Tobias Wolfram

Myoblast morphology and organization on biochemically micro-patterned hydrogel coatings under cyclic mechanical strain

Mechanical forces and geometric constraints play critical roles in determining cell functionality and tissue development. Novel experimental methods are essential to explore the underlying biological mechanisms of cell response. We present a versatile method to culture cells on adhesive micro-patterned substrates while applying long-term cyclic tensile strain (CTS). A polydimethysiloxane (PDMS) mold is coated with a cell repulsive NCO-sP(EO-stat-PO) hydrogel which in turn is covalently patterned by fibronectin using micro-contact printing. This results in two-dimensional, highly selective cell-adhesive micro-patterns. The substrates allow application of CTS to adherent cells for more than 4 days under cell culture conditions without unspecific adhesion. The applicability of our system is demonstrated by studying the adaptive response of C2C12 skeletal myoblasts seeded on fibronectin lines with different orientations relative to the strain direction. After application of CTS (amplitude of 7%, frequency of 0.5 Hz) we find that actin fiber organization is dominantly controlled by CTS. Nuclei shape is predominantly affected by the constraint of the adhesive lines, resulting in significant elongation. Morphologically, myotube formation was incomplete after 4 days of culture, but actin striations were observed exclusively on the 45 degrees line patterns subjected to CTS, the direction of maximum shear strain.

Site-specific presentation of single recombinant proteins in defined nanoarrays

The authors describe the deposition of single biomolecules on substrates at defined spacing by pure self-assembly. The substrate is equipped with an array of 8 nm large gold particles which form the template for biomolecule binding. The authors verified the successful binding of single biomolecules via specific antibody labeling and imaging by fluorescence microscopy. Scanning force microscopy provided evidence that every gold nanoparticle of the pattern is occupied by at least one biomolecule. Furthermore, gold conjugated secondary antibodies in combination with scanning electron microscopy proved that at least 75% of the nanoparticles carried only one active biomolecule. The precision given by such surface densities is molecularly defined and such considerably higher than in any other case reported so far.

Stimulation of Cell Adhesion at Nanostructured Teflon Interfaces

Design and control of physico-chemical surface properties of polymeric materials are one of the key challenges in biomedical engineering. Among these materials, polymers for the application as artifi cial vascular grafts are one of the most diffi cult and important applications in biomedical engineering these days. Their failure causes severe clinical complications. Besides polyesters and polyurethanes, polytetrafl uoroethylene (PTFE) is a commonly applied material for artifi cial vascular grafts. In general, two specifi c problems have to be overcome, especially when dealing with small artifi cial vascular grafts ( < 4 mm): thrombosis and restenosis. [ 1 ] The ultimate goal of vascular graft engineering is a graft with a stable and confl uent layer of endothelial cells. Such a monolayer of endothelial cells could potentially mimic the luminal surface of a real vessel and would solve the patency problems of small-diameter vascular grafts in the future. PTFE has benefi cial properties like small friction, great chemical stability and very good compatibility in vivo in general. Missing functional surface groups limits the application of known (bio)functionalization methods for PTFE based interfaces as it usually impedes the formation of covalent bonds. Two approaches can be utilized to overcome this dilemma. On the one hand, reactive groups can be introduced by procedures like plasma treatment, [ 2 ] UV treatment [ 3 ] and/or chemical reduction. [ 4 ] On the other hand, one can take advantage of non-covalent immobilization by unspecifi c adsorption. [ 5 ] Besides their advantages, both methods share common disadvantages as they are either limited to specifi c biomolecules and harsh procedures or they lack stability and control of biomolecule conformation. Therefore, biocompatibility of PTFE is limited. In order to enhance biocompatibility at interfaces most scientifi c approaches use ligands on the graft surface to strengthen endothelial cell adhesion compared to bare grafts.

Nanopatterns biofunctionalized with cell adhesion molecule DM-GRASP offered as cell substrate: Spacing determines attachment and differentiation of neurons

The density/spacing of plasma membrane proteins is thought to be crucial for their function; clear-cut experimental evidence, however, is still rare. We examined nanopatterns biofunctionalized with cell adhesion molecule DM-GRASP with respect to their impact on neuron attachment and neurite growth. Data analysis/modeling revealed that these cellular responses improve with increasing DM-GRASP density, with the exception of one spacing which does not allow for the anchorage of a cytoskeletal protein (spectrin) to three DM-GRASP molecules.

Cell adhesion to agrin presented as a nanopatterned substrate is consistent with an interaction with the extracellular matrix and not transmembrane adhesion molecules

BackgroundMolecular spacing is important for cell adhesion in a number of ways, ranging from the ordered arrangement of matrix polymers extracellularly, to steric hindrance of adhesion/signaling complexes intracellularly. This has been demonstrated using nanopatterned RGD peptides, a canonical extracellular matrix ligand for integrin interactions. Cell adhesion was greatly reduced when the RGD-coated nanoparticles were separated by more than 60 nm, indicating a sharp spacing-dependent threshold for this form of cell adhesion.ResultsHere we show a similar dependence of cell adhesion on the spacing of agrin, a protein that exists as both a secreted, matrix-bound form and a type-2 transmembrane form in vivo. Agrin was presented as a substrate for cell adhesion assays by anchoring recombinant protein to gold nanoparticles that were arrayed at tunable distances onto glass coverslips. Cells adhered well to nanopatterned agrin, and when presented as uniformly coated substrates, adhesion to agrin was comparable to other well-studied adhesion molecules, including N-Cadherin. Adhesion of both mouse primary cortical neurons and rat B35 neuroblastoma cells showed a spacing-dependent threshold, with a sharp drop in adhesion when the space between agrin-coated nanoparticles increased from 60 to 90 nm. In contrast, adhesion to N-Cadherin decreased gradually over the entire range of distances tested (uniform, 30, 60, 90, and 160 nm). The spacing of the agrin nanopattern also influenced cell motility, and peptide competition suggested adhesion was partially integrin dependent. Finally, differences in cell adhesion to C-terminal agrin fragments of different lengths were detected using nanopatterned substrates, and these differences were not evident using uniformly coated substrates.ConclusionThese results suggest nanopatterned substrates may provide a physiological presentation of adhesive substrates, and are consistent with cells adhering to agrin through a mechanism that more closely resembles an interaction with the extracellular matrix than a transmembrane adhesion molecule.

Live cell adhesion assay with attenuated total reflection infrared spectroscopy

Living confluent fish fibroblast cells RTG-P1 from rainbow trout adherent on diamond were examined by attenuated total reflection ATR infrared IR spectroscopy. In particular, IR spectra were recorded dynamically during the adsorption of the cells onto the diamond and during their biochemically induced structural responses to the subsequent addition of trypsin and cytochalasin D. It is shown that changes in the IR spectra result from changes in cell morphology and surface coverage. The results demonstrate the potential and the applicability ofATR IR spectroscopy for live cell adhesion assays.

Surface-Specific Interaction of the Extracellular Domain of Protein L1 with Nitrilotriacetic Acid-Terminated Self-Assembled Monolayers

We report a study on the interaction of the extracellular domain of trans-membrane proteins N-cadherin and L1 with nitrilotriacetic acid (NTA)-terminated self-assembled monolayers (SAMs) grown on silver and gold surfaces. Quartz crystal microbalance (QCM) and reflection absorption infrared spectroscopy (RAIRS) measurements reveal that upon addition of protein to an NTA-SAM there is a subsequent change in the mass and average chemical structure inside the films formed on the metal substrates. By using vibrational sum frequency generation (VSFG) spectroscopy and making a comparison to SAMs prepared with n-alkanethiols, we find that the formed NTA-SAMs are terminated by ethanol molecules from solution. The ethanol signature vanishes after the addition of L1, which indicates that the L1 proteins can interact specifically with the NTA complex. Although the RAIRS spectra display signatures in the amide and fingerprint regions, the VSFG spectra display only a weak feature at 866 cm(-1), which possibly indicates that some of the abundant phenyl rings in the complex are ordered. Although cell biology experiments suggest the directional complexation of L1, the VSFG experiments suggest that the alpha-helices and beta-sheets of L1 lack any preferential ordering.

Fabrication of multi-parametric platforms based on nanocone arrays for determination of cellular response

Summary Cellular response to both surface topography and surface chemistry has been studied for several years. However, most of the studies focus on only one of the two parameters and do not consider their possible synergistic effects. Here, we report on a fabrication method for nanostructured surfaces composed of highly ordered arrays of silica nanocones with gold tips. By using a combination of block copolymer nanolithography, electroless deposition, and reactive ion etching several parameters such as structure height and structure distance could easily be adjusted to the desired values. The gold tips allow for easy functionalization of the substrates through a thiol linker system. Improved neural cell adhesion can be obtained and is dependent on the nature of the nanocone surface, thus illustrating the influence of different surface topographies on the nanometer length scale, on a complex cellular behavior such as cell adhesion. Substrate and surface functionality are shown to last over several days, leading to the conclusion that the features of our substrates can also be used for longer term experiments. Finally, initial neural cell adhesion is found to be more prominent on substrates with short intercone distances, which is an important finding for research dealing with the reactions of neuron-like tissue in the immediate moments after direct contact with an implanted surface.