1 cell proliferation by MTS assay and cell differentiation as evidenced by elevated osteocalcin expression when compared with nonheparinized chitosan/PLAGA scaffold and chitosan/PLAGA scaffold with high heparin loading (14.1 microg/scaffold). This study demonstrated the potential of functionalizing chitosan/PLAGA scaffolds via heparinization with improved cell functions for bone tissue engineering applications.

Uropathogen Interaction With the Surface of Urological Stents Using Different Surface Properties

Ureteral stents commonly become infected or encrusted. Various coatings have been developed to decrease bacterial adherence. To our knowledge there has been no in vitro testing of coating with heparin to date. We determined the effects of heparin coating on bacterial adherence of common uropathogens and physical stent properties. Heparin coated Radiance ureteral stents (Cook) and noncoated Endo-Sof control stents were tested against triclosan eluting Triumph(R) stents and noneluting Polaris control stents for adherence of Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus aureus and Pseudomonas aeruginosa for 7 days. Adherent bacteria were determined and biofilms were visualized using fluorescent dyes. Radial, tensile and coil strength of the Radiance and Polaris stents was compared to determine the effect of heparin coating on physical stent characteristics. Heparin coating did not decrease bacterial adhesion compared to its control. E. coli adhesion was limited by all stents tested. The Polaris stent showed significantly greater resistance to bacterial adherence for Klebsiella, Pseudomonas and Enterococcus than the Endo-Sof and Radiance stents but was more susceptible to S. aureus adherence. The Triumph stent resisted all bacteria except Pseudomonas and Enterococcus. Mature biofilms were observed on all stents with lower viability on the Triumph stent. Radiance stents showed higher tensile and lower compression strength than its control. Heparin coating does not decrease bacterial adherence to ureteral stents. Drug eluting antimicrobials have an inhibitory effect on bacterial adherence and the Polaris stent showed the least bacterial adherence of the nondrug eluting ureteral stents tested.

Evaluation of Haemocompatibility of TLM Titanium Alloy with Surface Heparinization

A layer of TiO 2 film was prepared by sol-gel method on the surface of TLM(Ti-3Zr-2Sn-3Mo-25Nb) alloy. Then the coated samples were treated by hydroxyl solution and amination solution in order to introduce the OH − and NH 2 − active groups, and the heparin was linked on the surface of TiO 2 film through these active groups. The phase identification and the surface characteristics of the samples were successively undertaken using XRD, SEM and EDS. The in vitro blood compatibility of the TLM alloy specimens with and without heparinization treatments was evaluated by the contact angle test, the hemolysis test and investigation of their platelet adhesion behavior. The results showed that the haemocompatibility of the TLM alloy could be significantly improved by surface modification via heparinization.

Covalent Surface Heparinization Potentiates Porous Polyurethane Scaffold Vascularization

Porous scaffolds play an integral role in many tissue-engineering approaches, and the ability to improve vascularization, without eliciting an excessive inflammatory response, would constitute an important step towards achieving long-term healing and function of devices made from these materials. After having previously optimized the dimensional requirements of the well-defined pores, the present study aimed at a further shift from inflammation to vascularization via surface immobilization with heparin. Porous polyurethane disks were produced to contain well-defined pores (147 +/- 2 microm) with abundant interconnecting windows (67 +/- 2 microm). After heparinization via copolymer grafting and amination to contain 32 microg of heparin, the modification appeared as a uniform layer on all exposed surfaces, with no signs of pore obliteration or significant changes in pore size. After 28 days implantation in a rat subcutaneous model, the scaffolds were assessed for vascularization/arteriolization and inflammation using CD31/actin and ED-1 staining, respectively. Heparinization resulted in a significant increase in vascularization: capillaries increased by 62% in number (66.2 +/- 0.8 to 107.3 +/- 1.4 vessels/mm( 2); p < 0.03) and 56% in total area (0.9 +/- 0.1 to 1.4 +/- 0.02%; p<0.02). Arteriolization also increased in absolute terms (200% in number; p<0.03), but did not change significantly when normalized to capillary number. Heparinization did not significantly affect the inflammatory response at this time-point, as quantified by ED-1 positive macrophage and foreign body giant cell (FBGC) content. Thus, the in vivo vascularization of porous scaffolds could be increased without concomitant increase in the inflammatory response by employing a simple surface modification technique. This could be a valuable tool for in vivo tissue engineering applications where enhanced vascularization is required.

Islet Surface Heparinization Prevents the Instant Blood-Mediated Inflammatory Reaction in Islet Transplantation

In clinical islet transplantation, the instant blood-mediated inflammatory reaction (IBMIR) is a major factor contributing to the poor initial engraftment of the islets. This reaction is triggered by tissue factor and monocyte chemoattractant protein (MCP)-1, expressed by the transplanted pancreatic islets when the islets come in contact with blood in the portal vein. All currently identified systemic inhibitors of the IBMIR are associated with a significantly increased risk of bleeding or other side effects. To avoid systemic treatment, the aim of the present study was to render the islet graft blood biocompatible by applying a continuous heparin coating to the islet surface. A biotin/avidin technique was used to conjugate preformed heparin complexes to the surface of pancreatic islets. This endothelial-like coating was achieved by conjugating barely 40 IU heparin per full-size clinical islet transplant. Both in an in vitro loop model and in an allogeneic porcine model of clinical islet transplantation, this heparin coating provided protection against the IBMIR. Culturing heparinized islets for 24 h did not affect insulin release after glucose challenge, and heparin-coated islets cured diabetic mice in a manner similar to untreated islets. This novel pretreatment procedure prevents intraportal thrombosis and efficiently inhibits the IBMIR without increasing the bleeding risk and, unlike other pretreatment procedures (e.g., gene therapy), without inducing acute or chronic toxicity in the islets.

Study on antithrombogenicity and surface heparinization of small intestinal submucosa

目的 研究等离子体引发血管组织工程支架--小肠粘膜下层(SIS)表面肝素化及肝素化后SIS膜的抗凝血性能.方法 利用等离子体引发技术在SIS膜表面结合肝素,通过体外凝血时间检测及循环血流下观察肝素化SIS管腔的长期通畅性,以评价SIS膜的血液相容性.结果 肝素结合到低温等离子体引发的SIS膜表面,肝素化SIS膜表面水接触角降低,亲水性增强,表面自由能增大,其凝血酶原时间、部分凝血活酶时间及凝血酶时间明显延长,肝素化SIS小口径血管支架保持通畅达6周.结论 SIS膜表面通过等离子体进行肝素固化处理后具有良好而持久的抗凝血性能,表现出良好的血液相容性。

Surface Heparinization of Polyurethane Via Bromoalkylation of Hard Segment Nitrogens

Previous research from our group has demonstrated that bromoalkylation of polyurethane elastomers via base mediated activation of the urethane-hard segment nitrogen groups can be used to either attach bisphosphonate groups to confer calcification resistance or append cholesterol to promote endothelial cell adhesion. In the present studies we further explore the potential of this chemical approach by investigating bulk carboxylation of polyurethanes via bromoalkylation to enable surface heparinization for thromboresistance. Thus, polyurethane (PU) was modified with pendant 7-carboxy-5-thiaheptyl groups using a polymer-analogous reaction of bromobutylated PU with tetrabutylammonium 3-mercaptopropionate in mild conditions. The grafting of polyallylamine (PAA) onto the surface of carboxylated PU via direct coupling of amino and carboxy groups resulted in high levels of PAA (up to 8 mug/cm(2)). The surface-aminated PU was further covalently modified with unfractionated heparin as confirmed by FTIR. Fluorescence labeling of PAA hydrochloride and heparin with BODIPY-FL was used to quantify the extent of surface modifications. Heparin was covalently bound at a high level (1.11 +/- 0.06 mug/cm(2)) and was shown to be active, with demonstrable Factor Xa inhibition and platelet factor IV binding. It is concluded that surface amination of bulk-carboxylated PU represents a novel approach for heparinizing PU; carboxylation followed by surface amination represents another important dimension of bromo-alkyl activation of polyurethane hard segments, thereby enabling heparinization.

A prospective, double-blind study on the efficacy of the bioline surface-heparinized extracorporeal perfusion circuit

Background We evaluated the newly introduced Bioline heparin coating and tested the hypothesis that surface heparinization limited to the oxygenator and the arterial filter will ameliorate systemic inflammation and preserve platelets during cardiopulmonary bypass (CPB). Methods In a prospective double-blind study, 159 patients underwent coronary revascularization using closed-system CPB with systemic heparinization, mild hypothermia (33°C), a hollow-fiber oxygenator, and an arterial filter. The patients were randomly divided in three groups. In group A (controls, n = 51), surface heparinization was not used. In group B (n = 52), the extracorporeal circuits were totally surface-heparinized with Bioline coating. In group C (n = 56), surface heparinization was limited to oxygenator and arterial filter. Results No significant difference was noted in patient characteristics and operative data between groups. Operative (30-day) mortality was zero. Platelet counts dropped by 12.3% of pre-CPB value among controls at 15 minutes of CPB, but were preserved in groups B and C throughout perfusion ( p = 0.0127). Platelet factor 4, plasmin-antiplasmin levels, and tumor necrosis factor-α increased more in controls during CPB than in groups B or C ( p = 0.0443, p = 0.0238 and p = 0.0154 respectively). Beta-thromboglobulin, fibrinopeptide-A, prothrombin fragments 1 + 2, factor XIIa levels, bleeding times, blood loss, and transfusion requirements were similar between groups. Intensive care unit stay was shorter in groups B and C than in controls ( p = 0.037). Conclusions Surface heparinization with Bioline coating preserves platelets, ameliorates the inflammatory response and is associated with a reduced fibrinolytic activity during CPB. Surface heparinization limited to the oxygenator and the arterial filter had similar results as totally surface-heparinized circuits.

Comparison of haemocompatibility improvement of four polymeric biomaterials by two heparinization techniques

Two surface heparinization procedures, introduced by Bamford and Al-Lamee (Polymer 22 (1996) 4885; 13 (1994) 2844) and Seifert et al. (J. Mater. Sci.: Mater. Med. 7 (1996) 465), respectively, were applied to four commercially available biomaterials (silicone rubber, polyethylene, polypropylene and polyvinylchloride) in order to compare their efficiency in improving haemocompatibility. The indirect method (Bamford and Al-Lamee) produced a much better heparinization yield—10.5% maximum, compared to the direct one (Seifert et al.), of only 0.20% maximum. Both methods provided a better response of the heparinized biomaterials compared to the uncoated ones in terms of platelet retention and a significantly better response in terms of activation of the coagulation system, suggesting that heparin molecules remained biologically functional in both cases. The results were particularly interesting in the case of polyvinylchloride where the maximum immobilization yield was obtained by the indirect method resulting also to a pronounced haemocompatibility improvement. Scanning electron microscopy studies confirmed adhered platelet morphology whereas atomic force microscopy was used to examine surface morphology of heparinized and reference materials surface.

Effect in a Rat Model of Heparinized Peritoneal Dialysis Catheters on Bacterial Colonization and the Healing of the Exit Site

We performed a prospective, double-blind, randomized study to evaluate whether stable surface heparinization of silicone peritoneal dialysis (PD) catheters prevents bacterial colonization or biofilm formation and improves healing of the exit site. Heparinized catheters were implanted in 20 Sprague-Dawley rats (group H) and non heparinized catheters in another 20 (group C). The PD catheters, constructed of silicon tubing with two polyester cuffs, were patterned after the standard Tenckhoff catheter. A covalent multipoint method of attachment onto polymeric surfaces was used for stable, permanent chemical immobilization of heparin on the PD catheter. Dialysis exchanges (25-mL instillation volume) were performed twice daily for 4 weeks through the permanent catheter. Prophylactic antibiotics were not used. The exit sites were evaluated at 2-week intervals. The extent of biofilm coverage on the intraperitoneal portion of the catheter (obtained at the end of the experiment) was assessed, and sonicated fluid from the catheter tip was cultured for evaluating bacterial colonization of the catheter. Exit-site scores in group H were lower than in group C (p = 0.052) at the end of week 4. Bacterial colonization tended to be less common in group H [2 of 12 catheters (17%)] than in group C [8 of 15 catheters (53%); p = 0.058], but the extent of biofilm, the peritonitis rate, and the inflammation score of tissue adjacent to the cuff were not different between the groups. Those data suggest that heparinized PD catheters can be a practical approach to the prevention of bacterial colonization and can improve healing of the exit site.

Veno-venous bypass in liver transplantation: heparin-coated perfusion circuits reduce the activation of humoral defense systems in an in vitro model.

We studied the effects of bypass circuit surface heparinization on kallikrein-kinin, coagulation, fibrinolytic and complement activation in a closed model system for simulating veno-venous bypass (WBP) in orthotopic liver transplantation (OLT). The circuits were identical to those in routine use during clinical OLT in our institution. Fresh whole human blood diluted 1:2 with Ringer's acetate was circulated at a non-pulsatile flow (2 l/min) and at a constant temperature (37.5 degrees C) for 12 h. In 10 experiments, the entire inner surface of the circuits was coated with end-point attached heparin (HC). In the remaining 10, non-treated PVC tubing was used (NC). Components of the plasma kallikrein-kinin, coagulation, fibrinolytic and complement systems were analyzed using functional techniques (chromogenic peptide substrate assays) and enzyme immunoassays at baseline, 3 and 12 h. Significant activation of the initial (C3bc) and terminal (TCC) components of the complement system were found in both the NC and HC groups after 3 and 12 h: C3bc: NC: baseline = 4 (3.5-7.7), 3 h = 17.3* (12.5-27), 12h = 31* (17.7-63.6), HC: baseline = 4.9 (3.2-6.8), 3h = 9* (6-14.4), 12h = 13.7* (7.4-18.1). TCC: NC: baseline = 0.4 (0.2-0.6), 3h = 5*(0.8-11.9), 12 h: 13.1* (4.2-25.7). HC: baseline = 0.5 (0.1-0.6), 3 h = 0.6* (0.1-0.8), 12 h = 1.2* (0.3-2) AU/ml; median and range (*: p < 0.05). The C3bc and TCC concentrations were significantly higher in the NC group at 3 and 12 h, compared to the HC group: C3bc (NC vs. HC group): 3 h, p < 0.001; 12 h, p < 0.001. TCC (NC vs. HC group): 3h, p < 0.001; 12 h, p < 0.001. Significant increases in the values of thrombin-antithrombin complexes (p = 0.003), prothrombin fragment 1 + 2 (p = 0.006) and plasmin-alpha2-antiplasmin complexes (p = 0.016) were found in the non-coated group, but not in the heparin-coated group during the observation period, showing that the coagulation and fibrinolytic systems were activated in the non-coated circuits. We conclude that heparin-coating of the internal surface of the extracorporeal perfusion circuit used for WBP reduces activation of the plasma cascade systems in a closed venous system in vitro.

Anticoagulant surface prepared by the heparinization of ionic polyurethane film

We present a new method for heparinization on the surface of polyurethane. The segmented polyurethane was first modified with an epoxide monomer and followed by a ring-opening reaction with diethanolamine to introduce sufficient hydroxyl groups on the surface of cast film. On this film surface, a cationic monomer was grafted by using tetravalent Cerium salt as an initiator. Heparin was immobilized in high efficiency on the ionized surface through static interactions in aqueous solution. The structure of ionized and heparinized surfaces were characterized by attenuated total reflectance infrared spectroscopy (ATR–FTIR) and electron spectroscopy for chemical analysis (ESCA) spectra. The platelet-rich plasma (PRP) contacting test and the platelet-poor plasma (PPP) clotting time measurements showed that the immobilized heparin retained its strong anticoagulant property. The release of heparin from film into salt solution was also studied, and it was found that only a small portion of heparin (10–20%) was released over a period as long as 10 h. It is expected that this new method for surface heparinization can be used to prepare antithrombogenic materials with long-term stability. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 76: 382–390, 2000

Surface Heparinization Profoundly Decreases Acute Thrombosis on Crown and Mini-Crown Stents in the Baboon Arteriovenous Shunt Model

Surface Heparinization Profoundly Decreases Acute Thrombosis on Crown and Mini-Crown Stents in the Baboon Arteriovenous Shunt Model

Surface heparinization profoundly decreases acute thrombosis on crown and mini-crown stents in the baboon arteriovenous shunt model

Surface heparinization profoundly decreases acute thrombosis on crown and mini-crown stents in the baboon arteriovenous shunt model

Surface heparinization of central venous catheters reduces microbial colonization in vitro and in vivo

To evaluate in vitro and in vivo the efficacy of covalent end point-attached heparin to single-lumen polyurethane central venous catheters in reducing microbial adherence and colonization. In vitro study: A controlled bench study. In vivo study: A prospective, randomized, double-blind, clinical trial. Intensive care unit in a 1200-bed teaching hospital. In vitro study: Adhesion of 17 radiolabeled clinical isolates of Staphylococci to catheters was examined in vitro. In vivo study: The outcome of heparinized and control catheters was compared in vivo in patients receiving long-term parenteral nutrition. Fifty-five adult patients were prospectively, blindly randomized to heparinized or control central venous catheters. The catheters, removed on clinical grounds, were analyzed with semiquantitative and quantitative cultures. Blood cultures were done at catheter removal. In vitro study: Coagulase-negative Staphylococci adhered less in vitro to heparinized catheters than to control catheters (p < .05). In vivo study: Among 32 central venous catheters, or patients who completed the study, catheter-associated bacteremia or fungemia was observed in five patients in the control group (n = 19) and in no patient with a heparinized catheter (n = 13) (p = .047). Four of 13 catheters in the heparin group were colonized compared with 14 of 19 in the control group (p = .03). Coagulase-negative Staphylococci were the most frequent microorganisms in both groups. The numbers of organisms found on colonized catheters were larger in the control group than in the heparin group. Covalent end point surface heparinization appears to have a great impact on both in vitro and in vivo bacterial colonization of central venous catheters. Such heparinization can be a practical and economical approach to the prevention of catheter-associated bacteremia or fungemia.

Carmeda surface heparinization in neonatal ECMO systems: long-term experiments in a sheep model

The thromboresistance in three Carmeda (Stockholm, Sweden) heparin-coated neonatal ECMO systems with a runtime of 45, 56 and 96 hours, respectively, and three noncoated systems with a runtime of 12, 42 and 66 hours, respectively, were compared using a sheep model. The flow rate was 200 ml/min and the activated clotting time (ACT) was kept at approximately 120 seconds. At the end of the experiment, the heparin-coated systems only contained minimal clotting while the controls showed major clotting in the entire system. Fibrin monomers were not detected until after 24 hours in the heparin-coated group, but demonstrated within 60 minutes in the noncoated group. It is concluded that the Carmeda heparin coating has a thromboresistant effect, and may be used to reduce the need for systemic heparinization in ECMO treatment of neonates.

In situ ATR/FTIR studies of protein adsorption on polymeric materials: effectiveness of surface heparinization

The adsorption of two proteins from human plasma (human serum albumin (HSA) and human fibrinogen (HFG) onto six different polymeric surfaces (two of which are heparinized), has been studied byin situ ATR/FTIR spectroscopy. The different surface characteristics are reflected by different interfacial behaviours of the two proteins, but while both proteins unfold upon adsorption on all the different non-heparinized materials, they maintain the native conformation once adsorbed on the heparinized surfaces. These findings emphasize the effectiveness of surface heparinization.

Conformation of human plasma proteins at polymer surfaces: the effectiveness of surface heparinization

Studies were made on the adsorption of two human plasma proteins, albumin (HSA) and fibrinogen (HFg), onto three different polymeric surfaces: commercial pellethane R 2363-80AE (PU); pellethane crosslinked with a poly(amido-amine) (PUPA); and heparinized PUPA, using in situ ATR/FTIR spectroscopy (attenuated total reflection Fourier transform infrared spectroscopy). Conformational changes were found to occur on the two proteins upon adsorption onto bare PU and PUPA and the protein unfolding on bare PU was also found to be time dependent. On the contrary, the two proteins do not change conformation when they are adsorbed onto the heparinized surface, emphasising the effectiveness of surface heparinization. Biomaterials (1994) 15, (12) 955–962