Ulrich Beck

Cell architecture-cell function dependencies on titanium arrays with regular geometry

Knowledge about biocomplexity of cell behavior in dependence on topographical characteristics is of clinical relevance for the development of implant designs in tissue engineering. The aim of this study was to find out cell architecture-cell function dependencies of human MG-63 osteoblasts on titanium (Ti) arrays with regular geometry. We compared cubic pillar structures (SU-8, dimension 3 x 3 x 5 and 5 x 5 x 5 mum) with planar samples. Electrochemical surface characterization revealed a low amount of surface energy (including polar component) for the pillar-structured surfaces, which correlated with a reduced initial cell adhesion and spreading. Confocal microscopy of cells actin cytoskeleton revealed no stress fiber organization instead, the actin was concentrated in a surface geometry-dependent manner as local spots around the pillar edges. This altered cell architecture resulted in an impaired MG-63 cell function - the extracellular matrix proteins collagen-I and bone sialo protein (BSP-2) were synthesized at a significantly lower level on SU-8 pillar structures; this was accompanied by reduced beta3-integrin expression. To find out physicochemical factors pertaining to geometrically microstructured surfaces and their influence on adjoining biosystems is important for the development of biorelevant implant surfaces.

Mechanics and electrostatics of the interactions between osteoblasts and titanium surface.

Due to oxidation and adsorption of chloride and hydroxyl anions, the surface of titanium (Ti) implants is negatively charged. A possible mechanism of the attractive interaction between the negatively charged Ti surface and the negatively charged osteoblasts is described theoretically. It is shown that adhesion of positively charged proteins with internal charge distribution may give rise to attractive interaction between the Ti surface and the osteoblast membrane. A dynamic model of the osteoblast attachment is presented in order to study the impact of geometrically structured Ti surfaces on the osteoblasts attachment. It is indicated that membrane-bound protein complexes (PCs) may increase the membrane protrusion growth between the osteoblast and the grooves on titanium (Ti) surface and thereby facilitate the adhesion of osteoblasts to the Ti surface. On the other hand, strong local adhesion due to electrostatic forces may locally trap the osteoblast membrane and hinder the further spreading of osteointegration boundary. We suggest that the synergy between these two processes is responsible for successful osteointegration along the titanium surface implant.

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.

Mechanical integrin stress and magnetic forces induce biological responses in mesenchymal stem cells which depend on environmental factors

The control of mesenchymal stem cells (MSC) by physical cues is of great interest in regenerative medicine. Because integrin receptors function as mechanotransducers, we applied drag forces to β1 integrins on the apical surface of adherent human MSC. In addition to mechanical forces, the technique we used involved also the exposure of the cells to an inhomogeneous magnetic field. In order to assess the influence of the substrate on cell adhesion, cells were cultured on plain tissue culture polystyrene (TCP) or on coated well plates, which allowed only adhesion to embedded fibronectin or RGD peptides. We found that the expression of collagen I, which is involved in osteogenesis, and VEGF, a factor which stimulates angiogenesis, increased as a result of short‐term mechanical integrin stress. Whereas, collagen I expression was stimulated by mechanical forces when the cells were cultured on fibronectin and RGD peptides but not on TCP, VEGF expression was enhanced by physical stimulation on TCP. The study further revealed that magnetic forces enhanced Sox 9 expression, a marker of chondrogenesis, and reduced the expression of ALP. Concerning the intracellular mechanisms involved, we found that the expression of VEGF induced by physical forces depended on Akt activation. Together, the results implicate that biological functions of MSC can be stimulated by integrin‐mediated mechanical forces and a magnetic field. However, the responses of cells depend strongly on the substrate to which they adhere and on the cross‐talk between integrin‐mediated signals and soluble factors. J. Cell. Biochem. 111: 1586–1597, 2010.

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.

Automatic Actin Filament Quantification of Osteoblasts and Their Morphometric Analysis on Microtextured Silicon-Titanium Arrays

Microtexturing of implant surfaces is of major relevance in the endeavor to improve biorelevant implant designs. In order to elucidate the role of biomaterial’s topography on cell physiology, obtaining quantitative correlations between cellular behavior and distinct microarchitectural properties is in great demand. Until now, the microscopically observed reorganization of the cytoskeleton on structured biomaterials has been difficult to convert into data. We used geometrically microtextured silicon-titanium arrays as a model system. Samples were prepared by deep reactive-ion etching of silicon wafers, resulting in rectangular grooves (width and height: 2 µm) and cubic pillars (pillar dimensions: 2 × 2 × 5 and 5 × 5 × 5 µm); finally sputter-coated with 100 nm titanium. We focused on the morphometric analysis of MG-63 osteoblasts, including a quantification of the actin cytoskeleton. By means of our novel software FilaQuant, especially developed for automatic actin filament recognition, we were first able to quantify the alterations of the actin network dependent on the microtexture of a material surface. The cells’ actin fibers were significantly reduced in length on the pillared surfaces versus the grooved array (4–5 fold) and completely reorganized on the micropillars, but without altering the orientation of cells. Our morpho-functional approach opens new possibilities for the data correlation of cell-material interactions.

Preparation, microstructures, mechanical properties, and cytocompatibility of TiMn alloys for biomedical applications

The titanium-manganese (TiMn) alloys have been extensively used in aerospace and hydrogen storage. In this study, the TiMn alloys with various manganese contents ranging from 2 to 12 wt % were prepared by using mechanical alloying and spark plasma sintering (SPS) techniques. The microstructures, mechanical properties including hardness, elastic modulus and ductility, cytotoxicity and cell proliferation properties of the TiMn alloys were investigated to explore their biomedical applications. The addition of manganese to the titanium reduced the alpha to beta transformation temperature and was confirmed as a beta stabilizer element. The manganese increased the relative density of the alloy and thus high density TiMn alloys with alpha+beta structure were prepared by using SPS at 700 degrees C. The hardness increased significantly ranging from 2.4 GPa (Ti2Mn) to 5.28 GPa (Ti12Mn) and the elastic modulus ranging from 83.3 GPa (Ti2Mn) to 122 GPa (Ti12Mn), the ductility decreased ranging from 21.3% (Ti2Mn) to 11.7% (Ti12Mn) with increasing manganese content in the Ti. Concentrations of Mn below 8 wt % in titanium reveal negligible effects on the metabolic activity and the cell proliferation of human osteoblasts. The Mn could be used in lower concentrations as an alloying element for biomedical titanium. The Ti2Mn, Ti5Mn, and Ti8Mn alloys with supervisor mechanical properties and acceptable cytocompatibility have a potential for use as bone substitutes and dental implants.

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.

Cellular Activity and Biomaterial's Surface Topography

The contact of a cell on the biomaterial’s surface is mediated by its adhesion components. The topography of titanium surfaces influences these adhesion components of osteoblasts, e.g. the integrins, the adapter proteins and the actin cytoskeleton. In our current experiments we were interested in why osteoblasts were strongly aligned to the grooves of a structured pure titanium surface (grade 2). The titanium was characterized by EIS to get insights in the electro-chemically active surface. We used MG-63 human bone cells, cultured in DMEM with 10% FCS at 37°C. For protein adsorption the titanium discs were incubated for 24h with complete medium containing soluble fibronectin at 37°C. Interestingly, only in the grooves cells adhered and were aligned and this is not dependent on the gravitation. The cell adhesion seems to depend on the protein adsorption of fibronectin which we could find to be adsorbed exclusively in the valleys. We speculate that there are local differences in electro-chemical characteristics of this structured titanium surface.

A novel modular device for 3-D bone cell culture and non-destructive cell analysis.

Synthetic materials have emerged as bone substitutes for filling bone defects of critical sizes. Because bone healing requires a mechanically resistant matrix (scaffold) attractive to osteogenic cells and must allow revascularization for nutrient and oxygen supply, scaffold-based strategies focus on the further development of chemical and physical qualities of the material. Cellular ingrowth towards the scaffold center is critical; therefore selective information from inner regions, in particular from the central part, is essential. In this paper we introduce a novel modular in vitro system for three-dimensional (3-D) in vitro bone cell cultures. This 3-D system is developed exclusively for in vitro research purposes, with special emphasis on the geometrical scaffold design (pore size, pore design). The system is composed of a stack of titanium slices which are mounted on a clamp and which enable the separate monitoring of cell growth patterns on every single slice of the slide stack. In this way we are able to gain selective information about the regulation of the cell physiology in the inner part of the 3-D construct which can be used for the development of an optimized scaffold design for orthopedic implants.