Partially carboxymethylated feather keratins. 2. Thermal and mechanical properties of films.

Free cysteine thiol groups of keratin extracted from chicken feathers were partially carboxymethylated with iodoacetic acid (25-76% cysteine modification). Stable dispersions were used for the preparation of films by solution casting. Glycerol was used as a plasticizer (0.05-0.47 g/g of keratin), and films were stored at a constant relative humidity (20, 30, 50, 70, or 90%). The degree of crystallinity in the films was higher when more cysteine residues were carboxymethylated. The films displayed an optimum in mechanical properties at approximately 50% cysteine carboxymethylation. The tensile strength at this optimum was 25 MPa, the E modulus, 350 MPa, and the elongation at break, 50%. Probably, this optimum was the result of both a decreasing amount of disulfide bonds and an increasing degree of crystallinity for higher degrees of cysteine modification. The influences of a higher amount of glycerol and of different storage conditions on the mechanical properties of films from keratin with a defined degree of cysteine modification were also investigated.

Partially carboxymethylated feather keratins. 1. Properties in aqueous systems.

Feather keratins were extracted from chicken feathers with an aqueous solution of urea and 2-mercaptoethanol. The keratin solution obtained was dialyzed to remove the reagents. Upon dialysis, extensive protein aggregation occurred. To obtain stable solutions or dispersions in water, cysteine residues were modified prior to dialysis with iodoacetamide, iodoacetic acid, or bromosuccinic acid, thereby blocking free thiol groups and introducing hydrophilic groups. For the development of biodegradable materials with good mechanical properties from these biopolymers, disulfide bonds between the keratin molecules are needed. Therefore, cysteine residues were only partially modified by using different reagent/cysteine molar ratios. The reaction rate constants of iodoacetate with glutathione and 2-mercaptoethanol were successfully used to predict the degree of modification of keratin cysteine. It was shown that, for carboxymethylated keratin, fewer aggregates were formed for higher degrees of cysteine modification, while more protein was present as oligomers. Aggregates and oligomers were stabilized through intermolecular disulfide bonds.

In vivo testing of crosslinked polyethers. I. Tissue reactions and biodegradation.

The in vivo biocompatibility and biodegradation of cross-linked (co)polyethers with and without tertiary hydrogen atoms in the main chain and differing in hydrophilicity were studied by means of subcutaneous implantation in rats. After 4 days, 1 month, and 3 months postimplantation, the tissue reactions and interactions were evaluated by light microscopy (LM) and transmission electron microscopy (TEM). Poly(tetrahydrofuran) (poly(THF)), poly(propylene oxide) (poly(POx)), and poly(tetrahydrofuran-co-oxetane) (poly-(THF-co-OX)) were tested as relatively hydrophobic polyethers, and poly(ethylene oxide) (PEO) and a poly(THF)/ PEO blend were used as more hydrophilic materials. In general, all polyethers showed good biocompatibility with respect to tissue reactions and interactions, with low neutrophil and macrophage infiltration, a quiet giant cell reaction, and formation of a thin fibrous capsule. For the relatively hydrophobic polyethers studied, the biostability increased in the order poly(POx) < poly(THF-co-OX) < poly(THF), probably indicating that the absence of tertiary hydrogen atoms has a positive effect on the biostability. Concerning the more hydrophilic materials, crosslinked PEO showed the highest rate of degradation, probably due to the mechanical weakness of the hydrogel in combination with the highest presence of giant cells as a result of the high porosity. A frayed surface morphology was observed after implantation of the crosslinked poly(THF)/PEO blend, which might be due to preferential degradation of PEO domains.

In vivo testing of crosslinked polyethers. II. Weight loss, IR analysis, and swelling behavior after implantation.

As reported in Part I ("In vivo testing of crosslinked polyethers. I. Tissue reactions and biodegradation," J. Biomed. Mater. Res., this issue, pp. 307-320), microscopical evaluation after implantation of crosslinked (co)polyethers in rats showed differences in the rate of biodegradation, depending on the presence of tertiary hydrogen atoms in the main chain and the hydrophilicity of the polyether system. In this article (Part II) the biostability will be discussed in terms of weight loss, the swelling behavior, and changes in the chemical structure of the crosslinked polyethers after implantation. The biostability increased in the order poly(POx) < poly(THF-co-OX) < poly(THF) for the relatively hydrophobic polyethers. This confirmed our hypothesis that the absence of tertiary hydrogen atoms would improve the biostability. On the other hand, signs of biodegradation were observed for all polyether system studied. Infrared surface analysis showed that biodegradation was triggered by oxidative attack on the polymeric chain, leading to the formation of carboxylic ester and acid groups. It also was found that in the THF-based (co)polyethers, alpha-methylene groups were more sensitive than beta-methylene groups. For a hydrophilic poly(THF)/PEO blend, an increase in surface PEO content was found, which might be due to preferential degradation of the PEO domains.

Preparation of apolactoferrin with a very low iron saturation.

Iron(III) removal from lactoferrin by an iron(III)-chelating resin with immobilized 3-hydroxy-2-methyl-4(1H)-pyridinone ligands was studied at physiological pH in the presence of citrate. The resin had a marked effect on the extent of iron removal. By using the iron(III)-chelating resin, removal of iron from lactoferrin was nearly complete in < 24 h. Apolactoferrin with 4% iron saturation could be prepared conveniently from 100% or from 18% iron-saturated lactoferrin under mild conditions without affecting the iron-binding capacity of the protein. The iron saturation of the obtained apolactoferrin was much lower than that of the apolactoferrin prepared by reported methods.

Synthesis of thiophilic adsorbents, containing amine spacer arms, and adsorption of immunoglobulins from adult bovine serum.

In order to remove immunoglobulins from adult bovine serum (ABS), adsorptions were carried out in suspension with thiophilic adsorbents without (T-gel) and with (ST-gels) short amine spacer. The developed ST-gels could be cleaned in sodium hydroxide and specifically adsorbed immunoglobulins from ABS at a lower ionic strength of the adsorption buffer than a T-gel. The ST-gels are suited for the performance of adsorptions in a suspension with a high concentration of the adsorbent. For an almost complete removal of immunoglobulins from ABS in 0.5 M potassium sulfate and 0.1 M potassium phosphate (pH 7), up to 75 g ST-gel per 1 suspension may process up to 100 ml ABS. The synthesized ST-gels contained an equal amount of amine spacers and sulfone groups. The ratio between coupled 2-mercaptoethanol molecules and sulfone groups was about 0.4. In comparison with the T-gel, the chemical stability of the ST-gels in solutions of 0.1 to 1 M sodium hydroxide was higher. The capacity for adsorbing pure immunoglobulins of the T-gel was 0.329 g g-1 (10.9 g l-1) and of the ST2-gel, 0.677 g g1 (52.3 g l-1). The capacity for adsorbing IgG from ABS of the T-gel was 0.177 g g1 (5.88 g l-1) and of the ST2-gel, 0.152 g g-1 (11.7 g l-1). This reduced capacity was attributed to a low affinity binding to the adsorbents of other proteins from ABS. When an incubation of the adsorbent with ABS was performed in 0.5 M potassium sulfate and 0.1 M potassium phosphate (pH 7), the affinity constant (Ka) for IgG of the T-gel was 2.5 x 10(6) M-1 and of the ST-gel, 3.1 x 10(7) M-1. In this adsorption buffer at a protein concentration above 5 g l-1, salt-promoted precipitation of IgG took place. When an adsorption was performed at the minimal ionic strength of the adsorption buffer, the Ka of the T-gel for IgG was about 9 x 10(3) M-1 and of the ST-gel, about 2.3 x 10(5) M-1. In this case, the removal of Igs from ABS was achieved by means of a partition equilibrium, rather than by high affinity adsorption.

Iron removal from milk and other nutrient media with a chelating resin.

A water-insoluble iron(III)-chelating resin was used to study iron removal from milk and other nutrient media. Seventy to 85% of the iron could be removed from wine and beer with the resin, which was a crosslinked copolymer of 1-(beta-acrylamidoethyl)-3-hydroxy-2-methyl-4(1H)- pyridinone and N,N-dimethylacrylamide. Iron removal from milk was dependent on the pH of milk and on the concentration of soluble chelators added. Under the same conditions as used for the removal of iron from wine and beer, only 11 to 19% of the iron could be removed from milk. However, in combination with water-soluble chelators, the resin removed 60 to 75% of the iron from the milk. Preliminary results showed that the growth of spores of Clostridium tyrobutyricum in the treated milk was reduced. Moreover, addition of the resin and sodium bicarbonate to the milk completely inhibited the growth of the spores.

The binding of human blood platelets to fibrinogen-, fibronectin-, and Arg-Gly-Asp-derivatized Sephadex G-10.

The adhesive proteins fibrinogen (FG) and fibronectin (FN) were immobilized to glycine-Sephadex G-10. The derivatized Sephadex G-10 gels were used to bind human blood platelets. For comparison, Gly-Arg-Gly-Asp-Ser-Pro(GRGDSP)-derivatized Gly-Sephadex G-10 was used. FG-, FN-, and GRGDSP-Gly-Sephadex G-10 each bound a substantial number of activated blood platelets (> or = 5 x 10(8) ml-1 gel) while non-activated platelets were not bound. Binding of ADP-treated blood platelets to the affinity adsorbents was dependent on the ADP-concentration which was used, reaching a near-maximal value at about 10 microM ADP. Platelet binding to the three types of affinity gels could be completely inhibited by dissolved GRGDSP as well as monoclonal anti-platelet glycoprotein IIb/IIIa (GPIIb/IIIa) antibody CLB-C17, which demonstrates that platelet binding specifically involves the fibrinogen binding site on GPIIb/IIIa. Platelet binding to all three affinity gels required free Ca2+ and Mg2+ ions: platelets binding in the absence of these divalent cations was considerably lower than platelet binding in buffer containing 2 mM Ca2+ and 1 mM Mg2+. Moreover, activated ethylenediamine-tetraacetate (EDTA)-treated platelets did not bind at all to the affinity gels. The finding that non-activated platelets did not bind to the affinity gels is thought to be related to both the high hydrophilicity of the Sephadex basic material and to the native state of the gel-bound fibrinogen and fibronectin.

Effects of Tween 20 on the desorption of proteins from polymer surfaces.

The effects of Tween 20 on the desorption of proteins from polyethylene and polyurethane were studied, using single protein solutions of the human proteins fibrinogen (Fb), immunoglobulin G (IgG), serum albumin (HSA), high density lipoproteins (HDL), and plasma. The surfactant may partly or even completely desorb the proteins, depending on the type of polymer and protein. About 40% of adsorbed HSA and 80% of adsorbed HDL from the corresponding single protein solutions were desorbed by Tween 20 from polyethylene, whereas Tween 20 had a small effect on the desorption of adsorbed Fb and IgG under the same conditions. However, the desorption of Fb and IgG by Tween 20 was much higher in the case of a diluted plasma solution compared to a pure protein solution. These findings may be explained by the differences of the interaction strengths between polymers and the adsorbed proteins. The displacement of HSA from polyethylene by Tween 20 occurred in the first few minutes and did not increase in time. It was also observed that preadsorbed Tween 20 was able to prevent in a large extent the adsorption of HSA onto polyethylene. Thus, the effect of Tween 20 on the desorption of protein is due to either the displacement of protein or prevention of protein adsorption onto the surfaces.

Iron(III)-chelating resins. IX. Antibacterial activity of a water-insoluble iron(III)-chelating resin.

Antibacterial activity of a water-insoluble iron(III)-chelating resin with covalently bonded 3-hydroxy-2-methyl-4(1H)-pyridinone (HMP) groups was evaluated in a brain heart infusion (BHI) medium. The activity of the resin against Escherichia coli was lower than that of soluble HMP iron(III) chelators, whereas against Listeria inocua, an activity approximately equal to those of the soluble chelators was found. It was observed that the growth of E. coli and L. inocua was reduced by increasing the amounts of the resin from 2 to 40 mg of resin/mL of medium. Inhibition of bacterial growth in the presence of the resin (10 mg/mL) was abolished by addition of ferric ion to the medium, indicating that the growth of E. coli and L. inocua was dependent on the available iron in the medium. Reducing the iron concentration in the medium from 14.2 to 0.16 microM (by action of the resin) resulted in a decrease in the growth response from 100% to 19% for E. coli and from 100% to 10% for L. inocua. In addition, the influence of citrate was studied, but only small effects of citrate supplementation on the growth of bacteria and on the antibacterial activity of the resin were observed.

Preparation of cell-affinity adsorbents by immobilization of peptides to Sephadex G-10.

In this study, affinity adsorbents for the binding of activated blood platelets and endothelial cells were prepared from Sephadex G-10 by immobilization of peptides, derived from the cell-adhesive amino acid sequence RGD (arginine-glycine-aspartic acid). Derivatization of Sephadex G-10 was accomplished by sequential coupling of specific dipeptides (RG and DV) (V = valine) and by coupling of the RGD-containing hexapeptide GRGDSP (S = serine, P = proline). Two types of gel were prepared by sequential coupling, designated as G10 (acetone) and G10 (dimethylformamide) (G10(DMF)), containing peptides which had been coupled to the outer side of the beads and throughout the porous beads, respectively. The binding capacity of the prepared Sephadex-derivatives amounted up to 2 x 10(9) human blood platelets per millilitre GRGDV-Sephadex at immobilized peptide concentrations, that were in the nanomole range (per millilitre packed gel) and which differed a factor 10 between G10 (acetone) and G10 (DMF). In a second series of experiments, different amounts of the hexapeptide GRGDSP were coupled to carboxylated Sephadex G-10 and carboxylated Sepharose CL 6B. The binding of human umbilical vein endothelial cells to the resulting materials was studied. Up to 10(6) endothelial cells attached per ml GRGDSP-derivatized hydrogel at peptide concentrations of 15 nmol GRGDSP/ml Sephadex and at +/- 300 nmol GRGDSP/ml Sepharose. Substitution of the arginine residue of the RGD-sequence by glutamine abolished the cell-binding activity of the immobilized peptide towards activated blood platelets but not towards endothelial cells. From the results of this study it can be concluded that small peptides can be coupled to the outer side of the porous Sephadex beads, resulting in high effective ligand densities for cell-affinity applications. In this respect, Sephadex G-10, derivatized according to 'the acetone method' is a good alternative for polystyrene and other solid phase materials.

Iron (III)-chelating resins. 3. Synthesis, iron (III)-chelating properties, and in vitro antibacterial activity of compounds containing 3-hydroxy-2-methyl-4(1H)-pyridinone ligands.

The synthesis, iron (III)-chelating properties, and antibacterial activity of several compounds containing the 3-hydroxy-2-methyl-4(1H)-pyridinone (HMP) moiety are described. Using the HMP derivatives iron (III) could be mobilized from iron (III)-binding proteins at physiological pH with a rate order of transferrin > lactoferrin > ferritin. Addition of HMP-containing compounds to a growth medium at a concentration of 20 mM/L resulted in a complete inhibition of the growth of Escherichia coli and about 90% inhibition for Listeria inocua after 7 h of incubation at 37 degrees C. After inhibition of bacteria growth by the HMP derivatives growth started again when ferric ions were added to the medium, which implies that the antibacterial activity is due to a limitation of iron available to the organisms.

Colloidal carbon particles as a new label for rapid immunochemical test methods: quantitative computer image analysis of results.

Colloidal carbon particles can serve as label in sol particle immunoassays. The universal applicability of these particles in qualitative and (semi)quantitative immunoassays has been demonstrated. Sol particle and/or dipstick immunoassays, not yet optimized in terms of sensitivity, are discussed. The colloidal label has been used successfully in a mouse immunoglobulin isotyping kit. Human serum albumin spotted onto nitrocellulose in a concentration range of 7.8 to 1000 ng could be detected using anti-albumin antibody absorbed onto colloidal carbon particles. It was also possible to perform a competitive assay with this conjugate for a concentration range of free human serum albumin varying from 0.25 to 6.75 micrograms. The Kunitz-type trypsin inhibitor from soybean was determined by a colloidal carbon based immunoassay in a range of 2.5 to 160 ng. In this assay, free and colloidal carbon-bound inhibitor competed for binding specific antibodies spotted onto a nitrocellulose membrane. An image- and data-processing procedure has been developed that enables a rapid and simple quantification of colloidal carbon sol particle immunoassays. The average grey level of a spot is taken as a measure for quantitative purposes. This so-called Sol-particle Image Processed ImmunoAssay (SIPIA) procedure is equally well applicable to assays using other colloidal particles.

Tetrahydrofuran (co)polymers as potential materials for vascular prostheses.

Polyethers were studied as potential materials for vascular prostheses. By crosslinking poly(tetramethylene oxide)(PTMO) with poly(ethylene oxide)(PEO), hydrophilic networks were obtained containing PTMO as well as PEO. Attempts were made to reduce the crystallinity and melting point of PTMO because of the required elastomeric behaviour at body temperature. Compared to non-crosslinked PTMO, crosslinking in the melt resulted in a decrease in the melting point from 43.7 to 38.4 degrees C and a decrease of the crystallinity from 46 to 28%. By copolymerizing tetrahydrofuran with oxetane or dimethyloxetane, melting points below 38 degrees C were obtained, together with crystallinities lower than 20%.

Iron(III) chelating resins II. 3-Hydroxy-4(1H)-pyridinones-sepharose gels.

3-Hydroxy-4(1H)-pyridinones(HP)-Sepharose gels were prepared to study their iron(III) chelating properties. As ligands, derivatives of 3-hydroxy-4(1H)-pyridinone were coupled to CNBr-activated Sepharose gels. HP-Sepharose gels were obtained with HP densities of 23-28 mumol/ml gel and iron(III) chelating capacities of 19-23 mumol/ml gel at pH 6.8. From preliminary experiments, it was found that with the gels 19-27% iron could be removed from milk. In addition, 74% of iron(III) was removed from 100% iron(III) saturated lactoferrin within 24 h at pH 6.8 in the presence of citrate and a Sepharose gel, onto which 1-(2-aminoethyl)-3-hydroxy-2-methyl-4(1H)-pyridinone had been immobilized as a ligand. The properties of the gels make them potentially useful as water-insoluble iron(III) chelating agents.

Iron(III) chelating resins--I. Preparation and properties of Sepharose-desferrioxamine gels.

For the removal of iron(III), Sepharose-desferrioxamine gels were prepared by the coupling of CNBr-activated Sepharose with desferrioxamine (DFO) at pH 7.8-8.3. DFO densities of the gels were 12-23 mumol/ml gel with iron(III) chelating capacities of 8.5-18 mumol/ml gel. The Sepharose-DFO gels with a high affinity for iron(III) were used for the removal of iron(III) from aqueous iron(III) solutions, wine, milk and whey.

Improved adhesion and proliferation of human endothelial cells on polyethylene precoated with monoclonal antibodies directed against cell membrane antigens and extracellular matrix proteins.

Endothelial cell seeding may improve the patency of synthetic vascular grafts provided that platelet reactivity of nonendothelialized sites is not increased. We have investigated if surface-adsorbed monoclonal antibodies directed against endothelial cell membrane proteins and against extracellular matrix proteins promote the adhesion and proliferation of cultured human endothelial cells, without causing platelet deposition at non-endothelialized sites. Adhesion of endothelial cells onto polyethylene coated with monoclonal antibodies directed against endothelial cell-specific membrane antigens, integrin receptors and glycoprotein CD31 was equal to or higher than adhesion onto fibronectin-coated polyethylene. Endothelial cells did not proliferate on these surface-adsorbed antibodies. However, pre-coating of polyethylene with mixtures of endothelial cell-specific monoclonal antibodies and monoclonal antibodies directed against fibronectin or von Willebrand factor, resulted in relatively high adhesion and optimal proliferation. Platelet reactivity of the polyethylene surface was found to significantly increase after adsorption of fibronectin, endothelial cell-specific monoclonal antibody or its Fc fragments. In contrast, adsorption of F(ab')2 fragments of endothelial cell-specific monoclonal antibody did not promote platelet deposition. Therefore, it is concluded that coating of vascular graft materials with mixtures of F(ab')2 fragments of monoclonal antibodies specifically directed against endothelial cells and against extracellular matrix proteins may be an effective way to both promote the growth of seeded endothelial cells and limit platelet-graft interaction.

Adhesion of endothelial cells and adsorption of serum proteins on gas plasma-treated polytetrafluoroethylene.

From in vitro experiments it is known that human endothelial cells show poor adhesion to hydrophobic polymers. The hydrophobicity of vascular prostheses manufactured from Teflon or Dacron may be the reason why endothelialization of these grafts does not occur after implantation in humans. We modified films of polytetrafluoroethylene (Teflon) by nitrogen plasma and oxygen plasma treatments to make the surfaces more hydrophilic. Depending on the plasma exposure time, modified polytetrafluoroethylene surfaces showed water-contact angles of 15-58 degrees, versus 96 degrees for unmodified polytetrafluoroethylene. ESCA measurements revealed incorporation of both nitrogen- and oxygen-containing groups into the polytetrafluoroethylene surfaces, dependent on the plasma composition and exposure time. The thickness of the modified surface layer was approximately 1 nm. The adhesion of cultured human endothelial cells from 20% human serum-containing culture medium to modified polytetrafluoroethylene surfaces with contact angles of 20-45 degrees led to the formation of a monolayer of cells, which was similar to the one formed on tissue culture polystyrene, the reference surface. This was not the case when endothelial cells were seeded upon unmodified polytetrafluoroethylene. Surface-modified expanded polytetrafluoroethylene prosthesis material (GORE TEX soft tissue) also showed adhesion of endothelial cells comparable to cell adhesion to the reference surface. The amounts of serum proteins, including fibronectin, adsorbed from serum-containing medium to modified polytetrafluoroethylene surfaces were larger than those adsorbed to unmodified polytetrafluoroethylene. Moreover, the modified surfaces probably allow the exchange of adsorbed serum proteins with cellular fibronectin.

Poly(vinyl alcohol)-heparin hydrogels as sensor catheter membranes.

Poly(vinyl alcohol)-heparin hydrogels with varying water content were synthesized for use as sensor catheter membranes. Films were cast from aqueous mixtures of poly(vinyl alcohol) (PVA), a photosensitive cross-linker p-diazonium diphenyl amine polymer (PA), glutaraldehyde (GA) and heparin. After drying, the films were cross-linked by successive UV irradiation and heat treatment. To get an indication about the cross-linking density of the networks, the water content of the hydrogels was measured after equilibration in water. Hydrogels from PVA, PA, GA and heparin, with a water content of 35-95%, could be obtained if the components were dissolved in saline instead of water. The release of heparin from PVA-heparin or PVA-PA-heparin hydrogels was studied using different receiving phases. The cumulative amount of released heparin appeared to be dependent on the initial water content of the hydrogels and the composition of the receiving phase. For the PVA-PA-heparin hydrogels as well as the PVA-heparin hydrogels the cumulative amount of released heparin in water was about six times higher than in a Tris buffer. Using Tris buffer as receiving phase PVA-PA-heparin hydrogels with water contents of 53, 61 or 71% released heparin for at least 3 wk. The cumulative amount of released heparin increased with initial water content of these hydrogels. Recalcification times (RCT) of plasma exposed to PVA-PA-heparin hydrogels (water content 53%), which released heparin at a low rate (2 micrograms/cm2 per day), were markedly prolonged compared with the RCT values for PVA-PA hydrogels without heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of surface-adsorbed (lipo)proteins by means of a two-step enzyme-immunoassay: a study on the Vroman effect.

In view of reports on the involvement of high-molecular-weight (HMW) kininogen and high-density lipoprotein (HDL) in the Vroman effect, we studied the adsorption of fibrinogen, HMW kininogen, HDL and several other proteins from pooled human plasma and congenitally HMW kininogen-deficient plasma onto glass and low-density polyethylene, both as a function of the plasma concentration and the contact time. Mixtures of purified (lipo)proteins were also included in the study. Protein adsorption was determined by means of a two-step enzyme-immunoassay. Our results support the hypothesis that HMW kininogen is involved in the displacement of fibrinogen, which is almost instantly adsorbed from normal plasma onto glass. On hydrophobic polymers like polyethylene, the low amounts of adsorbed fibrinogen and HMW kininogen from plasma and concentrated plasma solutions may be due to a preferential adsorption of HDL.