Biomedical subjects

H Nygren

Publications and source records attributed to H Nygren.

At least 19 recordsLinked to original sources

PubMed2004

Silver deposition on freeze-dried cells allows subcellular localization of cholesterol with imaging TOF-SIMS.

Imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used for characterization and subcellular localization of organic ions in leucocytes adhering to glass surfaces. The cells were fixed by freeze drying in 0.15 m ammonium formate buffer at pH 7.2-7.4. The freeze-dried cells were sputter-coated with silver, and the silver surface was analysed with imaging TOF-SIMS. TOF-SIMS spectra were recorded by scanning the primary ion beam over the analysis area and acquiring positive mass spectra of the ions leaving the surface. The relative brightness of each pixel within the analysis area reflects the signal intensity of a selected ion in that pixel. Data were collected separately at high mass resolution m/delta m > 7000 and at high lateral resolution (= 0.5 micro m). The images were analysed by principal component analysis (PCA). The glass-adhering cells showed a well defined attachment area with a diameter of up to 20 micro m, and an equally well defined cell body, containing the nucleus, with a diameter of 8-10 micro m. On the raw data images, the obtained cholesterol distributions were consistent with a higher cholesterol content of the cell membrane in the attachment area than in the cell body. Using PCA analysis, silver-cationized molecular cholesterol was found localized mainly in the attachment area of the cells. Cholesterol was also seen at higher concentration in circular spots of </= 1 micro m in diameter, probably representing caveolae.

Cell Adhesion↗

PubMed2001

Respiratory burst response of peritoneal leukocytes adhering to titanium and stainless steel.

Titanium sheets, made hydrophilic by oxidative cleaning or hydrophobic by treatment with butanol, and stainless steel sheets with different patterns of pores (straight phi = 0.8 mm) were implanted into the peritoneal cavity of mice. The implants were removed after 2 h, and the surface-adhering leukocytes were stained with propidium iodide and fluorescein diacetate to quantitate cell adhesion and to indicate the presence of leaks in the cell membrane. The ability of the surface-adhering leukocytes to mount a respiratory burst response after stimulation with PMA or zymosan was measured by chemiluminiscence. The results show that stainless steel without pores induces membrane leakage in 80% of the surface-adhering leukocytes compared with 65% of cells adhering to porous steel. Hydrophilic titanium induces membrane leakage in 48% of the surface-adhering leukocytes compared with 19% of cells adhering to hydrophobic titanium. The respiratory burst response of the surface-adhering leukocytes stimulated with PMA was attenuated on stainless steel and hydrophilic titanium compared with hydrophobic titanium. Thus, butanol treatment of titanium and pores in stainless steel increase the biocompatibility of the materials.

Animals↗

PubMed2001

Cellular reactions and bone apposition to titanium surfaces with different surface roughness and oxide thickness cleaned by oxidation.

Titanium surfaces with three different surface characteristics were exposed to an intraperitoneal milieu in mouse or rat, or inserted into rabbit bone. The cleaning regimen of the TiO2 surfaces in this study included oxidation by heat or acid and a final rinsing and storage in water. Intraperitoneal exposure ranged from 1 to 64 min and the healing period in bone was 6 weeks. Cell recruitment to the surfaces was quantified by acridine orange staining and specific antibodies directed against cell membrane antigens. Removal torque, bone-to-metal contact, total bone area and histological evaluations were used to evaluate fixture stability and the healing-in of the implants. After the healing period of 6 weeks only a transient significant difference was seen in the total number of cells adherent on the surfaces. No significant differences were observed between any of the surfaces for removal torque, bone-to-metal contact, or bone area. The areas lacking bone-to-metal contact were filled with normal vascularised connective tissue with no signs of fibrous capsule formation or giant cells. These findings differ from findings published earlier of Ti implants that underwent a cleaning regimen with alcohol as the final rinsing step. The tissues around the implants were richly vascularised and there was continued bone growth toward the surfaces. The bone-to-metal contact in this study was lower than that seen with alcohol-cleaned TiO2.

Animals↗

PubMed2001

Interactions between human whole blood and modified TiO2-surfaces: influence of surface topography and oxide thickness on leukocyte adhesion and activation.

An in vitro model (Nygren et al., J Lab Clin Med 129 (1997) 35-46) was used to investigate interactions between leukocytes and four modified TiO2-surfaces. Surface topography was measured using scanning electron microscopy and optical profilometry while Auger electron spectroscopy was used to determine surface composition and oxide thickness. The surfaces were either smooth or rough with either thin or thick oxides. All surfaces consisted of TiO2 covered by a carbonaceous layer. The surfaces were incubated with capillary blood for time periods of between 8 min and 32 h. Immunofluorescence techniques together with computer aided image analysis and chemiluminescence technique were used to detect cell adhesion, expression of adhesion receptors and the zymosan-stimulated respiratory burst response. Leukocyte adhesion to the surfaces increased during the first hours of blood-material contact and then decreased. Polymorphonuclear granulocytes were the dominating leukocytes on all surfaces followed by monocytes. Cells adhering to rough surfaces had higher normalized expression of adhesive receptors than cells on smooth surfaces. Maximum respiratory burst response occurred earlier on the smooth than on the rough surfaces. In conclusion, topography had a greater impact than oxide thickness on most cellular reactions investigated, but the latter often had a dampening effect on the responses.

Antigens, CD↗

PubMed2001

Von Willebrand factor, a key protein in the exposure of CD62P on platelets.

When a biomaterial is introduced into the body water, electrolytes, and proteins adsorb to the surface. Platelets are then the first cells to interact with the surface adsorbed protein layer. We have studied the role of von Willebrand factor (vWF) for platelet-protein interaction by measuring different platelet responses to protein- and plasma-coated hydrophobic glass surfaces. A high exposure of CD62P on the platelet surface was seen after 10 min of incubation on platelets interacting with vWF and normal plasma-coated surfaces (79 and 67%, respectively). On the surfaces coated with albumin and factor VIII deficient plasma, the exposure was low (11 and 27%, respectively). A higher formation of filipodial extensions on the platelets was seen on the surfaces coated with vWF and normal plasma than on the surfaces coated with albumin or factor VIII deficient plasma. No significant differences were seen between the surfaces regarding the platelet release of PF4, ATP, or phospholipids. As shown by these results, vWF is a specific regulator of the exposure of CD62P by platelets and hence important for the interaction between platelets and later arriving neutrophils at biomaterial surfaces.

Adenosine Triphosphate↗

PubMed2001

Protection by glutathione of neutrophils against the toxic effects of peritoneal dialysis fluid.

The possibility of reducing the cytotoxic effect of heat-sterilized peritoneal dialysis (PD) fluid by addition of antioxidants/scavengers during incubation of titanium-adhering cells was investigated. Capillary blood from healthy donors was placed in drops on commercially available titanium pieces and incubated in a humidified chamber at 37 degrees C for 60min. After incubation the adherent polymorphonuclear leukocytes were immersed for 1-4h in PD-fluid, pH 7.4, containing 2.5% glucose with glutathione (GSH), superoxide dismutase, catalase or dithiothreitol (DTT). Luminol- or isoluminol-amplified chemiluminescence was used to measure the zymosan- and phorbol myristate acetate (PMA)-stimulated respiratory burst activity, as an indicator of the cytotoxicity of the PD-fluids. Heat sterilized PD-fluid had inhibitory effect on zymosan-induced respiratory burst and impaired both the extracellular and intracellular PMA-induced respiratory burst. Addition of GSH to the PD-fluid resulted in reduction of cytotoxical effects on the zymosan-induced and extracellular PMA-induced respiratory burst. The intracellular respiratory burst was not affected. The present results show that GSH and DTT have the ability to protect polymorphonuclear leukocytes against the cytotoxic effects of the PD-fluid by keeping the cell membrane in a reduced state.

Catalase↗

PubMed2001

The respiratory burst response of surface-adhering leukocytes. A key to tissue engineering.

Biomaterials implanted into tissue will participate in the complex signalling between cells during wound healing. Recent studies have revealed that crucial cellular signalling pathways are regulated by the extra- and intracellular redox states and that reactive oxygen species function as intercellular signal molecules. Biomaterials have been shown to affect the respiratory burst response of surface-adhering leukocytes, thus interfering with major regulatory functions of cells also in surrounding tissues. The respiratory burst of surface-adhering leukocytes may thus be a key event in the understanding of biomaterial interaction with tissues, and the aim of this review is to highlight this field of research.

Journal Article↗

PubMed2001

Adhesion receptors of polymorphonuclear granulocytes on titanium in contact with whole blood.

Titanium sheets were exposed to whole blood, and the TiO(2) surface was investigated regarding the presence of cells, receptor expression on adherent polymorphonuclear (PMN) granulocytes, and the ability of these cells to mount a respiratory burst when challenged with opsonized zymosan. The techniques used were immunofluorescence with computer-aided image analysis and chemiluminescence. Surface coverage of erythrocytes (9% to 10%), granulocytes (9% to 14%), and platelets (1% to 4%) dominated during the first 2 hours of blood contact. PMN granulocyte adhesion to titanium was associated with a rapid decrease in L-selectin expression within 16 minutes. Initially FcgammaIII receptor (CD16) expression dominated on the adherent cells. After 30 minutes, a shift toward integrin expression (CD11b) was found on the adherent cells. All investigated receptors were down-regulated within 1 hour of blood-titanium contact. Attempts were made to inhibit the initial adhesion of PMN granulocytes to titanium by adding specific antibodies or 2,3-diphosphoglyceric acid (phospholipase D inhibitor) to blood before surface contact. Adding anti-CD16 resulted in a 67% reduction in cell adhesion, whereas a 35% reduction was found with 2,3-diphosphoglyceric acid. No spontaneous respiratory burst was detected from adherent PMN granulocytes residing on the TiO(2) surface. The cells were, however, able to mount a respiratory burst in response to opsonized zymosan.

Biocompatible Materials↗

PubMed2001

Polymorphonuclear leukocytes in coagulating whole blood recognize hydrophilic and hydrophobic titanium surfaces by different adhesion receptors and show different patterns of receptor expression.

The mechanism of healing or rejection of implant materials is unknown, but the process always starts at the contact with coagulating blood. Here, the initial reactions of clean (hydrophilic) and alkylated (hydrophobic) titanium with blood were investigated by short-term exposure to human blood and detection of polymorphonuclear leukocyte (PMNL) surface antigens with an immunofluorescence technique. The fluorescence intensity was quantitated by computer-aided image analysis. Antibodies specific to CD11b, CD16, CD18, CD62L, and CD162 were used to block PMNL adhesion. The respiratory burst of adhering cells was stimulated with opsonized zymosan and measured by chemiluminiscence. The thrombin dependence of PMNL reactions was studied by using hirudin, a specific thrombin inhibitor. The expression of CD62L decreased with increasing exposure time, and the rate of decrease was faster at the hydrophilic surface. At the hydrophilic surface, the CD16 exposure was high after 8 minutes of blood contact, and it decreased with time. At the hydrophobic surface, a peak in CD16 expression was seen after 32 minutes of blood exposure. At the hydrophobic surface, the expression of CD11b increased slowly with increasing blood exposure time, whereas at the hydrophilic surface, a peak of CD11b expression was seen after 32 minutes of blood exposure. The expression of CD11b and that of CD16 were found to be thrombin dependent. At the hydrophilic surface, adhesion of PMNLs was blocked by CD16 antibodies, whereas adhesion to the hydrophobic surface was blocked by anti-CD162. Mixing blood with antibodies to CD11b, CD18, and CD62L amplified the adhesion of PMNLs to the hydrophilic surface.

Blood Coagulation↗

PubMed2000

Different kinetics of the respiratory burst response in granulocytes, induced by serum from blood coagulated in contact with polymer materials.

Tubes of different polymer materials were filled with blood collected by venous puncture. The blood was allowed to clot for 10 min, and the serum was collected. Complement activation was demonstrated through assessment of the C3-level by radial immunodiffusion. Phospholipid fingerprints were made after lipid extraction of serum and separation by thin-layer chromatography. The granulocyte fraction of venous blood was separated on a Percoll gradient and the cells were either loaded with a calcium probe, or incubated with luminol. These cells were used as a biological test for inflammatory mediators. Serum from blood coagulated in contact with different materials was added to the test cells. The intracellular calcium level was recorded by Calcium Green-1 fluorescence and the respiratory burst of the test cells was recorded by luminol-amplified chemiluminescence. Serum from blood coagulated in contact with glass tubes, methylised glass tubes and teflon (PTFE) tubes induced a transient increase of the cellular calcium level, indicating a G protein-coupled activation of the test cells. Serum from blood coagulated in contact with glass tubes, methylised glass tubes, and PTFE tubes primed the test cells for a subsequent f-MLP response. Serum from blood coagulated in contact with polyurethane and polypropylene induced a direct biphasic respiratory burst response in the test cells and serum from blood coagulated in contact with methylised glass induced a direct monophasic respiratory burst response in the test cells. Complement activation was demonstrated after blood contact with hydrophobic glass and PTFE. Different fingerprints of phospholipid content were found in sera after blood contact with different materials. The data show that different inflammatory mediators are released during blood coagulation in contact with different materials. The method may be valuable as a screening test for blood compatibility of materials.

Biocompatible Materials↗

PubMed1999

Surface characterization, protein adsorption, and initial cell-surface reactions on glutathione and 3-mercapto-1,2,-propanediol immobilized to gold.

Monolayers of glutathione (GSH) and 3-mercapto-1,2-propanediol (MG) on gold were tested for their bioreactivity by assessing the degree of inflammatory reaction as manifested by the adherence and activation of platelets and white blood cells (wbc) after exposure to blood ex vivo. Surface composition was characterized by XPS, and noncontact optical profilometry was used to determine surface roughness. The thickness and composition of the adsorbed protein layers were measured by ellipsometry/antibody techniques in vitro. Cell adhesion and activation were quantified by acridine orange staining, fluorescein-diacetate staining, and by specific antibodies against cell membrane antigens. Distinct differences among the surfaces were observed relative to the amounts and composition of adsorbed plasma proteins and the adhesion and activation of platelets (CD62P-exposure) and wbc (CD11b/CD18-exposure). GSH surfaces, which adsorbed the least amount of plasma protein, caused the least adherence and activation of platelets (CD62P), followed by the highest activation of wbc (CD11b/18). The MG surfaces caused a rapid recruitment and activation of platelets (CD62P), followed by a lower activation of wbc (CD11b/18). Thus it appears that measurements of the initial adsorption of plasma protein from anticoagulated plasma and of the adhesion and activation of platelets after 8 min of exposure to whole blood cannot be used to predict accurately the adhesion and activation behavior of inflammatory cells after longer periods (2 h) of exposure on different surfaces.

Adsorption↗

PubMed1999

Characterization of cellular response to thiol-modified gold surfaces implanted in mouse peritoneal cavity.

The early inflammatory reaction in vivo to three well defined surfaces-gold, gold coated with glutathione (GSH), and 3-mercapto-1, 2-propanediol (MG)-was assessed as manifested by the adherence and activation of inflammatory cells during implantation intraperitoneally in mice. Evaluation of cell adhesion and activation was done by immunohistochemistry using specific monoclonal antibodies directed against cell differentiation antigens CD11b/CD18, CD74, and CD25 or by measurement by chemoluminescence of reactive oxygen radical species produced by adhering cells. Cell recruitment and activation was slow on the GSH-coated gold surfaces. These surfaces also had the highest percentage of adhering cells with an intact cell membrane. The MG-coated surfaces, on the other hand, rapidly recruited and activated cells and also caused cell membrane leakage to propidium iodide, suggesting cell membrane damage or cell death. The respiratory burst of adhering cells was stimulated by phorbol-myristate acetate on the GSH-coated surface but not on the MG-coated surface and by opsonized zymosan on the Mg-coated surface but only to a small degree on the GSH-coated surface. The respiratory burst following zymosan activation of cells adhering to the MG-coated surface was inhibited by treatment with 2. 3-diphosphoglycerate, a phospholipase D inhibitor. The presented data suggest that peritoneal leukocytes adhering to foreign materials may raise a respiratory burst response via a phospholipase D-dependent and protein kinase C-independent pathway.

Animals↗

PubMed1998

Specific activation of platelets by surface-adsorbed plasma proteins.

Platelets were isolated from human blood by Percoll density gradient centrifugation in a low Ca2+/high Mg2+ buffer. The buffer reversibly inactivates the cells during separation. The purity of the isolated cells (> 99%) was determined by flow cytometry, their viability was confirmed by fluorescein diacetate hydrolysis, and their morphology was studied with TEM. Plasma proteins were adsorbed onto hydrophobic glass surface, and pure platelets were added and incubated for up to 30 min at 37 degree C. Platelet activation was determined by cell spreading, formation of microparticles and surface exposure of CD62P indicating the release of alpha-granules. Surface-immobilized IgG was shown to cause the release of microparticles and cell lysis, in accordance with data published by others. Surface-immobilized vWF was shown to induce CD62P exposure on the platelet cell surface. The specificity of this response was demonstrated by adsorbing plasma proteins from normal and factor VIII-deficient plasma.

Blood Platelets↗

PubMed1997

The initial reactions of TiO2 with blood.

The natural titanium oxide (TiO2) layer of commercial sheet titanium was dissolved in hydrofluoric acid. A new oxide layer was grown by oxidation in nitric acid or by annealing at 700 degrees C in air. At this temperature, reaction with nitrogen is unlikely. The purity of the oxidized sheet-titanium surfaces was investigated by Auger spectroscopy. The composition of both surfaces was TiO2 with carbon impurities. The carbon content of the acid-oxidized titanium was 20 +/- 2%, and the carbon content of the heat-oxidized titanium was 14 +/- 2% The initial reactions of the TiO2 surfaces with blood were investigated by short-time exposure to capillary blood and by detection of surface-adsorbed plasma proteins and cells with immunofluorescence. Antibodies specific to fibrinogen, complement factor C1q, prothrombin/thrombin, and platelet membrane antigen were used, and the fluorescence was quantitated by computer-aided image analysis. The results show that serine proteases are the dominating proteins adsorbed onto annealed titanium (C1q = 67 +/- 4.6; pt/t = 97 +/- 0.2; fib = 47 +/- 0.2). The adsorption of serine proteases was lower and the amount of fibrinogen was higher on the acid-oxidized surface (C1q = 46.3 +/- 2.6; pt/t = 25 +/- 2.9; fib = 64 +/- 0.7). Platelets adhered and spread on the annealed titanium surface within 5 sec of blood-material contact. The number of adhering platelets was higher on the acid-oxidized surface.

Biocompatible Materials↗

PubMed1997

The initial reactions of graphite and gold with blood.

The initial reactions of graphite and gold with blood were investigated by short-time exposure to capillary blood and detection of surface-adsorbed plasma proteins and cells with an immunofluorescence technique. Antibodies specific to fibrinogen, complement factors C1q and C3c, prothrombin/thrombin, von Willebrand factor, and platelet- and leukocyte-membrane antigens were used. The fluorescence intensity was quantitated by computer-aided image analysis. Fibrinogen was the most abundant plasma protein immobilized on either surface, and dense populations of platelets adhered to the protein layer. Complement factors and prothrombin/thrombin were found on the graphite surface, localized in fibrin clots or related to platelets. Platelets were activated (expression of selectin CD62) on both surfaces but more extensively so on the gold surface. Activation of polymorphonuclear granulocytes (PMNGs), measured as expression of integrin CD11b, was seen on both surfaces but with different kinetics. On the graphite surface, the CD11b expression was only transient whereas on gold it increased with time. Our data indicate that graphite is more thrombogenic than gold but less inflammatory.

Adsorption↗

PubMed1997

Adhesion and activation of platelets and polymorphonuclear granulocyte cells at TiO2 surfaces.

The initial reactions of two TiO2 surfaces with blood were investigated by short-time exposure to capillary blood and analysis of surface-adsorbed plasma proteins and surface-adhering cells by using immunofluorescence techniques. Antibodies directed against platelet membrane antigen and P-selectin were used to visualize platelet adhesion and activation. Acridine orange and anti-CD11b were used to detect adhesion and activation of polymorphonuclear granulocytes (PMNs). Antibodies against thrombospondin were used as markers for platelet alpha-granules. The fluorescence intensity was quantitated by computer-aided image analysis. Commercially pure, polished sheet titanium was oxidized in two different ways: (1) the natural oxide was dissolved with hydrofluoric acid and a new oxide layer was grown by oxidation in nitric acid, or (2) annealing was performed at 700 degrees C in air. Auger electron spectroscopy and x-ray photoelectron spectroscopy showed that both surfaces had similar composition consisting of TiO2 covered by a carbonaceous surface contamination layer. The thickness of the oxide layer was 4 nm on the acid-oxidized surface and 39 nm on the annealed surface. Optical profilometry and scanning electron microscopy showed that the acid-oxidized surface was rough and the annealed surface was smooth. The fibrinogen/prothrombin-thrombin ratio in the initial protein film differed between the surfaces. The number of adhering platelets was larger at the surface with a high surface concentration of adsorbed fibrinogen. Platelet activation (CD62) and priming of PMNs (CD 11b) were also significantly higher on the acid-oxidized surface. The results indicate that non-self recognition of biomaterials is an array of transient reactions comprising protein-material, protein-cell, and cell-cell interactions.

Biocompatible Materials↗

PubMed1996

Attractive adsorbate interaction in biological surface reactions.

Recent studies on ferritin adsorption have revealed strong adsorbate interactions during adsorption (H. Nygren, Biophys. J., 65 (1993) 1508). In the present study, efforts were made to quantify the attractive lateral adsorbate interaction and to describe the effect of such interactions on the time- and concentration dependence of biological surface reactions. Attractive forces between ligands increase the time that they are considered neighbours to other ligands. Thus, the inter-ligand interaction effects their distribution and this can be measured experimentally by the pair correlation function g(r). The attractive lateral interaction was shown to affect the surface adsorption of ferritin, virus particles and bacteria, but played a minor role in the binding of antibodies to surface-immobilised antigen.

Adsorption↗

PubMed1996

Platelet binding and protein adsorption to titanium and gold after short time exposure to heparinized plasma and whole blood.

Protein adsorption from human plasma and platelet binding and activation were studied at short blood-titanium/gold contact times. The protein adsorption was studied by ellipsometry-antibody techniques in situ, and adhering platelets were visualized with fluorescein isothiocyanate-labelled anti-CD 61 antibodies. Adhering platelets were quantified by counting labelled cells in microscopic image fields. The spreading of platelets was studied by scanning electron microscopy. The results show that after 1 min of plasma exposure, fibrinogen, IgG and albumin were detectable with antibodies on both surfaces. The amount of deposited fibrinogen and complement decreased with time on titanium, and the amount of adsorbed anti-high molecular weight kininogen increased. No complement was detected on gold surfaces after plasma incubation, and the antibody binding pattern also remained unchanged after prolonged plasma exposure. The surface-bound platelets were found to spread on the gold but not on titanium surfaces. C1q has been shown to induce the expression of P-selectin, i.e. cause secretion reactions in platelets. In this study secreted platelet-microvesicles were found on gold, but not on the titanium surfaces that bound significant amounts of C1q. Thus, the results of the present study indicate that the mixture of fibrinogen, C1q and kininogens, whilst causing adhesion and aggregation, does not result in the activation and microvesicle secretion of platelets. Platelet activation on biomaterial surfaces thus seems to be governed by the mixture of proteins present on that surface, and no one particular protein need cause a known reaction in platelets as obtained when platelets are exposed only to that particular protein.

Adsorption↗