Detection of surface bound complement at increasing serum anticoagulant concentrations.

Surface mediated immune complement activation can be detected by a variety of antibody utilizing methods such as ELISA, fluorescence- or radiolabelling techniques, QCM, and ellipsometry. In the present work we investigated how the common anticoagulants heparin, dalteparin, fondaparinux and sodium citrate affected the binding of anti-complement factor 3c (anti-C3c) on a model complement activator surface, immobilised IgG, after incubation in human blood serum. The results show, as expected, that different anticoagulants affect the antibody binding differently. Increasing amounts of heparin, dalteparin and sodium citrate in normal serum resulted in a decreasing anti-C3c binding. The antibody deposition was not sensitive for the fondaparinux concentration. Surprisingly high concentrations of anti-coagulantia were needed to completely eradicate the antibody binding. Experiments in EGTA-serum showed that anticoagulants interfered directly with both the classical and alternative pathways. Control C3a-des arg ELISA measurements show that the lowered antibody surface binding was not a result of complement depletion in serum. Kallikrein generation by hydrophilic glass surfaces was not affected by high anticoagulant concentrations.

Surface immobilized bisphosphonate improves stainless-steel screw fixation in rats.

An increase in the mechanical fixation in bone of metallic biomaterials is considered advantageous in joint replacement and fracture surgery. Different approaches to improve fixation may be e.g. surface roughening, Ca-mineral coating or surface immobilization of growth factors or drugs. In the present work, bisphosphonate, a class of drugs that inhibit bone resorption, was immobilized onto stainless-steel screws. The screws were first roughened and coated with immobilized and cross-linked fibrinogen. Subsequently, an N-bisphosphonate, pamidronate, was immobilized onto fibrinogen, and another N-bisphosphonate, ibandronate, adsorbed on top of this. The so coated screws were inserted into the tibiae of eight male Sprague-Dawley rats. Another eight rats received screws prepared in the same way, but without the bisphosphonate coating. Pullout strength tests were performed after 2 weeks of implantation. The results showed a 28% (p=0.0009) higher pullout force and 90% increased pullout energy for the bisphosphonate coated screws, and support the idea that surface immobilized bisphosphonates can be used to improve biomaterials fixation in bone.

Peptide functionalized poly(L-lysine)-g-poly(ethylene glycol) on titanium: resistance to protein adsorption in full heparinized human blood plasma.

The graft copolymer poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its RGD- and RDG-functionalized derivatives (PLL-g-PEG/PEG-peptide) were assembled from aqueous solutions on titanium (oxide) surfaces. The polymers were characterized by NMR in order to determine quantitatively the grafting ratio, g (Lys monomer units/PEG side chains), and the fraction of the PEG side chains carrying the terminal peptide group. The titanium surfaces modified with the polymeric monomolecular adlayers were exposed to full heparinized blood plasma. The adsorbed masses were measured by in situ ellipsometry. The different PLL-g-PEG-coated surfaces showed, within the detection limit of the ellipsometric technique, no statistically significant protein adsorption during exposure to plasma for 30 min at 22 degrees C or 37 degrees C, whereas clean, uncoated titanium surfaces adsorbed approximately 350 ng/cm2 of plasma proteins. The high degree of resistance of the PEGylated surface to non-specific adsorption makes peptide-modified PLL-g-PEG a useful candidate for the surface modification of biomedical devices such as implants that are capable of eliciting specific interactions with integrin-type cell receptors even in the presence of full blood plasma. The results refer to short-term blood plasma exposure that cannot be extrapolated a priori to long-term clinical performance.

Ellipsometric in vitro studies on blood plasma and serum adsorption to zirconium.

Ellipsometry/antibody techniques were used to study the adsorption of heparinized human blood plasma and serum onto spontaneously oxidized zirconium, and a colorimetric assay measured the formation of kallikrein by the surface in citrated plasma. After 10 min incubation in the blood plasma the protein film thickness was approximately 4.2 nm, and the film bound polyclonal antibodies mainly against high molecular weight kininogen (HMWK), immunoglobulin G (IgG) and fibrinogen. After 5 or 60 min of incubations in whole normal or EGTA sera, antibodies against complement factor 3 (C3) and complement factor 3d (C3d) deposited to the surface. Factor H and complement factor 1q (C1q) were detected similarly after 1 and 5 min of incubation in 1-10% normal serum in veronal buffer, respectively. The indications are that upon contact with blood plasma, zirconium activates the intrinsic pathway of coagulation and is opsonized with C3. The failure to detect properdin and transient presence of factor H at the surface suggest that complement binds to zirconium although the activation becomes quickly down-regulated.

Ellipsometric in vitro studies on the activation of complement by human immunoglobulins M and G after adsorption to methylated silicon.

Human serum immunoglobulin M (IgM) or human immunoglobulin G (IgG) were adsorbed to dichlorodimethyl silane (DDS) treated silicon. Subsequently, the model surfaces were incubated in normal-, complement factor 1q (C1q)-complement factor B or complement factor 2 (C2)-depleted human sera at 37 degrees C for up to 1.5 h. The serum deposition and binding of selected polyclonal complement antibodies into this layer were then quantified by null ellipsometry. Both types of precoated surfaces bound large amounts of anti-complement factor 3c (anti-C3c), anti-properdin and anti-C3d, after incubation in normal serum. In contrast to IgG coated surfaces, IgM coated surfaces bound no anti-C1q after the serum incubations and no anti-C3c deposition lag time was observed after incubations in EGTA serum. Upon immersions of IgM coated surfaces in the different sera, a rapid complement activation via a C1q factor B, and Ca(2+)-independent, but C2 dependent pathway, was indicated. When IgM was instead immobilized to APTES/glutaraldehyde surfaces, anti-C3c deposition was lower after incubations in EGTA than normal serum. The results suggest that, under the present experimental conditions, human IgM and IgG activate the complement system differently.

Inflammatory cell recruitment, distribution, and chemiluminescence response at IgG precoated- and thiol functionalized gold surfaces.

The role of complement activation by artificial surfaces relative to inflammatory response is not well understood. This study was performed to evaluate the inflammatory cell recruitment, distribution, and ex vivo metabolic activation of surfaces with different plasma protein adsorption and complement activation properties in vitro. The implants were (1) pure gold (reference), (2) albumin-precoated (3) IgG-precoated gold, and (4) 3-mercapto-1, 2-propanediol [mercaptoglycerol (MG)] and (5) glutathione (GSH) immobilized to gold. The implant disks were inserted subcutaneously in rats for 24 h, and the number of inflammatory cells that were recruited to the implant adjacent to the surrounding fluid phase (exudate) and the surfaces were quantified by DNA measurements. The oxidative burst was analyzed ex vivo using spontaneous and phorbol myristate acetate (PMA)-stimulated, luminol-enhanced chemiluminescence (CL). The in vitro surface-induced anti-rat C3 binding was evaluated by ellipsometry and antibody techniques after plasma incubations for 1 and 30 min. The ellipsometric results showed that immobilized mercaptoglycerol and IgG-coated, but not the immobilized glutathione or the reference Au, bound anti-C3. The in vivo results revealed that the largest amount of cells was associated with the IgG-coated surfaces, followed by immobilized GSH and MG, albumin-coated, and gold surfaces, respectively. No spontaneous ex vivo luminol-enhanced CL was recorded from the cells irrespective of surface functionality or localization. A down-regulation of surface-associated and exudate leukocyte CL was observed ex vivo, irrespective of surface functionality. The results do not indicate a clear relationship between the degree of complement activation in vitro and leukocyte recruitment and adhesion in vivo for differently functionalized surfaces.

Ellipsometric studies in vitro on kinetics of rat complement activation.

The role of complement activation may be important during the early interactions between implantable materials and blood and during the acute inflammatory phase, but it is not well understood. This applies especially to rats that are extensively used in in vivo animal models for materials and surface testing. Features of the kinetics of rat complement activation were studied and compared with human complement by the ellipsometry and antibody techniques. The results indicate that the rat classical pathway is rapidly activated, but it is not as fast as the human system. The activation of the alternative pathway was observed within 5 min in the rat system and within 15 min for the human. Thus, the observations indicate substantial differences in the kinetics between the two species. This may influence the choice of the rat experimental model and the tissue response to materials during in vivo conditions.

Studies on protein adsorption and activation of complement on hydrated aluminium surfaces in vitro.

Adsorption of human plasma and serum proteins onto hydrated aluminium was studied by ellipsometry/antibody techniques, and soluble complement components iC3b, Bb, and C4d with commercial ELISA plates. Aluminium that was incubated in plasma for 1 min bound significant amounts of anti-lipoproteins (anti-LP), no antibodies against contact activation of coagulation proteins, and no anti-fibrinogen (anti-Fib). Time course studies with serum revealed increasing deposition of anti-C3c with time. Complement factor 1q (C1q) was antibody detectable only after short-time serum incubations, but no anti-IgG and anti-properdin bound to the protein film at any time. Anti-C3c was not deposited after exposure of the surfaces to Clq-depleted serum. Intriguingly, and in spite of increasing deposition of C3 to the surface with time, the combined ellipsometry and ELISA results gave no unequivocal proof of activation of complement by hydrated aluminium.

Immobilized chicken antibodies improve the detection of serum antigens with surface plasmon resonance (SPR).

Surface plasmon resonance (SPR) and other refractive index and mass sensitive methods are, due to complement activation by mouse monoclonal antibodies and with concomitant high background signal, only rarely used for the detection of antibody-antigen interactions in the blood serum milieu. In the present study chicken IgY and mouse IgG were immobilized to a sensor chip CM5 dextran matrix and compared for their background signal and detection of serum antigen. Ellipsometry with antibodies adsorbed to methylated silicon surfaces was used as a complementary detection method. As expected, fundamental differences in binding properties between the two kinds of antibodies were observed. Mouse antibodies bound large quantities of human serum. Human C1q was detected on mouse IgG and the complement system was activated, as seen from the rapid C3 and properdin depositions. Chicken antibodies bound low quantities of human serum and no human C1q. Moreover, C3 and properdin deposited only after prolonged serum incubations. Addition of EDTA to serum reduced the background signal modestly for both IgG and IgY. Serum samples with different concentrations of human C3 were injected over surfaces with immobilized chicken anti-C3, and the response was measured by SPR. Small concentration differences (< 1.25 micrograms/ml) in a physiologically relevant range (1-40 micrograms/ml after 100 times dilution) could then be detected reproducibly. The SPR signal was totally obscured when a mouse monoclonal anti-C3 antibody was used for the detection.

Temporal studies on the deposition of complement on human colostrum IgA and serum IgG immobilized on methylated silicon.

The temporal deposition of selected complement proteins from human serum onto immobilized human colostrum immunoglobulin (Ig)A and human IgG on hydrophobic silicon was studied by ellipsometry-antibody techniques after incubations at 37 degrees C for up to 1 h. In parallel experiments the serum soluble iC3b, C4d, and Bb were detected by enzyme-linked immunosorbent assay techniques. The IgA-coated surfaces showed activation via the alternative pathway, and displayed a lag phase in the deposition of increased amounts of serum proteins, and anti-C3c and antiproperdin. Anti-IgG, -C1q, -C4, -factor H and -factor B were not deposited at any time to IgA surfaces. Upon coating of the surface with IgG, the classical pathway was rapidly activated and bound, then anti-C3c, antiproperdin, and after short serum incubation times, also anti-C1q and anti-IgG. When factor B-depleted or heat-treated sera were used, the observation was that properdin deposited onto IgG-coated surfaces from both. Ellipsometry and antibody techniques offer a convenient and rapid way to indicate the activation of the complement system on solid surfaces and facilitates a time-resolved determination of the activation pathway(s).

Imaging of the Early Events of Classical Complement Activation Using Antibodies and Atomic Force Microscopy

In the present report we use atomic force microscopy (AFM) combined with antibody techniques to study the lateral distribution of specific serum proteins adsorbed onto flat silicon surfaces precoated with immunoglobulin G (IgG). Null-ellipsometry was used as a complimentary technique to quantify the adsorbed protein layers. After 15 s of incubation in human blood serum a partial monolayer of randomly distributed serum proteins was observed. The following exposure to antibodies to complement factor 1q (anti-C1q) resulted in a development of enlarged protein aggregates and a significant increase in adsorbed mass. Conversely, exposure to antibodies to complement factor 3c (anti-C3c) resulted in only a few randomly distributed protein aggregates and a much smaller increase in adsorbed mass. After 60 s of serum incubation the entire surface was covered with a proteinaceous film with irregular topography. This layer bound large amounts of anti-C3c but showed significantly smaller affinity for anti-C1q. Prolonging the serum incubation to 30 min resulted in an increased thickness and roughness of the protein layer and caused a massive deposition of anti-C3c but no anti-C1q. The results suggests that the transient affinity of anti-C1q, seen on various classically complement activating surfaces, is due to a shielding of the initially adsorbed proteins by subsequently deposited layers of C3. The results also show that qualitative information of the lateral organisation of specific proteins in a heterogeneous mixture can be assessed using AFM in combination with immunological techniques.

Complement activation by IgG immobilized on methylated silicon.

Activation of the complement system by immobilized IgG on methylated silicon was studied by ellipsometry/antibody-, ELISA-, and RIA techniques after exposure to human serum at 37 degrees C for up to 1 h. The IgG-covered surfaces rapidly activated the complement system and the combined results suggest an initial classical pathway activation. Complement factor 1q (C1q) and IgG were antibody-detectable on the surfaces for serum incubations up to 5 min but not thereafter. Anti-C3c and anti-properdin bound to the surfaces at all serum incubation times. Experiments with 125I-IgG preadsorbed to surfaces, or added to normal-, EGTA-, and EDTA-sera, showed that IgG was not displaced from the protein film by serum.

Complement activation by 3-mercapto-1,2-propanediol immobilized on gold surfaces.

Thiol-modified surfaces are chemically well defined and suited for surface biological model experiments and biomaterials research. 3-Mercapto-1,2-propanediol (mercaptoglycerol, MG), immobilized on gold, spontaneously binds immunoglobulins from human serum and activates the complement system. The surface-bound complement factors were detected by ellipsometry-antibody techniques. The overall complement activation was subsequently corroborated independently with enzyme immunosorbent assay (EIA) and sheep and chicken erythrocyte haemolytic complement techniques. EIA experiments indicated elevated levels of C4d, but no significant increase of factor Bb was evident in the test serum from the MG system. The haemolytic assays show that MG surfaces consume complement factors from both pathways. Ellipsometry revealed that immunoglobulin G (IgG) and complement factor 1q (C1q) are transiently antibody detectable on MG after exposure to whole serum by the use of antibody techniques. Complement factor 3 (C3), C2, C4 and properdin could be detected on the surface, but not factors H and B. The total adsorbed mass and particularly C3 antibody deposition were suppressed by using EGTA-Mg2+ serum. The results suggest that MG surfaces initially activate complement via the classical pathway. Other IgG binding surfaces also appear to behave in a similar manner.

Blood protein interactions with titanium surfaces.

Protein adsorption and complement activation were studied on thin evaporated films of titanium (Ti). The surfaces were cleaned in either a radio frequency (RF) plasma unit, or washed sequentially in trichloroethylene, acetone, ethanol, and water. Both methods resulted in hydrophilic surface with low carbon contamination on the outermost oxide (approximately 11-13 at%). In situ ellipsometry suggested that Ti is an intrinsic coagulation activator in vitro, since significant amounts of factor XII (F XII) and high molecular weight kininogen (HMWK) were found on the surfaces after 1 min incubation in heparin plasma. Ellipsometry, performed after serum incubations ranging from 15 s to 30 min showed that the total amount of serum proteins and the deposition of antibodies to complement factor 3c (C3c) increased with serum incubation time. ELISA methods showed increased levels of free iC3b in serum after 10 min incubation of the surfaces, but no detectable amounts of C3 convertase fractions C4d or Bb. Ellipsometric results indicated, however, an increased deposition of antibodies to CIq and IgG on Ti after short serum incubation times. The combined results indicate that Ti-surfaces initially activate complement through the classical pathway. The activation then continues via a positive amplification loop where increased amounts of C3 are deposited on the surfaces via the alternative pathway.

Blood protein interactions with chromium surfaces.

Protein adsorption, contact activation, and complement activation were studied on thin evaporated films of chromium (Cr) in vitro. The surfaces were, prior to the experiments, cleaned in either ethanol and water, or in a basic peroxide solution (RCA standard clean 1, SC-1). Surface spectroscopic studies of the outermost oxides showed a significant reduction of carbon contaminants after washing in SC-1 but also suggested an increase in the oxidation state as compared with the ethanol-washed surfaces. In situ ellipsometry combined with antibody techniques was used to determine protein deposition and antibody binding onto surfaces after incubations in heparin plasma or in normal serum. Incubation times from 1 to 10 min in serum showed increased depositions of serum and antibodies to complement factor 3c (C3c) and was larger on ethanol-washed surfaces than on surfaces washed in SC-1. ELISA methods indicated increased amounts of iC3b in serum for both surfaces, but no presence of C3 convertases (C4d or Bb fractions). A low or transient complement activation via the classical pathway was indicated on ethanol washed Cr, since deposition of secondary antibodies to complement factor Iq (CIq) was observed only after short incubation times in serum. No procoagulant activity of Cr was indicated, since only low amounts of antibodies to factor XII (F XII), prekallikrein (PKK), and high molecular weight kiniogen (HMWK) bound to the surfaces after incubations in heparin plasma. These results were confirmed using a colorimetric assay where the relative amounts of free plasma kallikrein was assessed using a chromogenic substrate, H-D-Pro-Phe-Arg-pNA (S-2302).

Lens-on-surface method for investigating adhesion of Staphylococcus aureus to solid surfaces incubated in blood plasma.

Adhesion of Staphylococcus aureus was investigated on flat silicon oxide surfaces that had been incubated in human plasma at different concentrations. Adhesion of bacteria did not occur at high incubation concentrations of plasma or when the surface had been incubated in egg albumin. However, significant adhesion was observed when plasma was diluted. With the use of antibody method, it was noted that the adhesion of the bacteria coincided with adsorbed fibrinogen, and possibly also with IgG. We also investigated the effect of "narrow space" on the adsorption of blood plasma and subsequent adhesion of S. aureus. In these experiments, blood plasma was incubated under a convex lens placed upside-down on the silicon oxide surface. This method creates a continuous gradient of space from the contact point of the lens and outward. After rinsing off the plasma and the lens, the surface was incubated with a suspension of S. aureus followed by quantification of the attached bacteria by means of optical methods. Adhesion of bacteria occurred in several circular zones that were easily detectable with the naked eye or by the means of simple optical methods. In addition, in these experiments, adhesion coincided with adsorbed fibrinogen or IgG at the surfaces. The increased bacterial adhesion to surfaces incubated in diluted plasma, or plasma incubated in narrow space, is a variant of the so-called "Vroman effect." With a model protein system consisting of fibrinogen and IgG and the corresponding antibodies, we demonstrate that "dilution" and "incubation in narrow space" are two phenomenologically similar methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Antisera binding onto metals immersed in human plasma in vitro.

Titanium (Ti), silicon (Si), silver (Ag), vanadium (V), gold (Au), and chromium (Cr) surfaces were made hydrophilic by radiofrequency plasma treatment. The binding of antifibrinogen (a-fib) and anti-high-molecular-weight kininogen (a-HMWK) after incubation of surfaces in 10% human blood plasma was investigated with ellipsometry. Ti and Si surfaces bound a-HMWK but no detectable amounts of a-fib, whereas the other metals bound both types of antisera. Protein adsorption in a fluid gradient produced under a convex lens indicated that the small deposition of a-fib on Ti and Si surfaces is probably a result of protein displacement. This displacement was not evident at lens positions simulating low plasma concentrations where large amounts of a-fib adsorbed. Surfaces washed sequentially in trichloroethane, acetone, and ethanol produced more hydrophobic surfaces and resulted in slower rates of a-fib displacement. These studies would indicate that some metals form complexes with specific plasma proteins or, alternatively, that different metals may bind equal amounts of plasma proteins which express differing antigenicities toward their surroundings.

Studies of surface activated coagulation: antisera binding onto methyl gradients on silicon incubated in human plasma in vitro.

Human plasma proteins factor XII, high molecular weight kininogen, prekallikrein, factor XI and fibrinogen, participate in surface-initiated coagulation. Antisera binding to methyl gradients made on hydrophilic silicon was studied after immersion in normal and deficient human blood plasma. Scanning ellipsometry was used to quantify the adsorbed organic material. The hydrophilic part of the gradient deposited anti-factor XII and anti-high molecular weight kininogen, but low amounts of anti-fibrinogen. Increased amounts of anti-fibrinogen bound onto the hydrophobic part, and the intermediate gradient region with mixed polar-nonpolar surface characteristics bound low amounts of anti-factor XII, anti-high molecular weight kininogen and anti-fibrinogen. Tentatively, in this gradient region, simultaneous polar and non-polar surface characteristics result in a low-level of surface-activated coagulation. Surfaces immersed in heparinized-and EDTA-plasma indicate different antisera depositions.

'Lens-on-surface': a versatile method for the investigation of plasma protein exchange reactions on solid surfaces.

The exchange sequence of plasma proteins in narrow spaces on solid surfaces was studied by means of a modified 'lens-on-surface' method as originally described by Vroman and Adams. In our studies, lateral scanning ellipsometry was used as the detection method. With the use of antibodies it was demonstrated and confirmed that immunologically detectable plasma protein antigens appear and disappear in a time- and concentration-dependent sequence [IgG followed by fibrinogen followed by high-molecular weight kininogen (HMWK)] on silica surfaces. Plasma protein exchange reactions were also studied on hydrophilic titanium (Ti), vanadium (V), and silver (Ag) surfaces. Atypical exchange patterns were found on V and Ag surfaces as compared with hydrophilic silica (adsorbed fibrinogen was not removed).

Competition between adsorbed fibrinogen and high-molecular-weight kininogen on solid surfaces incubated in human plasma (the Vroman effect): influence of solid surface wettability.

The influence of the surface energy on the competition between fibrinogen and high-molecular-weight kininogen has been studied with the use of a recently described wettability gradient method. One finding is that the decrease in the antifibrinogen binding on plasma incubated gradient surfaces was not associated with an increase in anti-HMWK binding at all parts of the gradient surfaces.