Possible explanation for the white band artifact seen in clinically retrieved polyethylene tibial components.

Studies have focused attention on the appearance of a subsurface white band in clinically retrieved polyethylene components and the possible contribution of this phenomenon to early polyethylene delamination. Unconsolidated polyethylene particles and oxidation have been suggested as possible reasons for the appearance of the white band. Calcium stearate and other additives used in processing ultra-high molecular weight polyethylene may also contribute to formation of the white band. A quantitative investigation was conducted on 11 retrieved tibial components that exhibited a subsurface white band to determine whether the amount of calcium stearate particles and additives were greater in the white band region when compared with the mid-portion of the same section of polyethylene. Calcium stearate particles and other additives were quantified using backscattered electron imaging with correlated elemental analysis. The particles were identified based on morphology and elemental patterns similar to reference calcium stearate particles and known additives. Significantly more (p < 0. 0001) calcium stearate particles and additives were present in the white band region (4578 +/- 418 particles/mm(2); mean +/- standard error) than the mid-portion region (1250 +/- 147 particles/mm(2)) of the sectioned tibial inserts. The percent area occupied by calcium stearate particles and additives was five times higher (p < 0.0001) within the white band region (0.81 +/- 0.10%) than the mid-portion region (0.16 +/- 0.03%). The increased presence of calcium stearate and other additives in the white band region suggests that they may play a role in the formation of the white band. In future investigations it may be important to consider how calcium stearate and other additives in polyethylene resins affect white band formation and the possible contribution to crazing, early delamination, and osteolysis in total joint replacement.

Elimination of the effects of stray light in measurements by total internal reflection aqueous fluorescence (TIRAF).

Total internal reflection aqueous fluorescence has been shown to be capable of achieving spatial resolution in surface contours of about 1 nm. When used with highly structured objects, errors in measurements can arise from light scattered either by the object or within the body of the microscope. We describe how these errors can be eliminated when studying surface contours of human platelets.

Nitric oxide donors inhibit platelet spreading on surfaces coated with fibrinogen but not with fibronectin.

We have studied the effect of nitric oxide (NO) on the interaction of washed human platelets with fibrinogen or fibronectin adsorbed on glass. Platelet contacts were visualized by interference reflection microscopy at 37 degrees C in Tyrode's solution. The areas of spread platelets were measured, using digital video image processing techniques, at times up to 30 minutes after initial surface platelet contact. On fibrinogen spreading was inhibited by NO donors in the order of potency: S-nitroso-acetylpenicillamine > sodium nitroprusside > S-nitroso-glutathione. The inhibitory action of NO donors was prevented by Oxy-haemoglobin, confirming that this inhibition was due to the release of NO. In contrast, NO donors had virtually no effect on platelet spreading on fibronectin. Platelet adhesion to fibrinogen is mediated by GP IIb IIIa and the vitronectin receptor (VNR), while that to fibronectin is via GP IIb IIIa, VNR, Ic IIa and VLA-6. Our results thus indicate that NO inhibits adhesion mediated by GP IIb IIIa and/or VNR but not by the two other receptors. The mechanism of this receptor-specific inhibition of platelet adhesion remains to be elucidated.

Adsorption of a novel fluorescent derivative of a poly(ethylene oxide)/poly(butylene oxide) block copolymer on octadecyl glass studied by total internal reflection fluorescence and interferometry.

We have used total internal reflection fluorescence (TIRF) to measure the adsorption kinetics of a newly synthesized fluorescent derivative of a triblock copolymer comprising two poly(ethylene oxide) arms connected by a poly(butylene oxide) segment. The composition is (EO)400 (BO)55 (EO)400, in which EO represents ethylene oxide, BO represents butylene oxide, and one or both of the terminal OH groups of the two (EO)400 arms are labeled with tetramethylrhodamine. The poly(butylene oxide) segment binds to hydrophobic octadecyl glass, used as a substratum. The TIRF signal is shown to be derived almost entirely from surface-adsorbed polymer. This facilitates calculation of adsorption isotherms from 0.1-0.005% bulk polymer solution by means of diffusion kinetics. Information about the effective thickness of the adsorbed polymer, determined by optical interference microscopy, corresponds with what is known about the conformation of similar molecules at interfaces and indicates monolayer adsorption on the glass.

Inhibition of platelet spreading from plasma onto glass by an adsorbed layer of a novel fluorescent-labeled poly(ethylene oxide)/poly(butylene oxide) block copolymer: characteristics of the exclusion zone probed by means of polystyrene beads and macromolecules.

We have investigated the anti-adhesive properties of a newly synthesized fluorescent triblock copolymer containing poly(ethylene oxide). This adsorbs from aqueous solution onto glass that has been rendered hydrophobic. When the polymer-treated surface was exposed to human platelet-rich plasma (PRP) or whole blood at 37 degrees C, platelet adhesion and spreading were prevented. Avid adhesion and rapid platelet spreading occurred along tracks scraped in the adsorbed polymer coating, as seen by video-enhanced interference reflection microscopy. Leukocytes from whole blood are eventually able to adhere to the polymer-treated surface and were seen to remove labeled polymer from their vicinity and accumulate it at the cell body. Interferometry using polystyrene spheres showed that they do not adhere to polymer-coated glass and are unable to approach closer than 70-95 nm. On scraped tracks, beads make molecular contacts with the glass. Because the fully extended solvated (EO)400 arms may extend up to 100 nm from the glass, this suggests that the polymer forms a monolayer with the hydrophilic arms projecting into the water, whereas the hydrophobic (BO)55 segment binds the molecule to the hydrophobic surface. Another tri-bloc copolymer with shorter hydrophilic arms allows particles to approach more closely.

Contact signalling and cell motility.

There is now clear evidence that signals can be generated when cells make adhesions. This happens when contacts are made with the extracellular matrix, with other cells or with experimental substrata. It is shown that intracellular signals are triggered in a variety of situations by the aggregation of transmembrane glycoproteins. Such aggregation occurs at adhesion sites, and it is suggested that adhesion molecules which are capable of lateral diffusion become trapped at developing adhesion sites because their headgroups bind to arrays of external ligands. Signals generated by molecular clustering can influence cell spreading and motility via familiar transduction pathways. Although it is not yet possible to reconstruct with confidence the entire sequence of events between initial glycoprotein clustering and motility, there is a lot of information which points to the identities of the molecular ingredients. I shall focus the discussion on the thin peripheral lamellar extensions produced by cell types ranging from amoebae to vertebrate nerve growth cones, and discuss the experimental evidence relating to their cytoskeletal architecture and dynamics. Finally I shall try to construct models of motility consistent with the experimental data, and suggest how lamellar motility may be modulated by signals generated by clustered adhesion molecules.

Contact-mediated triggering of lamella formation by Dictyostelium amoebae on solid surfaces.

Amoebae of the slime mould Dictyostelium discoideum form broad ultrathin cytoplasmic lamellae by a centripetal contractile process soon after they have spread on certain solid surfaces. We have investigated the surface requirements for initial triggering of this contact-mediated signalling system. The lamellar response is not normally evoked by glass, but is seen on glass covalently derivatized with paraffinic chains, as well as on glass covalently derivatized with amine groups and on glass bearing adsorbed polylysine. We have recorded the frequency of the lamellar response on these surfaces as a function of ionic strength and pH, and have measured the electrostatic potentials of the surfaces by the streaming potential method. Using these data we have concluded that the general trigger for the lamellar response is not a 'simple' physical or chemical property of the substrata: it is not dependent on specific chemical groups, degree of hydrophobicity, electrostatic potential, or charge density, taken as isolated factors. It seems likely that triggering is dependent on the overall energetics of cell-substratum interaction.

Cell adhesion to hydroxyl groups of a monolayer film.

We have studied cells on chemically defined monomolecular films of the long-chain alcohol docosanol. Langmuir-Blodgett films of the alcohol were deposited on glass coverslips, previously made hydrophobic with octadecyl groups. This gives films in which the alcohol headgroups face outwards to the water. Molecular orientation and film integrity were shown by a fluorescence adsorption test. Cell contacts on the films were observed in media without proteins by interference reflection microscopy (IRM) and the mechanics of detachment were examined by hydrodynamic shearing in a flow chamber. Cell contact with docosanol was compared with that on an adjacent area of octadecyl glass without a monolayer. Dictyostelium amoebae settled and spread on both docosanol and octadecyl glass, but little or no locomotion was seen on docosanol. On octadecyl glass the amoebae moved actively, forming ultrathin cytoplasmic lamellae, which look dark under IRM, and left distinctive trails of membranous debris. Hydrodynamic shearing showed that the amoebae stuck strongly to both surfaces and could not be removed from either at the maximum attainable wall shear stress of 6Nm-2. Red blood cells also adhered to both surfaces and removal from both occurred between 1 and 3Nm-2. IRM and scanning electron microscopy (SEM) studies indicated that this force leads to a minimal measure of red cell adhesion, since removal often involved the breakage of cytoplasmic tethers. Our results show that alcoholic -OH groups, in a two-dimensional array, provide a surface that is strongly adhesive for cells. No other method has made it possible to demonstrate cell adhesion purely to -OH groups, in a known orientation and density, and in the absence of any other functional groups on the interface.

Contacts of chick fibroblasts on glass: results and limitations of quantitative interferometry.

We have examined the contacts made by explanted chick heart and limb bud fibroblasts after 24-48 h on glass, using quantitative interference reflection microscopy (IRM). Contacts beneath very thin cytoplasmic lamellae were avoided because the images of such contacts depend on the thickness of the lamellae. Plaque-like focal contacts, distinguished on the basis of shape and low irradiance (darkness), are intimate adhesions to the substratum. These images can be interpreted if it is assumed that microfilaments associated with the lower membrane increase the local cytoplasmic refractive index. The range of irradiances measured for focal contacts was found to be rather wide, and our modelling shows that the most likely explanation for this is that the images receive variable contributions from the adjacent cytoskeleton. For this reason it is particularly difficult to assign a characteristic thickness for these contacts from IRM data. Close contacts, seen principally as 'grey' regions under migrating cells at the edges of the explants, also show a wide range of irradiances. Unlike focal contacts, it is not necessary to postulate any involvement of the cytoskeleton in their images and they can be modelled as regions where an aqueous glycocalyx zone about 20-30 nm thick separates the membrane bilayer from the glass. Paler grey regions that also look like close contacts are apparently formed where the cell surface has lifted several tens of nanometres from the glass.

Mapping cell-glass contacts of Dictyostelium amoebae by total internal reflection aqueous fluorescence overcomes a basic ambiguity of interference reflection microscopy.

The widespread ability of eukaryotic cells to produce thin cytoplasmic sheets or lamellae 100-200 nm thick can give rise to uncertainties in the interpretation of interference reflection microscopy (IRM) images when cell-substratum topography is the key interest. If allowed to spread upon a poly-L-lysine-coated surface, Dictyostelium discoideum amoebae typically form ultrathin lamellae of approximately equal to 100 nm thickness by cytoplasmic retraction. Whereas the cell body is grey, the lamellae appear very dark under IRM optics. These dark areas could be misinterpreted as stemming from a closer cell-substratum apposition beneath the lamellae than the cell body. This ambiguity can be avoided if the technique of total internal reflection aqueous fluorescence (TIRAF) is used in conjunction with a high refractive index glass (n = 1.83) as substratum. Contributions to the image generated by thin cytoplasm and also variable cytoplasmic refractive index are thereby minimized due to the extremely short range of the 'illuminating' evanescent wave. From our comparative IRM and TIRAF study of the ultrathin lamellae of Dictyostelium amoebae it is concluded that the cell-glass gap is relatively uniform beneath the entire cell. We briefly discuss the sensitivity of several cell types to TIRAF, the generation of ultrathin lamellae and the nature of the cell-glass gap.

General electromagnetic theory of total internal reflection fluorescence: the quantitative basis for mapping cell-substratum topography.

Total internal reflection fluorescence (TIRF) has recently been used to look at the contacts made between cells and a glass surface on which they are spread. Our method utilizes the fluorescence of a water-soluble dye that acts as an extracellular aqueous volume marker. Fluorescence is stimulated by the short-range electric field near the glass surface that exists under conditions of total internal reflection. Since fluorescence is normally generated beneath a spread cell and not beyond it, the fluorescence of the image is related to the size of the cell-glass water gap. The images obtained are remarkable for their detail, contrast and the absence of confusing granularity due to cytoplasmic heterogeneity, which is commonly seen in interference reflection (IRM) images. We here develop a rigorous electromagnetic theory of total internal reflection in layered structures appropriate for cell contacts and apply it to quantitative TIRF. We show that: (1) TIRF, unlike IRM, can report cell-glass gaps in a way that is practically independent of the detailed physical properties of the cell; (2) TIRF is also far more sensitive than IRM for measuring cell-glass water gaps up to approximately equal to 100nm. These striking results explain the image quality seen by TIRF. As the initial step towards verifying our theory we show that measurement of the fluorescence stimulated by total internal reflection at a simple glass-water interface matches theoretical predictions.

Inhibition of cell adhesion by a synthetic polymer adsorbed to glass shown under defined hydrodynamic stress.

A co-polymer with hydrophobic and hydrophilic segments was allowed to adsorb from aqueous solution onto glass previously made hydrophobic by derivatization with octadecyl dimethylchlorosilane. The polymer is thought to adsorb via its hydrophobic segments, leaving the hydrophilic segments free to extend into the water. After allowing cells to settle on the treated surface, the shear stress at the chamber wall required to remove red blood cells, Dictyostelium discoideum amoebae and Escherichia coli was determined in a calibrated laminar flow chamber. On octadecyl glass a shear stress of 2-3 Nm-2 evicts 50% of adherent red cells and E. coli. No D. discoideum amoebae could be removed at 5Nm-2. In striking contrast, the lowest experimentally obtainable shear stress of 0.03 Nm-2 removes 97.0-99.5% of cells of all three types from the polymer-treated surface, even after a cell residence time of 1 h without flow in the absence of free polymer. The minimum shear stress of 0.03Nm-2 corresponds to only approximately equal to 20 times the force of gravity on a red cell. The mechanism of action of the polymer and the implications of the results are discussed.

Direct measurement of cell detachment force on single cells using a new electromechanical method.

We describe a new device in which an accurately measured force is applied to individual adherent cells while the topography of the adhesion zone is simultaneously monitored. The force is applied via a flexible glass micropipette, attached by suction to the cell under study, and is calculated directly from the measured pipette deflection. Regions of close contact in the adhesion zone are observed using interference reflection microscopy. We have used the device to measure the force required to detach human red blood cells from hydrophobic and hydrophilic glass surfaces, and to detach Dictyostelium discoideum amoebae from a hydrophobic glass surface. The measured forces per unit length of contact perimeter are within an order of magnitude of the tensions required for membrane rupture.

Red blood cells experience electrostatic repulsion but make molecular adhesions with glass.

We have studied the detachment of unfixed red cells from glass coverslips under unit gravity and by centrifugation in buffered isotonic solutions over a range of ionic strengths. Cell-glass contact areas and separation distances were measured by quantitative interference reflection microscopy. Detachment under unit gravity is highly dependent on ionic strength: dilution increases electrostatic repulsion and greatly reduces the proportion of adherent cells. However, even at 1.5 mM some cells stick. Over the range 3-110 mM such adherent cells are progressively removed by increasing centrifugal forces, but in a manner virtually independent of ionic strength. This fact, together with the irreversibility of pre-adherent cells as ionic strength is progressively reduced, as well as the resistance of cells to lateral shearing forces, provide evidence sufficient to reject the notion of secondary minimum adhesion for unfixed cells at any ionic strength down to 1.5 mM. We conclude that all unfixed cells that stick at ionic strengths from 157 to 1.5 mM make molecular contacts with glass. Comparison with long range force calculations suggests that to penetrate the electrostatic repulsion barrier the contact regions are unlikely to have average surface properties. A new method that compares frequency distributions of contact areas with responses to detachment forces shows that detachment forces are not linearly related to contact areas. This lack of relationship is less clearly evident for rigid glutaraldehyde-fixed cells and may therefore depend on the degree of cellular deformability.

Topography of cell-glass apposition revealed by total internal reflection fluorescence of volume markers.

We have developed a new method based on total internal reflection fluorescence to map the shape of the region between glass and the lower surface of a living cell spread upon it. Fluorescently labeled nonadsorbing volume marker molecules that cannot penetrate into the cell are locally stimulated so that they fluoresce only very near the glass/medium interface. The total fluorescence intensity at any point beneath the cell depends on the cell-to-glass separation. Focal contacts appear as dark areas owing to dye exclusion, whereas when the gap exceeds approximately 150 nm, fluorescence asymptotes to the bright background level. Our technique provides greater contrast than does interference reflection microscopy and is free from errors due to cytoplasmic thickness and refractive index inhomogeneities arising from cytoplasmic inclusions. We have shown that sufficiently large molecules suffer steric exclusion from regions accessible to small molecules, which gives new information about lateral penetrability in the apposition region.

Lessons for the study of membrane fusion from membrane interactions in phospholipid systems.

'Fusion' in model systems usually refers to the decay of membrane configurations that are inherently unstable because of the method of preparation. Natural fusion is a controlled event during which the underlying forces and instabilities are subject to the additional effects of biochemical reactions. To understand biological fusion one must be able first to assess the interplay among these physical and chemical factors. This paper reviews traditional measurements of electrostatic double layer and electrodynamic van der Waals forces acting between bilayer membranes. It also describes the much stronger hydration forces that have now been systematically studied. An essential part of any fusion event is the ability of membrane surfaces to overcome or circumvent the hydration barrier in order to make contact. This may be accomplished through applied force, through bridging substances that displace water from the membrane surface, or through biochemical modification of surfaces. In model systems, destruction of the hydration layer can cause violent adhesion, membrane deformation, and rupture. Natural fusion proceeds by more subtle processes whereby interfacial forces are harnessed in ways not yet understood.

Conformational response of the glycocalyx to ionic strength and interaction with modified glass surfaces: study of live red cells by interferometry.

We have measured separation distances between live human red blood cells and simple or modified glass surfaces, using the finite aperture technique of microscope interferometry. In general, separation increases as the ionic strength falls, in isotonic solutions. Restriction on movement parallel to the glass in all except the most dilute salt solutions, coupled with the absence of Brownian motion, indicates direct molecular contact with the substratum. Thus increased separation must be due to swelling of the glycocalyx under electrostatic forces. However, at approximately less than to 2mM adherent cells show a separation greater than 100 nm, execute Brownian motion and the restriction on lateral motion is less evident. This suggests that secondary minimum adhesion by long-range forces with little or no direct molecular connection occurs at extreme dilution only. Treatment of cells with trypsin reduces separation by up to 40 nm, but the extent to which this reflects reduced double-layer repulsion due to loss of surface charge, as opposed to the reduced opportunity for swelling in a trimmed-down glycocalyx, is unclear. Adhesion at a separation approximately 100 nm in 1 mM buffer after trypsinization supports the view that adhesion can occur without very long glycoprotein connections, but does not prove it. Adhesion to unwettable methylated glass and completely wettable unmethylated glass, with an identical ionic strength dependence of the separation, shows that hydrophilicity is not an absolute requirement. Red cells interact closely at all ionic strengths with glass made polycationic with poly-L-lysine, owing to electrostatic attraction. The interference technique also shows that adherent cells can be spaced from the glass by an intervening layer of previously absorbed serum albumin.