Stimulation of monocytes and macrophages: possible influence of surface roughness.

The aim of this study was to understand the mechanisms of interaction of monocytes/macrophages and foreign body giant cell (FBGC) with implant materials, with respect to the roughness and the solubility of calcium phosphate based coatings. Anderson et al. (Bone Engineering, J.E. Davies, ed., Toronto, 2000, pp. 81-93) showed that the presence of FBGC's and monocytes/macrophages influenced the strength of the implant-tissue integration and that more monocytes/macrophages rested on smooth surfaces compared to rough surfaces. We seeded human bone marrow cells on uncoated ultrasmooth polished TiAl6V4 samples as well as on coated TiAl6V4 discs of the same diameter with two different calcium phosphates coatings, monetite (DCP) and hydroxyapatite (OHAp), both with rougher surfaces. On uncoated ultrasmooth polished TiAl6V4 discs (UUTi, diameter 16 mm, thickness 2 mm) and on TiAl6V4 discs of same diameter coated with OHAP or DCPA, human bone marrow cells (HMBC) were seeded and cultivated under standard culture conditions for 90 days without addition of inducing substances like ascorbic acid, Na-beta-glycerophosphate or dexamethasone. The roughnesses of the virgin samples were assessed with atomic force microscopy and light profilometry. After 90 days of cultivation a fraction of the samples, with cells and extracellular matrix, were stained with hematoxylin eosin (HE) and examined in light microscopy. R(a) roughness values of virgin uncoated TiAl6V4 samples were 0.001 microm, of DCP coated discs 4 microm and of OHAp coated discs 3 microm. The examination of HE stained samples showed a high number of FBGC and monocytes/macrophages on the UUTi samples. On the DCP coated samples there were less FBGC and monocytes/macrophages and on the OHAp coated samples we could not find any FBGC and monocytes/macrophages. The extracellular matrix (ECM) we found on the UUTi samples was finer and thinner than on the coated samples. The ECM was vastly spread and not dense on the UUTi samples in contrast to the calcium phosphate coated samples, where the ECM was much thicker and stronger. The ultrasmooth surface of the uncoated TiAl6V4 samples, a material which is accepted to be biocompatible, evidently induced the differentiation of cells of the monocytic lineage and the formation of FBGC out of the cell populations present in the human bone marrow.

Radiographic contrast media alterate the localization of actin/band4.9 in the membrane cytoskeleton of human erythrocytes.

Different radiographic contrast media (RCM) were shown to induce morphological changes of blood cells (e.g. erythrocytes or thrombocytes) and endothelial cells. The echinocytic shape change of erythrocytes, particularly, affords alterations of the membrane cytoskeleton. The cytoskeleton plays a crucial role for the shape and deformability of the red blood cell. Disruption of the interaction between components of the red blood cell membrane cytoskeleton may cause a loss of structural and functional integrity of the membrane. In this study band4.9 and actin as components of the cytoskeletal junctional complex were examined in human erythrocytes after suspension in autologous plasma or in plasma RCM mixtures (30% v/v Iodixanol-320 or Iopromide-370) followed by a successive double staining with TRITC-/FITC-coupled monoclonal antibodies. After adding Iopromide-370 to the plasma in practically none of the cells the rounded conformation of the membrane cytoskeleton - as it appeared in cells suspended in autologous plasma - was found. In addition, Iopromide-370 induced thin lines and coarse knob-like structures of band4.9 at the cell periphery while most cell centers were devoid of band4.9, and a box-like arrangement of bands of band4.9. A dissociation between colours red (actin) and green (band4.9) occurred as well. In contrast, erythrocytes suspended in a plasma/Iodixanol-320 mixture showed a membrane cytoskeleton comparable to cells suspended in autologous plasma, Similar results were found with respect to the distribution of actin. This study revealed for the first time RCM-dependent differences in band4.9 activities as possible pathophysiological mechanism for the chemotoxicity of radiographic contrast media.

Effect of radiographic contrast media (Iodixanol, Iopromide) on the spectrin/actin-network of the membranous cytoskeleton of erythrocytes.

Red blood cells demonstrate a unique ability for repeated large deformation. Under the influence of a variety of agents, shapes other than the discocyte--e.g. stomatocytes or echinocytes--can be observed. Some radiographic agents induce shape changes from discocytic to echinocytic cells. Especially the echinocyte formation is associated with a rigidification of the cells bearing the risk of a hindered capillary passage of the echinocytes. The mechanisms leading to the formation of echinocytes are not well understood assuming that the membrane cytoskeleton is a key player. That is why this examination was focused on the participation of components of the membrane cytoskeleton in the formation of echinocytes and the protrusions accompanying the formation of echinocytes. Two radiographic contrast media approved for intra-arterial application were used to study echinocyte formation (Iodixanol320; Iopromide370). In the in vitro study serious changes in the membrane cytoskeleton were only found in those erythrocytes incubated in plasma supplemented with Iopromide370 (30%v/v). The shape of the spectrin net was completely altered; from the more homogeneous distribution--typical of cells in autologous plasma and also of cells in plasma supplemented with Iodixanol320--to a distribution of spectrin concentrated in the membrane-near regions with the appearance of spectrin-actin co-localization. Co-localized spectrin with actin was also found around the membranous roots of protrusions which resemble exocytotic processes. In central parts of the cells there was a pronounced dissociation of spectrin and actin; green coloured condensed spectrin bundles originating from the cell membrane reached up to the root of the protrusions. Separate from this there were also fine long actin fibres passing through the whole cell. The incubation of erythrocytes in plasma supplemented with Iopromide370 induced rounded bubble-like protrusions from the cell membrane containing almost completely long bundles of actin fibres. The examination confirmed earlier studies showing that some radiographic contrast media are able to induce echinocyte formation. Furthermore, subcellular mechanisms were revealed explaining the different effects of Iodixanol in comparison to Iopromide.

Distribution of actin of the human erythrocyte membrane cytoskeleton after interaction with radiographic contrast media.

A type-dependent chemotoxic effect of radiographic contrast media on erythrocytes and endothelial cells was reported several times. While mechanisms of toxicity are still unclear the cellular reactions e.g. echinocyte formation in erythrocytes and the buckling of endothelial cells coincided with deterioration of capillary perfusion (in patients with coronary artery disease) and tissue oxygen tension (in the myocardium of pigs). Whether the shape changes in erythrocytes coincide with changes in the arrangement of actin, the core of the actin-spectrin cytoskeletal network and possible actor in membrane stresses and deformation is not known until now. To get specific informations actin was stained using two different staining methods (antibodies to ß-actin staining oligomeric G-actin and polymeric F-actin and Phalloidin-Rhodamin staining polymeric F-actin only). In addition, an advanced version of confocal laser scanning microscopes was used enabling the display of the actin arrangement near substrate surfaces. Blood smears were produced after erythrocyte suspension in autologous plasma or in two different plasma/RCM mixtures. In this study an even homogenous distribution of fine grained globular actin in the normal human erythrocyte could be demonstrated. After suspension of erythrocytes in a plasma/Iodixanol mixture an increased number of membrane protrusions appeared densely filled with intensely stained actin similar to cells suspended in autologous plasma, however, there in less numbers. Suspension in Iopromide, in contrast, induced a complete reorganization of the cytoskeletal actin: the fine grained globular actin distribution disappeared and only few, long and thick actin filaments bundled and possibly polymerized appeared, instead, shown here for the first time.

Monoclonal antibody to polycyclic aromatic hydrocarbons based on a new benzo[a]pyrene immunogen.

Benzo[a]pyrenebutyric acid (B[a]PBA) has been synthesized and covalently coupled to bovine serum albumin to generate monoclonal antibodies (Mab). A competitive indirect enzyme-linked immunosorbent assay (ELISA) for polycyclic aromatic hydrocarbons (PAH) has been developed with Mab B[a]P-13. It was shown by testing with 21 parent PAH and 10 compounds carrying methyl, hydroxy, or butyric acid functions that the antibody had broad specificity. Highest affinity was found for four- to six-ring PAH. Different organic co-solvents were tested. No loss in sensitivity, compared with controls in PBS, were found with methanol, dimethyl sulfoxide, and glycerol at final concentrations of 5 to 10%. Further, an observation was made that a modification (fine-tuning) of the affinity and specificity of the antibodies was possible by changing the type of the added organic co-solvent. The high susceptibility of the ELISA with regard to inorganic ions might be an indication of a more hydrophilic binding pocket e.g. involving a pi-cation interaction. Investigation of the effect of pH revealed that for pH between 6 to 9 there was no noticeable impairment. With an LOD as low as 30 pg per well for B[a]P the sensitivity of the ELISA is sufficient for analyses of solvent extracts of many environmental samples. As an example, the determination of a PAH sum parameter, given as B[a]P-equivalents, in crude aerosol extracts by both ELISA and HPLC revealed good correlation (r2=0.717) but approximately five-fold overestimation by the immunochemical method, obviously as a result of cross-reacting analytes.