The nanomorphology of cell surfaces of adhered osteoblasts.

The functionality of living cells is inherently linked to subunits with dimensions ranging from several micrometers down to the nanometer scale. The cell surface plays a particularly important role. Electric signaling, including information processing, takes place at the membrane, as well as adhesion and contact. For osteoblasts, adhesion and spreading are crucial processes with regard to bone implants. Here we present a comprehensive characterization of the 3D nanomorphology of living, as well as fixed, osteoblastic cells using scanning ion conductance microscopy (SICM), which is a nanoprobing method that largely avoids mechanical perturbations. Dynamic ruffles are observed, manifesting themselves in characteristic membrane protrusions. They contribute to the overall surface corrugation, which we systematically study by introducing the relative 3D excess area as a function of the projected adhesion area. A clear anticorrelation between the two parameters is found upon analysis of ca. 40 different cells on glass and on amine-covered surfaces. At the rim of lamellipodia, characteristic edge heights between 100 and 300 nm are observed. Power spectral densities of membrane fluctuations show frequency-dependent decay exponents with absolute values greater than 2 on living osteoblasts. We discuss the capability of apical membrane features and fluctuation dynamics in aiding the assessment of adhesion and migration properties on a single-cell basis.

Coagulation using organic carbonates opens up a sustainable route towards regenerated cellulose films.

Due to their biodegradability, biocompatibility and sustainable nature, regenerated cellulose (RC) films are of enormous relevance for green applications including medicinal, environmental and separation technologies. However, the processes used so far are very hazardous to the environment and health. Here, we disclose a simple, fast, environmentally friendly, nontoxic and cost-effective processing method for preparing RC films. High quality non-transparent and transparent RC films and powders can be produced by dissolution with tetrabutylphosphonium hydroxide [TBPH]/[TBP]+[OH]- followed by coagulation with organic carbonates. Investigations on the coagulation mechanism revealed an extremely fast reaction between the carbonates and the hydroxide ions. The high-quality powders and films were fully characterized with respect to structure, surface morphology, permeation and selectivity. This method represents a future-oriented green alternative to known industrial processes.

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.

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.

Impact of plasma chemistry versus titanium surface topography on osteoblast orientation.

Topographical and chemical modifications of biomaterial surfaces both influence tissue physiology, but unfortunately little knowledge exists as to their combined effect. There are many indications that rough surfaces positively influence osteoblast behavior. Having determined previously that a positively charged, smooth titanium surface boosts osteoblast adhesion, we wanted to investigate the combined effects of topography and chemistry and elucidate which of these properties is dominant. Polished, machined and corundum-blasted titanium of increasing microroughness was additionally coated with plasma-polymerized allylamine (PPAAm). Collagen I was then immobilized using polyethylene glycol diacid and glutar dialdehyde. On all PPAAm-modified surfaces (i) adhesion of human MG-63 osteoblastic cells increased significantly in combination with roughness, (ii) cells resemble the underlying structure and melt with the surface, and (iii) cells overcome the restrictions of a grooved surface and spread out over a large area as indicated by actin staining. Interestingly, the cellular effects of the plasma-chemical surface modification are predominant over surface topography, especially in the initial phase. Collagen I, although it is the gold standard, does not improve surface adhesion features comparably.

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.

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.

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 cell's 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.

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.

Interface interactions of osteoblasts with structured titanium and the correlation between physicochemical characteristics and cell biological parameters.

Cellular behavior at the interface of an implant is influenced by the material's topography. However, little is known about the correlation between the biological parameters and the physicochemical characteristics of the biomaterial. We therefore modified pure titanium surfaces by polishing, machining, blasting with glass spheres, blasting with corundum particles, and vacuum plasma spraying to give progressively higher surface roughness. The material surface was characterized by SEM, surface profiling, and electrochemical methods. We revealed a correlation for integrin expression and formation, adhesion, spreading, proliferation, and bone sialo protein expression with the physicochemical parameters of the titanium surfaces.

The influence of surface roughness of titanium on beta1- and beta3-integrin adhesion and the organization of fibronectin in human osteoblastic cells.

Mechanisms of cell adhesion and extracellular matrix formation are primary processes in the interaction with the material surface of an implant which are controlled by integrin receptors. The aim of our study was to find out whether beta1- and beta3-integrins of osteoblastic cells sense the surface topography of titanium, and if structural alterations of integrin adhesions were involved in the organization of fibronectin. Pure titanium surfaces were modified by polishing (P), machining (NT), blasting with glass spheres (GB), and blasting with corundum particles (CB) resulting in increasing roughness. Confocal microscopic investigations revealed fibrillar adhesions of beta1- and alpha5-integrins on P, NT, and GB, but on CB with its sharp edges these integrin subunits did not form fibrillar adhesions. beta3 generally appeared in focal adhesions. We observed aligned fibrillar structures of fibronectin on NT not only on the basal site but interestingly, also on the apical cell surface. In contrast, on CB, fibronectin appeared apically clustered. We suggest that this alignment of fibronectin fibrils depends on the directed actin cytoskeleton and in particular, on the capability of the beta1-integrins to form fibrillar adhesions, which is affected by the surface roughness of titanium.

Ozone-induced gene expression occurs via ethylene-dependent and -independent signalling.

Recent studies suggest that ethylene is involved in signalling ozone-induced gene expression. We show here that application of ozone increased glucuronidase (GUS) expression of chimeric reporter genes regulated by the promoters of the tobacco class I beta-1,3-glucanases (GLB and Gln2) and the grapevine resveratrol synthase (Vst1) genes in transgenic tobacco leaves. 5'-deletion analysis of the class I beta-1,3-glucanase promoter revealed that ozone-induced gene regulation is mainly mediated by the distal enhancer region containing the positively acting ethylene-responsive element (ERE). In addition, application of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, blocked ozone-induced class I beta-1,3-glucanase promoter activity. Enhancer activity and ethylene-responsiveness depended on the integrity of the GCC boxes, cis-acting elements present in the ERE of the class I beta-1,3-glucanase and the basic-type pathogenesis-related PR-1 protein (PRB-1b) gene promoters. The minimal PRB-1b promoter containing only the ERE with intact GCC boxes, was sufficient to confer 10-fold ozone inducibility to a GUS-reporter gene, while a substitution mutation in the GCC box abolished ozone responsiveness. The ERE region of the class I beta-1,3-glucanase promoter containing two intact GCC boxes confered strong ozone inducibility to a minimal cauliflower mosaic virus (CaMV) 35S RNA promoter, whereas two single-base substitution in the GCC boxes resulted in a complete loss of ozone inducibility. Taken together, these datastrongly suggest that ethylene is signalling ozone-induced expression of class I beta-l,3-glucanase and PRB-1b genes. Promoter analysis of the stilbene synthase Vst1 gene unravelled different regions for ozone and ethylene-responsiveness. Application of 1-MCP blocked ethylene-induced Vst1 induction, but ozone induction was not affected. This shows that ozone-induced gene expression occurs via at least two different signalling mechanisms and suggests an additional ethylene independent signalling pathway for ozone-induced expression of genes involved in phytoalexin biosynthesis.