Spermidine-Induced Attraction of Like-Charged Surfaces Is Correlated with the pH-Dependent Spermidine Charge: Force Spectroscopy Characterization.

The ubiquitous molecule spermidine is known for its pivotal roles in the contact mediation, fusion, and reorganization of biological membranes and DNA. In our model system, borosilicate beads were attached to atomic force microscopy cantilevers and used to probe mica surfaces to study the details of the spermidine-induced attractions. The negative surface charges of both materials were largely constant over the measured pH range of pH 7.8 to 12. The repulsion observed between the surfaces turned into attraction after the addition of spermidine. The attractive force was correlated with the degree of spermidine protonation, which changed from +3 to +1 over the measured pH range. The force was maximal at pH 7.8. To explain the observed pH and spermidine concentration dependence, two different theoretical approaches were used: a chemical model of the charge equilibrium of spermidine and Monte-Carlo simulations of the orientation of the rodlike spermidine molecules in the gap between the borosilicate and mica surfaces. Monte-Carlo simulations of the orientational ordering of the rodlike spermidine molecules suggested the induction of attractive interactions between the surfaces if the gap was bridged by the molecules. For larger gaps, the orientational distribution function of the spermidine molecules predicted a considerable degree of parallel attachment of the molecules to the surfaces, resulting in reduced effective surface charge densities of both surfaces, which reduced their electrostatic repulsion.

Cellular Nutrition in Complex Three-Dimensional Scaffolds: A Comparison between Experiments and Computer Simulations.

Studies on bone cell ingrowth into synthetic, porous three-dimensional (3D) implants showed difficulties arising from impaired cellular proliferation and differentiation in the core region of these scaffolds with increasing scaffold volume in vitro. Therefore, we developed an in vitro perfusion cell culture module, which allows the analysis of cells in the interior of scaffolds under different medium flow rates. For each flow rate the cell viability was measured and compared with results from computer simulations that predict the local oxygen supply and shear stress inside the scaffold based on the finite element method. We found that the local cell viability correlates with the local oxygen concentration and the local shear stress. On the one hand the oxygen supply of the cells in the core becomes optimal with a higher perfusion flow. On the other hand shear stress caused by high flow rates impedes cell vitality, especially at the surface of the scaffold. Our results demonstrate that both parameters must be considered to derive an optimal nutrient flow rate.

Low fibronectin concentration overcompensates for reduced initial fibroblasts adhesion to a nanoscale topography: single-cell force spectroscopy.

Using single-cell force spectroscopy, we compared the initial adhesion of L929 fibroblasts to planar and nanostructured silicon substrates as a function of fibronectin concentration. The nanostructures were periodically grooved with a symmetric groove-summit period of 180 nm and a groove depth of 120 nm. Cell adhesion strength to the bare nanostructure was lower (79%± 13%) than to the planar substrate, which we attribute to reduced contact area. After pre-incubation with a low fibronectin concentration (5 µg/ml) the adhesion strengths to both surfaces increased, with adhesion strength on the nanostructure outweighing that of the planar substrate by 133%± 14%. At a high fibronectin concentration (25 µg/ml) the adhesion strengths on both surfaces further increased and showed wide variations. In parallel, the nanostructure lost its clear advantage over the planar substrate. Our results demonstrate that cell adhesion is influenced by substrate topography and fibronectin, which mediate the interplay between specific interactions, non-specific interactions, and cell mechanics. Two parallel processes govern the initial adhesion strength: the detachment of the cell body from the substrate and the extraction of tethers from the cell membrane. The duration of the latter process is determined by tether lifetimes, and is a major contributor to the overall work required for cell-substrate detachment. Cell body detachment and tether lifetimes are affected by surface topography and may be strongly modulated by the presence of adsorbed proteins, whereas the tether extraction forces remained unchanged by these factors.

Atomic force microscopy studies of the influence of convex and concave nanostructures on the adsorption of fibronectin.

Atomic force microscopy (AFM)-based force spectroscopy was used to analyze the adsorption of bovine plasma fibronectin on periodically grooved nanostructures (groove/summit width: 90 nm; depth: 120 nm). We present a simple procedure that allowed us to directly compare the local protein density and conformation for the convex summits, the concave grooves and planar reference regions of the substrate. At a bulk fibronectin concentration of 5 µg/ml, the amount of adsorbed protein per surface area was significantly higher in all regions of the nanostructure than on the planar reference, and fibronectin tended to adsorb preferentially in the concave grooves. The increased surface concentration resulted in an additional stabilization of the molecules by protein-protein interactions and a lower degree of denaturized fibronectin in the nanostructured regions. The stabilization was less pronounced in concave regions, indicating that the increased contact area in the grooves counteracted the stabilization by increased protein-substrate interactions and must be compensated for by additional protein-protein interactions. Less favorable sites were occupied at higher bulk fibronectin concentrations (25 µg/ml, 100 µg/ml), and a high degree of native folded fibronectin was observed in both the nanostructured and planar regions. Our results demonstrate that the amount of adsorbed fibronectin per surface area can be increased if a substrate is provided with a topographic nanostructure. Our results also show that the local conformational state of fibronectin is determined by the locally different interplay of protein-protein and protein-substrate interactions.

Hard implant coatings with antimicrobial properties.

Infection of orthopaedic implants often leads to inflammation immediately after surgery and increases patient morbidity due to repetitive operations. Silver ions have been shown to combine good biocompatibility with a low risk of inducing bacterial resistance. In this study a physical vapour deposition system using both arc deposition and magnetron sputtering has been utilized to produce silver ion doped TiN coatings on Ti substrates. This biphasic system combines the advantages of silver induced bactericidity with the good mechanical properties of TiN. Crystallographic analysis by X-ray diffraction showed that silver was deposited as well in its elementary form as it was incorporated into the crystal lattice of TiN, which resulted in increasing hardness of the TiN-coatings. Elution experiments revealed a continuous release of Ag ions in phosphate buffered saline. The coatings showed significant inhibitory effects on the growth of Staphylococcus epidermidis and Staphylococcus aureus and practically no cell-toxicity in cytocompatibility tests.

Electrostatic and dispersion interactions during protein adsorption on topographic nanostructures.

Recently, biomaterials research has focused on developing functional implant surfaces with well-defined topographic nanostructures in order to influence protein adsorption and cellular behavior. To enhance our understanding of how proteins interact with such surfaces, we analyze the adsorption of lysozyme on an oppositely charged nanostructure using a computer simulation. We present an algorithm that combines simulated Brownian dynamics with numerical field calculation methods to predict the preferred adsorption sites for arbitrarily shaped substrates. Either proteins can be immobilized at their initial adsorption sites or surface diffusion can be considered. Interactions are analyzed on the basis of Derjaguin-Landau-Verway-Overbeek (DLVO) theory, including electrostatic and London dispersion forces, and numerical solutions are derived using the Poisson-Boltzmann and Hamaker equations. Our calculations show that for a grooved nanostructure (i.e., groove and plateau width 8 nm, height 4 nm), proteins first contact the substrate primarily near convex edges because of better geometric accessibility and increased electric field strengths. Subsequently, molecules migrate by surface diffusion into grooves and concave corners, where short-range dispersion interactions are maximized. In equilibrium, this mechanism leads to an increased surface protein concentration in the grooves, demonstrating that the total amount of protein per surface area can be increased if substrates have concave nanostructures.

The influence of topographic microstructures on the initial adhesion of L929 fibroblasts studied by single-cell force spectroscopy.

Single-cell force spectroscopy was used to investigate the initial adhesion of L929 fibroblasts onto periodically grooved titanium microstructures (height ~6 µm, groove width 20 µm). The position-dependent local adhesion strength of the cells was correlated with their rheological behavior. Spherical cells exhibited a significantly lower Young's modulus (<1 kPa) than that reported for spread cells, and their elastic properties can roughly be explained by the Hertz model for an elastic sphere. While in contact with the planar regions of the substrate, the cells started to adapt their shape through slight ventral flattening. The process was found to be independent of the applied contact force for values between 100 and 1,000 pN. The degree of flattening correlated with the adhesion strength during the first 60 s. Adhesion strength can be described by fast exponential kinetics as C1[1-exp(-C2·t] with C1 = 2.34 ± 0.19 nN and C2 = 0.09 ± 0.02 s⁻¹. A significant drop in the adhesion strength of up to 50% was found near the groove edges. The effect can be interpreted by the geometric decrease of the contact area, which indicates the inability of the fibroblasts to adapt to the shape of the substrate. Our results explain the role of the substrate's topography in contact guidance and suggest that rheological cell properties must be considered in cell adhesion modeling.

Nanoscaled periodic surface structures of medical stainless steel and their effect on osteoblast cells.

Nanoscaled lamellar surface structures have been prepared on medical stainless steel AISI 316LVM surfaces by chemical etching of the decomposed phases. The effect of this structure on osteoblastic cells has been investigated. Long filopodia were developed by the cells perpendicular to the lamellar structure while almost no or only short filopodia were formed parallel to the lamellae. These results are explained in terms of a topographical influence of the nanostructure. During the growth process of the filopodia a nearly flat surface was recognized parallel to the lamellae while a topographical change was sensed perpendicular to the structure, which was preferred by the cells.