Regina Lange

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.

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.

Third-body abrasive wear of tibial polyethylene inserts combined with metallic and ceramic femoral components in a knee simulator study

Aim Total knee arthroplasties have reached a high grade of quality and safety, but most often fail because of aseptic implant loosening caused by polyethylene wear debris. Wear is generated at the articulating surfaces, e.g. caused by third-body particles. The objective of this experimental study was to determine the wear of tibial polyethylene inserts combined with metallic and ceramic femoral components under third-body wear conditions initiated by bone cement particles. Methods and Materials Wear testing using a cemented unconstrained bicondylar knee endoprosthesis (Multigen Plus CR knee system) was performed in a knee wear simulator. Tibial polyethylene inserts were combined with the identical femoral component design, but made of two different materials (cobalt-chromium and ceramic). Bone cement debris including zirconium oxide particles was added every 500,000 cycles between the articulating surfaces. After 5 million load cycles, the amount of wear was determined gravimetrically and compared with results from standard wear test conditions. The surfaces of tibial inserts were also analyzed. Results The average gravimetrical wear of the tibial polyethylene inserts in combination with cobalt-chromium and ceramic femoral components under third-body wear conditions amounted to 31.88 ± 4.53 mg and 13.06 ± 1.88 mg after 5 million cycles, respectively, and was higher than under standard wear test conditions in both cases. Conclusions The wear simulator test demonstrates that wear of polyethylene inserts under third-body wear conditions, in combination with ceramic femoral components, was significantly lower than with metallic femoral components.

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.

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.

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.

Osteoblast Sensitivity to Topographical and Chemical Features of Titanium

The titanium-osteoblast-interaction can be influenced both by surface roughness and by chemical modifications. We have ascertained that a positively charged titanium surface boosts osteoblast cells adhesion due to their negatively charged cellular hyaluronan coat. In current experiments, chemical surface modifications were combined with different topographies. Titanium disks of technical purity were modified (i) in their roughness by polishing (P), machining (M) and corundum blasting (CB), and (ii) by subsequently chemical functionalization by a thin film (d≤0.1 µm) of microwave plasma polymerized allylamine (PPAAm). In addition, collagen I was immobilized on PPAAm via the bifunctional linker polyethylene glycol diacid or glutar dialdehyde, respectively. The cell shape and materials contact of human osteoblasts was analyzed by FE-SEM and time dependent cell adhesion measured by flow cytometry. The cell dynamic of the adhesion component vinculin was observed in living cells. Amino-functionalization (PPAAm) considerably enhances the adhesion of osteoblasts in combination with topographical features, which was in contrast to collagen modified surfaces. PPAAm allows the cells to literally melt into the groove structure of the titanium. The bone cells lie over a large area and very close to the surface, so that the edges of the cells can hardly be distinguished from the structure of the surface. The combinatory effect of topography and plasma modification could improve bonding of the implant to the bone tissue.

PROLIFERATION AND MIGRATION OF HUMAN OSTEOBLASTS ON POROUS THREE DIMENSIONAL SCAFFOLDS

Porous tantalum (Ta) biomaterial is designed to function as a scaffold for osseous ingrowths and has found applications in orthopedics. Integration of this Ta foam into the neighboring bone requires that osteoprogenitor cells attach to the implant, grow into the scaffold, proliferate and differentiate to osteoblasts. The aim of the present study was to create an in vitro 3D model system to investigate the interaction of human osteoblasts with porous Ta in the depth of the corpus. To explore active migration of osteoblasts into the Ta scaffold two porous Ta discs (Zimmer, Poland) were horizontally fixed within a clamping ring. Thereby a 3D Ta module with 4 levels is generated, which is placed into a cell culture well with the appropriate medium. Osteoblast-like cells were seeded apical onto the Ta module and cultured for 7 days in humidified atmosphere. Active migration of cells into the scaffold was monitored by field emission scanning electron microscopy (FESEM) imaging of the apical, medial and basal layers. A problem in 3D cell culture is the nutrition of cells inside of the scaffold. Therefore morphological changes and differentiation of the cells in distinct layers were analyzed.