There is currently great interest in material science to mimic cellular microenvironments with controlled bioactive features and biomolecule presentation at the nanometer length scale. Target cells exposed to those 2- or 3D-substrates are stimulated to manifest a variety of cellular functions like cell adhesion, migration and differentiation.

Based on micellar nanolithography, we developed an experimental setup to present single biomolecules on a 2D-nanoarray over large areas on cell-adhesive or cell-repellent substrates. The spacing between biomolecules can be adjusted to 30-250 nm. To mimic a more complex cellular micro-environment with cell-cell contacts as well as cell-extracellular matrix (ECM) contacts, we used biomolecules from different cell adhesion molecule families, like cadherins, immunglobulin-superfamily, integrins and laminins. Via a Ni2+- nitrilo triacetic acid (NTA) system, the amount and the orientation of proteins is chemically controlled such that it resembles the native in vivo settings in the cellular microenvironment. By using poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG) molecules, we generated 2D-protein-nanoarrays with a cell repellent background matrix. After modification of this matrix we incorporated small bioactive peptides to create a microenvironment which allows cell adhesion. These approaches resulted in substrates with specific presentation of biomolecules to mimic cell-cell contacts and cell-ECM interactions simultaneously.

With these substrates we investigated primary fibroblast and neuronal cell adhesion mediated by different biomolecules and with different nanometer spacing. We quantified neurite outgrowth of dorsal root ganglion (DRG) neurons as a differentiation paradigm on nanostructured substrates. Gold nanoparticles were functionalized with peptides, containing the arginine-glycine-aspartic acid (RGD) motif, an integrin activation sequence. Cell adhesion as well as differentiation showed a dependency on the nanometer length scale and on the presented biomolecules.

A key attribute missing from current state-of-the-art biomaterials is the ability to be remodeled by the host after implantation. In contrast, the natural extracellular matrix (ECM) is constantly being remodeled by proteases secreted from cells in response to local environmental changes. Mimicking this strategy, we have designed a new protein-based scaffold that can be degraded and remodeled on demand by the growth cones of regenerating neurites. Using recombinant protein techniques, we synthesized a family of biodegradable and biologically active scaffold materials. The scaffolds include peptide sequences derived from natural ECM proteins. Interspersed with these ECM domains are proteolytic sequences readily degraded by tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), two proteases secreted by the growth cones of extending neurites. By altering the primary amino acid sequences of the protease cleavage domains, we can tune the degradation rates of otherwise identical engineered proteins in a controlled and predictable manner over approximately two orders of magnitude. These recombinant proteins are crosslinked to form bulk, protein-based scaffolds with mechanical properties that can be tuned to match that of the spinal cord. Initial cell experiments have shown that the proteins support growth and differentiation of the model PC-12 neuronal-like cell line. By tailoring the scaffold degradation rate to the tPA and uPA secretion levels of specific neuronal populations, we aim to fabricate a scaffold that will promote neurite extension through the matrix by allowing local degradation to occur specifically around the neuronal growth cone while maintaining the bulk integrity of the overall scaffold.

Experimental and computational studies on mechanical unfolding of proteins suggest that rupture forces approach a limiting value of a few hundred pN at vanishing pulling velocities. We develop a fracture mechanics based theoretical framework that considers the free energy competition between entropic elasticity of polypeptide chains and rupture of peptide hydrogen bonds, which we use here to provide an explanation for the intrinsic strength limit of proteins. Our analysis predicts that individual protein domains stabilized by hydrogen bonds can not exhibit rupture forces larger than approximately ≈200 pN, regardless of the presence of a large number of hydrogen bonds. This result explains a wide range of experimental and computational observations.

Films made of a recombinant spider silk protein and silk fibroin were prepared by spincoating and casting. Therefore the solubility of these substances was investigated in hexafluoroisopropa-nol, ionic liquids and concentrated salt solutions. The roughness and the thickness of the protein films were determined by the Atomic Force Microscopy (AFM) and by mechanical profilometry. The micromechanical behaviour was investigated by acoustic impedance analysis using a quartz crystal microbalance (QCMB) as well as by microindentation. Films with thickness less than 350 nm revealed an almost ideal elastic behaviour in the range of 5 to 75 MHz. At a higher film thickness the half-band-half width increases considerably and the films show a viscoelastic be-haviour with a considerable dissipation. The relative humidity significantly influences the me-chanical behaviour of protein films. Hence the micro-hardness and the ability of water adsorption were determined in dependence on the relative humidity.

We report our findings with theoretical arguments on chiral symmetry breaking on the surface of charged cylinders. We use a model for periodic patterns of charges constrained over a cylindrical surface. In particular we focus on patterns of oriented lamellar patterns, such as, chiral helices, achiral rings or vertical lamellae, with the constraint of global electroneutrality. We study the dependence of the patterns' size and pitch angle on the radius of the cylinder and salt concentration. We obtain a phase diagram by using numerical and analytic techniques. For pure Coulomb interactions, we find a ring phase for small radii and a chiral helical phase for larger radii. We extend the findings to discrete triangular lattices wrapped over a cylindrical geometry. We find no symmetry breaking chiral helical phase in the discrete wrapping when using just an electrostatic potential and the minimum energy configuration is an achiral lattice matching the six-fold symmetry of triangular lattice. Conversely, with the addition of an elastic potential between the charges on the surface of the cylinder we find a stable chiral configuration. We discuss possible consequences and generalizations of our model.

Collagen is the most abundant protein in the human proteome. The post-translational modification of collagen by the enzyme prolyl 4-hydroxylase increases markedly the conformational stability of the collagen triple helix. We have discovered that a previously unappreciated force—stereoelectronic effects—is responsible for this increased stability. By exploiting these stereoelectronic effects (e.g., the gauche effect and n→π* interaction) and reciprocal steric effects, we have created synthetic collagen of unprecedented stability. We have also used the molecular self-assembly of triple-helical fragments to create synthetic collagen of unprecedented length. These synthetic collagens have numerous applications in biotechnology and biomedicine.