Cecilia Eriksson

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.

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.

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.

A cell preparation method allowing subcellular localization of cholesterol and phosphocholine with imaging TOF-SIMS

Imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used for characterization and subcellular localization of membrane lipids in leukocytes adhering to glass surfaces. The cells were prepared by freeze drying in 0.15 M ammonium formiate at pH 7.2–7.4. Imprints of the cells were made on silver foil, and the silver surface was analyzed by 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 was collected separately at high mass resolution m/Δm>7000 and at high lateral resolution (≥0.5 μm). This cell preparation procedure allows localization of cholesterol and phosphocholine in surface-adhering cells.

Time-of-flight secondary ion mass spectrometric analysis of the interface between bone and titanium implants

Implant healing into bone tissue is a process where the mature bone grows towards and eventually fuses with the implant. In this study we investigated implant healing during 4 weeks with focus on the implant-tissue interface. Our main interest was to study the mineralization process around the implant. Titanium discs were implanted in rat tibia for 2 and 4 weeks. After implantation cross sections of bone and implant were made using a low-speed saw equipped with a diamond wafering blade. One section from each sample was stained with basic fuchsin and micrographed by light microscopy (LM). The other section was analyzed with imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) using a Bi(3)(+) cluster ion source. This ion source has recently been shown to enable identification of high-mass hydroxyapatite (HA) fragment ions (m/z 291-653) in bone samples. The LM images were used to identify areas suitable for TOF-SIMS analysis. Three areas were selected for mass spectral analysis, corresponding to interface region, bone and soft tissue, from which positive ion spectra were recorded. In the areas identified as bone, high-mass HA fragments ions were found after both 2 and 4 weeks. In the soft tissue area, no high-mass ions were found after 4 weeks. However, after 2 weeks HA-related ions were identified in mineralized spots in areas defined as soft tissue. After 4 but not after 2 weeks, high-mass HA fragment ions were found in the interface region. In conclusion, differences were observed regarding mineralization between 2 and 4 weeks of implantation and between different regions surrounding the implants. Imaging TOF-SIMS analysis using a Bi(3)(+) cluster as ion source enables identification of high-mass HA fragment ions at implant-tissue interfaces in bone. This technique might therefore be useful for biocompatibility assessment and for studying the mineralization process at implant surfaces.

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.

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.

REACTIONS OF TWO HYDROPHILIC SURFACES WITH DETERGENTS, PROTEIN AND WHOLE HUMAN BLOOD

Abstract The surface chemistry of moscovite mica and hydrophilic glass was characterized by ESCA. The adsorption of detergents and lysozyme was measured by surface force methods, ellipsometry or ESCA. The results show that the protein has a higher affinity to the mica surface. A biological characterization of the surfaces was performed by exposure to whole blood, followed by detection of surface-adsorbed plasma proteins and cellular antigens by immunofluorescence. The brightness of the fluorescence was measured by computer-aided image analysis. The results show that hydrophilic glass was the most efficient activator of serine protease cascade enzymes. Platelets were activated at both surfaces, but were shown to leave the glass surface with time. The glass surface was more inflammatogenic than the mica surface as determined by the CD11b-integrin expression of PMN-cells. The results indicate that the nonself recognition of the surfaces is an array of subreactions, the kinetics of which determine the outcome of the blood-material contact.

Dental plaque pH and ureolytic activity in children and adults of a low caries population

Abstract Objectives. The aim of this study was to evaluate the plaque pH level and ureolytic activity among children and adults of Karen Hill tribes. Methods. Thirty-four children aged 6–10 years and 46 adults aged 20–38 years were interviewed regarding oral hygiene practices, sucrose intake and betel chewing. Caries experience (DMFT and DT), calculus, bleeding on probing (BoP) and Plaque index (PlI) were registered. Ureolytic activity in supragingival plaque was tested at two interproximal sites (11/12 and 41/42) with the rapid urease test (RUT). Registration of plaque pH was performed at two interproximal sites (15/16 and 31/41) before, during and 30 min after rinsing with an urea solution (0.25%). Four interproximal plaque samples (one from each quadrant) per individual were collected to test the bacterial composition using the checkerboard technique. Results. Children and adults had similarly low DMFT and DT values. Children had a higher baseline pH and a higher ureolytic activity in the maxilla (p < 0.05) compared with adults. A significant correlation (r 2 = 0.63) was found between baseline pH and urease activity in the mandibular anterior teeth. Caries-free individuals had a higher baseline pH compared with caries active individuals in the anterior mandibular region (p < 0.01). The microbiological composition was characterized by an anaerobic low acidiogenic microbiota. Conclusions. Dental plaque pH is related to the ureolytic activity, which explains the low acidogenic plaque microflora and the low caries levels in the Karen population.

Experimental approach to a biological characterization of materials

Titanium oxide surfaces with thin (4 to 5 nm) or thick (30–40 nm) TiO2 layers and rough (Rrms 1.9 μm) or smooth (Rrms 0.45 μm) surface structure were exposed to capillary blood for 5 s through 64 min. The adsorption of main plasma proteins (albumin, IgG, fibrinogen), binding of cascade enzymes (serine proteases) and adhesion and activation of blood cells was measured with immunofluorescence techniques using specific antibodies, the binding of which was quantitated by computer-aided image analysis. The activation of cascade enzymes was detected with antibodies against prothrombin/thrombin (terminal step in the coagulation cascade), C1q and C3c (initial complement activation), terminal complement complex (TCC), and plasmin (terminal step in fibrinolysis). The activation of cells was measured by expression of selectin (platelet CD62), expression of integrins (neutrophil CD11b), or respiratory burst (NBT test) of peritoneal leucocytes adhering to the surfaces after in vivo exposure. The results show different...