| 1. |
- Benesch, Johan, et al.
(author)
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Blood protein adsorption onto chitosan
- 2002
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In: Biomaterials. - 0142-9612 .- 1878-5905. ; 23:12, s. 2561-2568
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Journal article (peer-reviewed)abstract
- Chitosan was recently indicated to enhance osteogenesis, improve wound healing but to activate the coagulation and the complement systems. In the present study approximately 10nm thick chitosan film were prepared on aminopropyltriethoxysilane (APTES) coated silicon. The surfaces were incubated in serum or plasma and subsequently in antibodies towards key complement and contact activation of coagulation proteins. The deposited amounts were compared with those on hydrophilic and hydrophobic silicon, APTES and IgG coated reference samples. Although large amounts of serum deposited to chitosan only a weak transient activation of the complement system and no activation of the intrinsic pathway was observed. Upon acetylation the chitosan layer became a strong activator of the alternative pathway of the complement. After incubation in human plasma anti-fibrinogen deposited onto chitosan but not onto the acetylated chitosan, a finding that may explain previous observations of procoagulant activity by chitosan. Copyright © 2002 Elsevier Science Ltd.
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| 2. |
- Benesch, Johan, 1969-
(author)
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Null Ellipsometry and Protein Adsorption to Model Biomaterials
- 2001
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Doctoral thesis (other academic/artistic)abstract
- When implants are inserted into the human body cascades of events become started that will determine the outcome of wound healing and ultimately the success of the implantation. The events start with the adsorption of small molecules, and proteins that may be activated (enzymes) or are able to activate cells of the immune defense and the healing process. In the biomaterials research that is conducted in our group we often ask two questions: "How much?" and "What?" proteins adsorb to a specific surface after incubation in serum or plasma. In the first two papers in this thesis I studied how well we are able to determine the answer to the first question. In the latter two works I tried to answer both questions for two model biomaterial surfaces: oligo(ethylene glycol) terminated self assembled monolayers on gold and chitosan coated silicon.In many null ellipsometric studies the protein film refractive, Nfilm, is assumed to be close to 1.5. In the first paper we analyzed if the assumption of Nfilm = 1.465 is satisfactory for the determination of the surface mass density of a submonolayer thin protein film. Human serum albumin (HSA) was labeled with 125I and mixed with non-labeled HSA, and hydrophobic and hydrophilic silicon pieces were incubated in the solutions. The surface mass densities on all pieces determined by both ellipsometry and gamma counter measurements, and was pair-wise compared. The above assumption regarding the value of Nfilm for the agreement between the methods was satisfactory, although precautions have to be made not to overestimate the surface mass density when studying radiolabelled proteins, especially at rough surfaces.Are the assumptions made in Paper I also true for up to 100 nm thick protein films or do we have to use a different protein film refractive index and do the ellipsometric model still hold? Human serum albumin (HSA) and polyclonal anti-HSA were labeled with 125I and mixed with unlabelled proteins. Hydrophobic silicon pieces were alternatingely incubated in the two protein solutions. Again the surface mass density was quantified with null ellipsometry and a gamma counter and the methods compared. The thickest protein layers were also gently scratched and the thickness measured by AFM. It appeared that a protein film refractive index Nfilm = 1.5 was a good choice for the determination of the protein film thickness. However, in order to obtain a good methods agreement for the adsorbed mass density a linear correction term was needed in the Cuypers surface mass density formula for ellipsometry. The physical interpretation of the correction term is presently unclear.Self assembled monolayers (SAMs) containing oligo (ethyleneglycol) end groups (OEG) have been successfully used to minimize protein adsorption from single protein solutions. We investigated the protein resistance in a fibrinogen solution, serum and plasma of OEG-SAMs with an increasing number of OEG units and with different end groups. It turned out that the adsorbed amounts after 10 minutes of plasma incubation and 15 minutes of fibrinogen incubation decreased with an increasing number of EG units. In serum, the total deposition and subsequent deposition of antibodies towards complement proteins (C3c, C3d and properdin) did not depend on the number of EG units. In summary, the investigated OEG-SAMs were not protein resistant in complex solutions, although the adsorbed amounts varied with the number of EG units and the terminal chemical group. Complement deposition was observed at OEG surfaces.For the last 50 odd years different polysaccharides, such as heparin and cellulose have been used for clinical applications and in recent years also chitin and its deacetylated form chitosan have gained increasing attention as potential biomaterials. Previous studies on complement activation by chitosan derivatives have focused on the soluble complement factors and not the surface bound ones that may be important for the binding of cells to surfaces. In our study about 10 nm thick chitosan films were incubated in plasma or serum and subsequently in polyclonal antibodysolutions. The films did not activate complement and the intrinsic pathway of coagulational though fibrinogen was detectable after plasma incubations. When the chitosan film was acetylated it became a strong alternative complement pathway activator in serum and fibrinogen was then no longer antibody detectable after plasma incubations.
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| 3. |
- Benesch, Johan, et al.
(author)
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Protein adsorption to oligo(ethylene glycol) self-assembled monolayers : Experiments with fibrinogen, heparinized plasma, and serum
- 2001
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In: Journal of Biomaterials Science. Polymer Edition. - : Taylor & Francis. - 0920-5063 .- 1568-5624. ; 12:6, s. 581-597
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Journal article (peer-reviewed)abstract
- Low protein adsorption is believed advantageous for blood-contacting materials and ethylene glycols (EG)-based polymeric compounds are often attached to surfaces for this purpose. In the present study, the adsorption of fibrinogen, serum, and plasma were studied by ellipsometry on a series of well-defined oligo(EG) terminated alkane-thiols self-assembled on gold. The layers were prepared with compounds of the general structure HS-(CH2)15-CONH-EGn, where n = 2, 4, and 6. Methoxy-terminated tri(EG) undecanethiol and hydroxyl-terminated hexadecanethiol self-assembled monolayers (SAMs) were used as references. The results clearly demonstrate that the adsorption depends on the experimental conditions with small amounts of fibrinogen adsorbing from a single protein solution, but larger amounts of proteins from serum and plasma. The adsorption of fibrinogen and blood plasma decreased with an increasing number of EG repeats and was temperature-dependent. Significantly less serum adsorbed to methoxy tri(EG) than to hexa(EG) and more proteins remained on the latter surface after incubation in a sodium dodecyl sulfate (SDS) solution, indicating a looser protein binding to the methoxy-terminated surface. All surfaces adsorbed complement factor 3(C3) from serum and plasma, although no surface-mediated complement activation was observed. The present study points to the importance of a careful choice of the protein model system before general statements regarding the protein repellant properties of potential surfaces can be made.
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| 4. |
- Benesch, Johan, et al.
(author)
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Quantification of adsorbed human serum albumin at solid interfaces : A comparison between radioimmunoassay (RIA) and simple null ellipsometry
- 2000
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In: Colloids and Surfaces B. - 0927-7765 .- 1873-4367. ; 18:2, s. 71-81
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Journal article (peer-reviewed)abstract
- Radioimmunoassay (RIA) and null ellipsometry are two common methods to quantify adsorbed proteins. However, the accuracy of null ellipsometry with a constant protein refractive index (n=1.465, k=0) at ?=632.8 nm has this far not been explored. The present study compared the methods, and the degree of agreement between the simplified single wavelength null ellipsometry and RIA to quantify adsorbed proteins was explored on different surfaces. The quantification methods agreed well when Angstrom smooth hydrophilic or hydrophobic silicon surfaces, and freshly radio-labelled proteins were used. Some discrepancies were noted when either rough surface or stored and aged labelled proteins were used. The differences decreased when the aged protein solution was equilibrated with freshly dissolved proteins at room temperature (RT) for a few hours prior to the surface incubations. Significant differences were also noted between the methods when albumin was adsorbed at it's iso-electric point (pH 4.8). Copyright (C) 2000 Elsevier Science B.V.
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| 5. |
- Benesch, Johan, et al.
(author)
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The determination of thickness and surface mass density of mesothick immunoprecipitate layers by null ellipsometry and protein 125iodine labeling.
- 2002
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In: Journal of colloid and interface science. - : Elsevier BV. - 0021-9797 .- 1095-7103. ; 249:1, s. 84-90
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Journal article (peer-reviewed)abstract
- The aim of the present study was to ellipsometrically determine the thickness and surface mass density in air for up to 110-nm-thick organic layers made of alternatingly deposited layers of HSA and polyclonal anti-HSA on hydrophobic silicon. The ellipsometrically determined thickness was compared to that obtained by AFM and the deposited surface mass density calibrated with (125)I-labeled proteins. The results indicate a good agreement in protein layer thickness between AFM and ellipsometry when the protein film refractive index N(film)=1.5-0i, although then the calculated surface mass density from the ellipsometry data became grossly overestimated by the Cuypers one-component formula. A good agreement in the surface mass density was obtained when the M/A ratio in this formula was lowered from 4.14 to 2.35. This approach indicates a convenient means of determining the refractive indices and surface mass densities of mesothick organic layers proteins on solid supports.
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| 6. |
- Hansson, Kenny, 1972-, et al.
(author)
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Surface plasmon resonance detection of blood coagulation and platelet adhesion under venous and arterial shear conditions.
- 2007
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In: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 23:2, s. 261-8
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Journal article (peer-reviewed)abstract
- A surface plasmon resonance (SPR) based flow chamber device was designed for real time detection of blood coagulation and platelet adhesion in platelet rich plasma (PRP) and whole blood. The system allowed the detection of surface interactions throughout the 6mm length of the flow chamber. After deposition of thromboplastin onto a section of the sensor surface near the inlet of the flow chamber, coagulation was detected downstream of this position corresponding to a SPR signal of 7 to 8 mRIU (7 to 8 ng/mm2). A nonmodified control surface induced coagulation 3.5 times slower. Platelet adhesion to gold and fibrinogen coated surfaces in the magnitude of 1.25 and 1.66 mRIU was also shown with platelets in buffer, respectively. SPR responses obtained with PRP and whole blood on surfaces that were methylated or coated with von Willebrand factor (vWF), fibrinogen, or collagen, coincided well with platelet adhesion as observed with fluorescence microscopy in parallel experiments. The present SPR detection equipped flow chamber system is a promising tool for studies on coagulation events and blood cell adhesion under physiological flow conditions, and allows monitoring of short-range surface processes in whole blood.
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| 7. |
- Vikinge, Trine P., et al.
(author)
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Blood plasma coagulation studied by surface plasmon resonance
- 1999
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In: BIOMEDICAL SENSORS, FIBERS, AND OPTICAL DELIVERY SYSTEMS, PROCEEDINGS. - : SPIE - International Society for Optical Engineering. ; , s. 107-114
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Conference paper (peer-reviewed)abstract
- A surface plasmon resonance (SPR) apparatus was used to investigate blood plasma coagulation in real-time as a function of thromboplastin and heparin concentrations. The physical reason for the SPR signal observed is discussed and 3 different models are proposed. The response curves were analyzed by multivariable curve fitting followed by feature extraction. Interesting parameters of the sigmoid curves were lag time, slope and maximum response. When thromboplastin concentrations were increased, the lag-time decreased and the slope of the curve increased. A prolonged clotting time was mostly followed by increased maximum response, with exception for samples with no or very little thromboplastin added. High heparin concentrations changed the clotting kinetics, as seen from the lag-time vs. slope relation. Atomic force microscopy (AFM) pictures of sensor surfaces dried after completed clotting, revealed differences in fibrin network structures as a function of thromboplastin concentration, and fiber thickness increased with lower thromboplastin concentration. The results correlate well with present common methods.
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| 8. |
- Vikinge, Trine P., et al.
(author)
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Blood plasma coagulation studied by surface plasmon resonance
- 2000
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In: Journal of Biomedical Optics. - : SPIE-Intl Soc Optical Eng. - 1083-3668 .- 1560-2281. ; 5:1, s. 51-55
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Journal article (peer-reviewed)abstract
- A surface plasmon resonance (SPR) apparatus was used to investigate blood plasma coagulation in real time as a function of thromboplastin and heparin concentrations. The response curves were analyzed by curve fitting to a sigmoid curve equation, followed by extraction of the time constant. Clotting activation by thromboplastin resulted in increased time constant, as compared to spontaneously clotted plasma, in a dose dependent way. Addition of heparin to the thromboplastin-activated plasma counteracted this effect. Atomic force microscopy (AFM) pictures of sensor surfaces dried after completed clotting, revealed differences in fibrin network structures as a function of thromboplastin concentration, and the fiber thickness increased with decreased thromboplastin concentration. The physical reason for the SPR signal observed is ambiguous and is therefore discussed. However, the results summarized in the plots and the fibrin network properties observed by AFM correlate well with present common methods used to analyze blood coagulation.
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