Origin of the nonadhesive properties of fibrinogen matrices probed by force spectroscopy

The deposition of a multilayered fibrinogen matrix on various surfaces results in a dramatic reduction of integrin-mediated cell adhesion and outside-in signaling in platelets and leukocytes. The conversion of a highly adhesive, low-density fibrinogen substrate to the nonadhesive high-density fibrinogen matrix occurs within a very narrow range of fibrinogen coating concentrations. The molecular events responsible for this transition are not well understood. Herein, single-cell and molecular force spectroscopy were used to determine the early steps in the formation of nonadhesive fibrinogen substrates. We show that the adsorption of fibrinogen in the form of a molecular bilayer coincides with a several-fold reduction in the adhesion forces generated between the AFM tip and the substrate as well as between a cell and the substrate. The subsequent deposition of new layers at higher coating concentrations of fibrinogen results in a small additional decrease in adhesion forces. The poorly adhesive fibrinogen bilayer is more extensible under an applied tensile force than is the surface-bound fibrinogen monolayer. Following chemical cross-linking, the stabilized bilayer displays the mechanical and adhesive properties characteristic of a more adhesive fibrinogen monolayer. We propose that a greater compliance of the bi- and multilayer fibrinogen matrices has its origin in the interaction between the molecules forming the adjacent layers. Understanding the mechanical properties of nonadhesive fibrinogen matrices should be of importance in the therapeutic control of pathological thrombosis and in biomaterials science.     

Interaction of fibrinogen with n-alkylagaroses and its purification by critical hydrophobicity hydrophobic interaction chromatograpy

A rational application of hydrophobic interaction chromatography (HIC) to the purification of proteins has remained an enigma in spite of over 30 years of research. The critical hydrophobicity parameter, which can be determined from a concentration series of n-alkyl Sepharose 4B (Seph-Cn) offers the possibility of adapting the HIC gel to the needs of purification. To this end a library of HIC gels (Seph-C4 to Seph-C6) of different immobilized alkyl residue concentrations was synthesized and tested with purified bovine fibrinogen. Binding of fibrinogen to such a concentration series resulted in sigmoidal binding curves. Analysis of the Seph-C5 data according to the lattices-site binding model yielded adsorption coefficients (nS) between 5 and 10 indicating that 5-10 lattice-sites (alkyl residues) interact multivalently with a fibrinogen molecule for adsorption at low ionic strength. The apparent lattice-site half-saturation constant of dissociation lies between 21 and 25 micromol/ml packed gel. For each alkyl chain length a critical hydrophobicity could be determined. For fibrinogen purification the critical hydrohobicity gel, Seph-C5 (13 micromol/ml packed gel), was selected. With the help of the cosolvents NaCl or glycine a fully reversible adsorption of fibrinogen could be facilitated on the critical hydrophobicity gel. Application of the method to human and bovine blood plasma resulted in a single step purification of fibrinogen in high yields. A comparison of the classical purification of fibrinogen with the critical hydrophobicity HIC (CHIC) method demonstrates a reduction in preparation time from several days to ca. 1 h. The subunit structure of HIC-purified human fibrinogen is identical to the classically purified protein. In the case of bovine fibrinogen however HIC-purified fibrinogen displayed a different subunit structure in that the Aalpha chain of fibrinogen had a ca. 5 kDa higher molecular mass. This may be due to the rapidity of the new one-step method and an avoidance of proteolysis.     

Photocontrol of cell adhesion and proliferation by a photoinduced cationic polymer surface

In this study photoinduced cation generation, based on the photochemical properties of malachite green (MG), was used for the surface design and in vitro photochemical control of cell adhesion and proliferation. The MG-derivatized surface was prepared by coating a photoreactive polymer as a substrate onto a poly(ethylene terephthalate) (PET) sheet. The photoreactive polymer was radical copolymer of styrene with the MG-derivatized monomer diphenyl(4-vinylphenyl)methane leucohydroxide (degree of substitution of MG unit: 12.4 mol%). Water contact angle measurements and X-ray photoelectron spectroscopy revealed high hydrophobicity and homogeneous distribution of the MG groups on the outermost surface of the coated film, respectively. When the coated film was exposed to ultraviolet light (UV) irradiation at wavelengths of 290-410 nm, a time-dependent color change of the film was observed from pale yellow, before irradiation, to green. These results indicated generation of cations on the film surface by photochemical cation generation of the MG groups, which was quantitatively characterized by force versus distance curves measurements in atomic force microscopic (AFM) observation using a carboxylated AFM tip. The seeding and culture of endothelial cells showed a marked reduction in adhesion on the nonirradiated coated film surface, whereas the UV-irradiated surface promoted cell adhesion and proliferation except for incubation in serum-free medium, which was similar to commercial tissue culture PET sheet. These observations may be due to adsorption of cell adhesive proteins, typified by fibronectin, in serum-containing medium onto the cationized photoreactive copolymer surface by electrostatic interactions.     

On the regulation of the interaction of synthetic polymers with blood

The interaction of polymeric hydrogels containing immobilized trypsin and heparin with serum, blood and model solutions of serum proteins has been studied. It has been shown that these binary systems have high haemocompatibility due to specific fibrinogen binding by immobilized heparin and the subsequent lysis of bound substrate by immobilized protease. The lysis is accelerated in the presence of serum albumin and such materials may be regarded as biologically active ternary polymers.     

PET光学膜紫外光固化硬涂层的制备

本文以改性聚氨酯丙烯酸酯为主体,制备了一种PET光学膜用透明硬涂层材料.讨论了光固化树脂、活性稀释剂、光引发剂等因素对PET光学膜加硬后的硬度、耐磨性、附着力等性能的影响,并从反应机理方面分析探讨了实验结果.经测试:实验制备的涂层硬度5H,附着力100%,经RCA纸带耐磨擦试(500g,1000cycles),PET膜可见光透过率无损失,同时具有低表面张力,出色的柔韧性和良好的耐化学品性.     

Bacterial attachment on reactive ceramic ultrafiltration membranes

Bacterial attachment is an initial stage in biofilm formation that leads to flux decline in membrane water filtration. This study compares bacterial attachment among three photocatalytic ceramic ultrafiltration membranes for the prevention of biofilm formation. Zirconia ceramic ultrafiltration membranes were dip-coated with anatase and mixed phase titanium dioxide photocatalysts to prevent biofilm growth. The membrane surface was characterized in terms of roughness, hydrophobicity, bacterial cell adhesion, and attached cell viability, all of which are important factors in biofilm formation. The titanium dioxide coatings had minimal impact on the membrane roughness, reduced the hydrophobicity of membranes, prevented Pseudomonas putida attachment, and reduced P. putida viability. Degussa P25 is a particularly promising reactive coating because of its ease of preparation, diminished cell attachment and viability in solutions with low and high organic carbon concentrations, and reduced flux decline. These reactive membranes offer a promising strategy for fouling resistance in water filtration systems.     

Colloid probe AFM investigation of interactions between fibrinogen and PEG-like plasma polymer surfaces

Interaction forces between surfaces designed to be protein resistant and fibrinogen (Fg) were investigated in phosphate-buffered saline with colloid probe atomic force microscopy. The surfaces of the silica probes were coated with a layer of fibrinogen molecules by adsorption from the buffer. The technique of low-power, pulsed AC plasma polymerization was used to make poly(ethylene glycol) (PEG)-like coatings on poly(ethylene teraphthalate) by using diethylene glycol vinyl ether as the monomer gas. The degree of PEG-like nature of the films was controlled by use of a different effective plasma power in the chamber for each coating, ranging from 0.6 to 3.6 W. This produced a series of thin films with a different number of ether carbons, as assessed by X-ray photoelectron spectroscopy. The interaction force measurements are discussed in relation to trends observed in the reduction of fibrinogen adsorption, as determined quantitatively by (125)I radio-labeling. The plasma polymer coatings with the greatest protein-repelling properties were the most PEG-like in nature and showed the strongest repulsion in interaction force measurements with the fibrinogen-coated probe. Once forced into contact, all the surfaces showed increased adhesion with the protein layer on the probe, and the strength and extension length of adhesion was dependent on both the applied load and the plasma polymer surface chemistry. When the medium was changed from buffer to water, the adhesion after contact was eliminated and only appeared at much higher loads. This indicates that the structure of the fibrinogen molecules on the probe is changed from an extended conformation in buffer to a flat conformation in water, with the former state allowing for stronger interaction with the polymer chains on the surface. These experiments underline the utility of aqueous surface force measurements toward understanding protein-surface interactions, and developing nonfouling surfaces that confer a steric barrier against protein adsorption.     

Effect of surface hydrophobicity on the adhesion of S. cerevisiae onto modified surfaces by poly(styrene-ran-sulfonic acid) random copolymers

The hydrophobicity of solid surfaces has been regarded as a controlling factor in microbial adhesion phenomena. In this study, the surface hydrophobicity was modified by coating with a poly(styrene-ran-sulfonic acid) random copolymer (PS-x-SA, charge density (x): 0-15.3%), and the adhesion rate, J0, of S. cerevisiae performed with a direct observation technique. The results indicated that the degree of sulfonation of PS-x-SA greatly influenced the hydrophobicity of substrates and the adhesion of yeast cells. The J0 of PS-x-SA substrates were gradually decreased as increasing charge density. The interactions between cells and substrates explained by the XDLVO theory, predicted that the decrease of J0 as increasing charge density was not due to the increase of electric double layer repulsion, but mainly due to the hydrophobic acid-base interactions. Also, it predicted that microbial adhesions of PS-x-SA were mostly reversible, while some of PS and PS-5.1-SA adhered cells were hardly removed. Based on these results, XDLVO theory was effective for predicting adhesion phenomena of S. cerevisiae onto the PS-x-SA-coated substrates.     

Physical and chemical effects of quartet structure (bamboo / zinc borate / shellac / surfactant) on organic coatings

Bamboo was used owing to its properties as environmentally friendly and lightweight material. The purpose of this study was to increase hydrophobicity, flammability, gloss etc. by using additives (bamboo, shellac, zinc borate and surfactant) with different amounts (0–6%, w/w). Taguchi method was applied to reduce in the number of experiments by carrying out 16 experiments. As a result of the optimization studies, it was seen that the hydrophobicity of the water-based acrylic organic coating has increased from 39.52° (reference coating) up to 104.32° (optimum coating, Exp. No:15, Levels: 4-3-2-4). Shellac increased its homogenization and coating properties with its gloss property. In addition, zinc borate was preferred to increase its properties of flame retardancy, antimicrobial and nonwettable. Surfactant based methacrylate was used for dispersion of coatings. Thermogravimetric Analysis-Differential Thermal Analysis (TG-DTA), Scanning Electron Microscope (SEM), Contact Angle Measurement and physical test (adhesion, gloss, aging) results showed that the additives enhanced the thermal, morphological and physical properties of coatings and it could be evaluated in different areas of the construction industry.     

Functionality and stability of heparin immobilized onto poly(dimethylsiloxane)

Poly(dimethylsiloxane) (PDMS) has become an attractive material when working in the field of microfluidics, mainly because of the rapid prototyping process it involves. The increased surface volume ratio in microchannels makes the interaction between sample and material surface highly important, evident when handling complex biological samples such as plasma or blood. This study demonstrates a new grade of non-covalent heparin surface that adds efficient anticoagulant property to the PDMS material. The surface modification is a simple and fast one-step process performed at neutral pH, optimal when working with closed microsystems. The heparin formed a uniform and functional coating on hydrophobic PDMS with comparatively high level of antithrombin-binding capacity. In addition, long-term studies revealed that the immobilized heparin was more or less stable in the microchannels over a time of three weeks. Recalcified plasma in contact with native PDMS showed complete coagulation after 1h, while no fibrin formation was detected in plasma incubated on heparin-coated PDMS within the same time. In conclusion, we see the heparin coating developed and evaluated in this study as a tool that greatly facilitates the use of PDMS in microfluidics dealing with plasma or blood samples.     

Protein adsorption and platelet adhesion of polysulfone membrane immobilized with chitosan and heparin conjugate

Polysulfone (PSF) membranes were treated with ozone to introduce peroxides, and then grafted with either acrylic acid or chitosan, followed by the immobilization of heparin. The effect of spacer arm on blood compatibility was investigated using three chitosans of different molecular weight [1170 (water soluble), 160 000, and 400 000] and similar degrees of deacetylation (75%). The hydrophilicity was evaluated by measuring the contact angle of water. Blood compatibility was evaluated using the activated partial thromboplastin time (APTT) as well as the adhesion of platelets. The protein affinity was determined by the absorption of human serum albumin (HSA) and human plasma fibrinogen (HPF). The results show that by the coupling of chitosan, the amount of heparin immobilized can be increased by four times. Water contact angle (from 78 ° to 41 °) decreased with the increase of the amount of heparin immobilized, showing increased wettability. The heparinized PSF membrane showed longer APTT and decreasing platelet adhesion, compared to that of unmodified PSF membrane. The adsorption of HSA and HPF were reduced to 17 and 6%, respectively. This suggests that longer spacer binding to heparin can increase the opportunity of anti‐coagulation on contacting blood. These results demonstrated that the hydrophilicity and blood compatibility of PSF membrane could be improved by chitosan and heparin conjugate. Copyright © 2003 John Wiley & Sons, Ltd.     

Surface Modification of Polyurethanes with Covalent Immobilization of Heparin

Thrombus formation and blood coagulation is a major problem associated with blood contacting products such as catheters, vascular grafts, arteries, artificial hearts and heart valves. An intense research is being conducted towards the synthesis of new hemocompatible materials and modifications of surfaces with biological molecules. In this study, polyurethane (PU) films were synthesized in medical purity from diisocyanate and polyol without using any other ingredients and their surfaces were modified by covalent immobilization of heparin. Two types of heparin, unfractionated (UFH) and low molecular weight heparin (LMWH), were immobilized to investigate their effect on cell adhesion. The surface properties of the modified PUs were examined with ESCA, ATR-FTIR and AFM. ESCA results demonstrated sulfur peaks indicating the presence of heparin and AFM results showed the alteration of surface structure after coating with heparin. Cell adhesion studies were conducted with heparinized whole human blood. The surfaces of the UFH immobilized films resulted in lesser red blood cell adhesion in comparison to LMWH demonstrating strong anti-thrombogenic activity of the latter.     

Bioactive heparin immobilized onto microfluidic channels in poly(dimethylsiloxane) results in hydrophilic surface properties

A new composition of heparin coating for microfluidic systems made out of poly(dimethylsiloxane) (PDMS) was developed and evaluated. The coating that consists of a conditioning polyamine layer followed by two heparin/glutaraldehyde layers, resulted in channel surfaces with sufficient wettability to obtain flow of human normal plasma by capillary force alone. Hydrophilic channel walls are a desirable characteristic in microfluidic devices, since alternative pumping mechanisms must otherwise be included into the system. The immobilized heparin showed high antithrombin-binding capacity and a low degree of blood–material interaction. Plasma in contact with heparin-coated PDMS formed no detectable fibrin in a spectrophotometric assay by which plasma in contact with non-treated PDMS showed complete coagulation. The quartz crystal microbalance technique with energy dissipation monitoring (QCM-D) was utilized to obtain detailed information regarding adsorption kinetics and structural properties of the different layers composing the heparin coating.     

A theory of protein-resin interaction in hydrophobic interaction chromatography

Docking simulations were performed in order to investigate surface area of interaction between several ribonucleases and a reduced model for the hydrophobic moiety used in Phenyl Sepharose using the program AutoDock 3.0. For each ribonucelase, 80 independent simulations with populations consisting of 100 random structures were performed and from these the most probable docked protein-ligand conformations were obtained. A new methodology was used to select the most probable conformations, based on qualitative and quantitative considerations. The interacting amino acids in each protein were identified. The average surface hydrophobicity of the interfacial zone (local hydrophobicity, LH) was determined. The LH showed a high correlation level (r2 = 0.99) with the "hydrophobic contact area" (HCA) experimentally determined for the different ribonucleases as well as with the dimensionless retention time (r2 = 0.90). This study allowed us to identify the zones on the protein surface most probably involved in protein retention in HIC, without tedious experimental work. Given the good correlation level obtained, this new methodology may constitute a novel approach that could be used to predict protein behavior in HIC.     

Vibrational spectroscopic analysis of a metal/carbon nanotube coating interface and the effect of its interaction with albumin

Multi-walled carbon nanotubes (MWCNTs) as a modifying phase on the titanium support can be potentially used for medical purposes as a material for the production of implants or implantable electrodes or for applications for cardiac surgery. Developing better blood compatible biomaterials must be connected with the condition of their anti-thrombogenic characteristic. A carbon nanotube layer was formed on a titanium plate coated in half with MWCNTs to have admission to: MWCNTs coating, to the Ti/MWCNTs interface region at the MWCNTs coating edge and finally to the Ti support. The Raman measurements were performed in two different locations: in the interface/edge region of the titanium and MWCNTs coating and in the center of the MWCNTs layer. For each of these positions, measurements in two different depths were performed: on the sample top surface of the MWCNT layer and near the bottom of the MWCNTs layer, i.e. at the titanium support interface. The studied sample regions differ in G-, D- and D′-mode structural characteristics as dispersion, crystallinity, the size of the arranged domains, and the distance between the point defects. The phase boundary region is more disordered and exposed to a greater surface tension. These features influence the interactions with albumin which represented the material behavior in contact with the tissue. The MWCNTs coating is hydrophilic (contact angle ∼55°), in the border area this value increases to ∼60°, then Ti support is hydrophobic (∼98°). Two dimensional correlation analysis allows us to unravel albumin-MWCNTs' interaction. The cross-peaks show a contribution from G⁺ and G⁻ carbon nanotubes bands and protein secondary structure demonstrating the formation of a film on the surface of the test sample and indicate the change of the albumin conformation during adhesion.     

Synthesis and characterization of poly(ethylene glycol) methyl ether endcapped poly(ethylene terephthalate)

Linear and branched poly(ethylene terephthalate) (PET) copolymers with polyethylene glycol) (PEG) methyl ether (700 or 2000 g/mol) end groups were synthesized using conventional melt polymerization. DSC analysis demonstrated that low levels of PEG end groups accelerated PET crystallization. The incorporated PEG end groups also decreased the crystallization temperature of PET dramatically, and copolymers with a high content of PEG (>17.6 wt%) were able to crystallize at room temperature. Rheological analysis demonstrated that the presence of PEG end groups effectively decreased the melt viscosities and facilitated melt processing. XPS and ATR-FTIR revealed that the PEG end groups tended to aggregate on the surface, and the surface of compression molded films containing 34.0 wt% PEG were PEG rich (85 wt% PEG). PEG end-capped PET (34.0 wt% PEG) and PET films were immersed into a fibrinogen solution (0.7 mg/mL BSA) for 72 h to investigate the propensity for protein adhesion. XPS demonstrated that the concentration of nitrogen (1.05%) on the surface of PEG endcapped PET film was statistically lower than PET (7.67%). SEM analysis was consistent with XPS results, and revealed the presence of adsorbed protein on the surface of PET films.     

The influence of pre‐treatments of aluminium alloys on bonding of PET coatings

Application of organic coatings on aluminium alloys is commonplace for corrosion protection. The adhesion of coatings is of great importance to the final protection properties. It is therefore necessary to understand on a molecular level the mechanisms with which a coating is able to bond. In this paper, we explore the possibilities of combining model molecules for a poly(ethylene terephthalate) (PET) type coating, di‐methyl terephthalate (DMT), with differently pre‐treated samples of AA1050 and AA5182 alloys. Bonding is studied by means of Fourier‐transform infrared (FTIR) spectroscopy. Because the type of bonding gives a direction for adhesion of a coated system, we also test (macroscopically) the adhesion of PET coatings with a novel technique: asymmetrical double cantilever beam (ADCB). In this method, a thin knife is used as a wedge on the interface of the alloy and the polymer. The displacement of the crack front as measured from the knife's contact point with the coating is used as an input parameter to obtain the adhesion energy for various systems. We show that there is a relationship between the character of bonding of DMT molecules and adhesion energies of PET on both alloys after pre‐treatments in alkali and acid and boiling in water. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparison of the adhesion forces in single and double-layer coatings on the MEMS surfaces by JKR and DMT models

In many medical and industrial applications, some strategies are needed to control the adhesion forces between the materials, because surface forces can activate or hinder the function of the device. All actual surfaces present some levels of roughness and the contact between two surfaces is transferred by the asperities on the surfaces. The force of the adhesion, which depends on the operating situations, can be influenced by the contact region. The aim of the present study is to predict the adhesion force in MEMS surfaces using the JKR and DMT models. The surfaces of the coating material in this research consisted of the single-layer coating of Gold and Silver, and the double-layer coating of TiO₂/Gold and TiO₂/Silver on the silicon (100) substrates. The depositing was done by the thermal evaporation method. The results showed that the double-layer coating developed by the new deposition method helped the reduction of the adhesion forces between the probe tip and the specimen surface. The predicted adhesion forces between the probe and the specimens with DMT and JKR models were compared with the experimental results. For all specimens, the simulated data by applying the JKR theory were in a good agreement with the adhesion force experimental values.     

Preparation and characterization of controlled heparin release waterborne polyurethane coating systems

In this study, to improve hemocompatibility of biomedical materials, a waterborne polyurethane (WPU)/heparin release coating system (WPU/heparin) is fabricated via simply blending biodegradable WPU emulsions with heparin aqueous solutions. The surface compositions and hydrophilicity of these WPU/heparin blend coatings are characterized by attenuated total reflectance infrared spectroscopy (ATR-FTIR) and water contact angle measurements. These WPU/heparin blend coatings show effectively controlled release of heparin, as determined by the toluidine blue method. Furthermore, the biocompatibility and anticoagulant activity of these blend coatings are evaluated based on the protein adsorption, platelet adhesion, activated partial thromboplastin time (APTT), thrombin time (TT), hemolysis, and cytotoxicity. The results indicate that better hemocompatibility and cytocompatilibity are obtained due to blending heparin into this waterborne polyurethane. Thus, the WPU/heparin blend coating system is expected to be valuable for various biomedical applications.