[en] The design of electrode interfaces has a strong impact on cell-based bioelectronic applications. We present a new type of microelectrode array chip featuring a nanoporous alumina interface. The chip is fabricated in a combination of top-down and bottom-up processes using state-of-the-art clean room technology and self-assembled generation of nanopores by aluminum anodization. The electrode characteristics are investigated in phosphate buffered saline as well as under cell culture conditions. We show that the modified microelectrodes exhibit decreased impedance compared to planar microelectrodes, which is caused by a nanostructuring effect of the underlying gold during anodization. The stability and biocompatibility of the device are demonstrated by measuring action potentials from cardiomyocyte-like cells growing on top of the chip. Cross sections of the cell–surface interface reveal that the cell membrane seals the nanoporous alumina layer without bending into the sub-50 nm apertures. The nanoporous microelectrode array device may be used as a platform for combining extracellular recording of cell activity with stimulating topographical cues. (paper)

[en] Fibrinogen has become highly attractive for tissue engineering scaffolds since it is a naturally occurring blood protein, which contains important binding sites to facilitate cell adhesion. Here, we introduce a novel biofabrication technique to prepare three-dimensional, nanofibrous fibrinogen scaffolds by salt-induced self assembly. For the first time, we were able to fabricate either free-standing or immobilized fibrinogen scaffolds on demand by tailoring the underlying substrate material and adding a fixation and washing procedure after the fiber assembly. Using scanning electron microscopy we observed that different buffers including phosphate buffered saline and sodium phosphate reproducibly yielded dense fiber networks on bare and silanized glass surfaces, gold as well as polystyrene upon drying. Fibrillogenesis could be induced with a fibrinogen concentration of at least 2 mg ml⁻¹ in a pH regime of 7–9. Fiber diameters ranged from 100 to 300 nm, thus resembling native fibrin and ECM protein fibers. By adjusting the salt concentration we could prepare fibrinogen scaffolds with overall dimensions in the centimeter range and a thickness of 3 to 5 μm. Using FTIR analysis we observed peak shifts of the amide bands for fibrinogen nanofibers in comparison to planar fibrinogen, which indicates changes in the secondary structure. Since fibrillogenesis was only induced upon drying when salt ions were present we assume that protein molecules were locally oriented in the respective buffers, which—in combination with the observed conformational changes—led to the assembly of individual molecules into fibers. In summary, our novel self assembly process offers a simple and well controllable method to prepare large scale 3D-scaffolds of fibrinogen nanofibers under physiological conditions. The unique possibility to chose between free-standing and immobilized scaffolds makes our novel biofabrication process highly attractive for the preparation of versatile tissue engineering scaffolds. (paper)

[en] Current knowledge about cell-biomaterial interactions is often based on two-dimensional (2D) cell culture systems like protein-coated glass slides. However, such smooth surfaces cannot mimic the nanofibrous environment of the native extracellular matrix (ECM). It is therefore a major challenge to transfer the results from 2D surfaces to 3D protein scaffolds with biomimetic nanofiber architecture. To understand the influence of different protein topographies on the cell response we introduce a new process to fabricate binary collagen scaffolds of variable thickness with spatially controlled regions of nanofibrous and smooth topography. We used pH-induced self-assembly to prepare collagen nanofibers with diameters between 130 and 150 nm on glass surfaces, which were partly covered with a polymer mask. After cross-linking with glutaraldehyde, smooth collagen films were prepared on the remaining glass regions. Atomic force microscopy revealed a much lower surface roughness of smooth collagen compared to nanofibers. Subsequently, we studied the viability, morphology and migration of 3T3 fibroblasts on both collagen topographies. We found small, elongated fibroblasts with few, long filopodia on collagen nanofibers whereas large, flat fibroblasts with many short filopodia were observed on smooth collagen. Actin stress fibers on collagen nanofibers were substantially reduced in comparison to smooth collagen. Live cell tracking revealed that fibroblasts on thin nanofibrous collagen migrated faster than on smooth collagen. In summary, binary collagen scaffolds enabled us for the first time to study cell responses to topographical cues on a single protein scaffold. In future, it will be intriguing to transfer our patterning process to other proteins to study fundamental principles of topography-dependent cell recognition processes. (paper)

[en] Nanostructured surfaces have recently gained in importance for electrochemical applications because of an enhanced surface area compared to planar substrates. Due to this property, structured substrates are well suited for electrochemical (bio-)sensors, capacitive coupling with electrogenic cells, and other bioelectronic applications. However, the relationship between electrolytically exposed and redox-active surface areas of nanostructured electrodes compared to planar electrodes is still under discussion. Here, we performed a series of comparative studies to elucidate processes taking place at the electrochemically active surface of gold nanopillars. The pillars, approximately 200 nm in height and 50 nm in diameter, were fabricated using template-assisted nanostructuring. The surface area increase compared to planar electrodes was determined by scanning electron microscopy (SEM), and the redox-active surface area of the same sample was derived from cyclovoltammetric studies. We found consistency between the SEM results and the electrochemically active surfaces determined by cyclic voltammetry of immobilized ferrocenylhexanethiol, immobilized cytochrome c, and oxidation/reduction of gold for small scan rates. Similar values were derived from the capacitance measured by cyclic voltammetry, whereas impedimetric measurements revealed values twice as high. Commonly used diffusion-controlled systems, such as hexacyanoferrate, showed a smaller increase of the electroactive surface area. Finally, we conclude that the sterically restricted diffusion of redox-active species leads to an inaccurate determination of the electroactive surface area of nanosized electrodes

[en] Highlights: • Fibrinogen nanofibers formed by salt-induced self-assembly cover an area of 9 cm². • Nanofibrous fibrinogen scaffolds exhibit ductile failure under wet conditions. • Self-assembled fibrinogen nanofibers support fibroblast adhesion and proliferation. • Fibrinogen nanofibers induce changes in the cell morphology and actin cytoskeleton. • E. coli bacteria do not migrate through nanofibrous fibrinogen scaffolds. Fibrinogen nanofibers hold great potential for wound healing applications since they mimic the native blood clot architecture and offer important binding sites to support fibroblast adhesion and migration. Recently, we introduced a new method of salt-induced self-assembly to prepare nanofibrous fibrinogen scaffolds. Here, we present our results on the mechanical properties of these scaffolds and their interaction with 3T3 fibroblasts and E. coli bacteria, which we used as model systems for wound healing. Hydrated, nanofibrous fibrinogen scaffolds showed a Young's modulus of 1.3 MPa, which is close to the range of native fibrin. 3T3 fibroblasts adhered and proliferated well on nanofibrous and planar fibrinogen up to 72 h with a less pronounced actin cytoskeleton on nanofibers in comparison to planar fibrinogen. Fibroblasts on nanofibers were smaller with many short filopodia while larger cells with few long filopodia were found on planar fibrinogen. Live cell tracking revealed higher migration velocities on nanofibers in comparison to planar fibrinogen. The growth of E. coli bacteria on nanofibrous fibrinogen was significantly reduced as compared to agar controls with no bacteria migrating through the nanofibers. In summary, we conclude that self-assembled fibrinogen nanofibers could become highly attractive as future scaffolds for wound healing applications.