Influence of Divalent Metal Ions on the Precipitation of the Plasma Protein Fibrinogen.

Fibrinogen nanofibers are very attractive biomaterials to mimic the native blood clot architecture. Previously, we reported the self-assembly of fibrinogen nanofibers in the presence of monovalent salts and have now studied how divalent salts influence fibrinogen precipitation. Although the secondary fibrinogen structure was significantly altered with divalent metal ions, morphological analysis revealed exclusively smooth fibrinogen precipitates. In situ monitoring of the surface roughness facilitated predicting the tendency of various salts to form fibrinogen fibers or smooth films. Analysis of the chemical composition revealed that divalent salts were removed from smooth fibrinogen films upon rinsing while monovalent Na+ species were still present in fibrinogen fibers. Therefore, we assume that the decisive factor controlling the morphology of fibrinogen precipitates is direct ion-protein contact, which requires disruption of the ion-surrounding hydration shells. We conclude that in fibrinogen aggregates, this mechanism is effective only for monovalent ions, whereas divalent ions are limited to indirect fibrinogen adsorption.

Controlling the Multiscale Structure of Nanofibrous Fibrinogen Scaffolds for Wound Healing.

As a key player in blood coagulation and tissue repair, fibrinogen has gained increasing attention to develop nanofibrous biomaterial scaffolds for wound healing. Current techniques to prepare protein nanofibers, like electrospinning or extrusion, are known to induce lasting changes in the protein conformation. Often, such secondary changes are associated with amyloid transitions, which can evoke unwanted disease mechanisms. Starting from our recently introduced technique to self-assemble fibrinogen scaffolds in physiological salt buffers, we here investigated the morphology and secondary structure of our novel fibrinogen nanofibers. Aiming at optimum self-assembly conditions for wound healing scaffolds, we studied the influence of fibrinogen concentration and pH on the protein conformation. Using circular dichroism and Fourier-transform infrared spectroscopy, we observed partial transitions from α-helical structures to ß-strands upon fiber formation. Interestingly, a staining with thioflavin T revealed that this conformational transition was not associated with any amyloid formation. Toward novel scaffolds for wound healing, which are stable in aqueous environment, we also introduced cross-linking of fibrinogen scaffolds in formaldehyde vapor. This treatment allowed us to maintain the nanofibrous morphology while the conformation of fibrinogen nanofibers was redeveloped toward a more native state after rehydration. Altogether, self-assembled fibrinogen scaffolds are excellent candidates for novel wound healing systems since their multiscale structures can be well controlled without inducing any pathogenic amyloid transitions.

Phosphorylation of Fibronectin Influences the Structural Stability of the Predicted Interchain Domain.

As a key player in cell adhesion, the glycoprotein fibronectin is involved in the complex mechanobiology of the extracellular matrix. Although the function of many modules in the fibronectin molecule has already been understood, the structure and biological relevance of the C-terminal cross-linked region (CTXL) still remains unclear. It is known that fibronectin is only phosphorylated in the CTXL domain, but no results have been presented to date, which indicate a biological function based on this phosphorylation. For the first time, we introduce a structural model of the CTXL region in fibronectin, which we obtained by exhaustive replica exchange molecular dynamics simulations (TIGER2hs). The sampling revealed a conformational landscape of the dimerization module, and the global minimum state showed an umbrella-like module body and conspicuous structural region with two feet. We observed that the CTXL foot region exhibits a structural stability in its physiological state, which disappears upon changes in the phosphorylation state. Thus, our in silico studies enabled us to show that the flexibility of the CTXL region is guided by phosphorylation. These results indicate an in vivo function of the CTXL domain in protein binding and cell adhesion, which is controlled by phosphorylation.

Nanopore Diameters Tune Strain in Extruded Fibronectin Fibers.

Fibronectin is present in the extracellular matrix and can be assembled into nanofibers in vivo by undergoing conformational changes. Here, we present a novel approach to prepare fibronectin nanofibers under physiological conditions using an extrusion approach through nanoporous aluminum oxide membranes. This one-step process can prepare nanofiber bundles up to a millimeter in length and with uniform fiber diameters in the nanometer range. Most importantly, by using different pore diameters and protein concentrations in the extrusion process, we could induce varying lasting structural changes in the fibers, which were monitored by Förster resonance energy transfer and should impose different physiological functions.

Minimal synthetic cells to study integrin-mediated adhesion.

To shed light on cell-adhesion-related molecular pathways, synthetic cells offer the unique advantage of a well-controlled model system with reduced molecular complexity. Herein, we show that liposomes with the reconstituted platelet integrin αIIb ß3 as the adhesion-mediating transmembrane protein are a functional minimal cell model for studying cellular adhesion mechanisms in a defined environment. The interaction of these synthetic cells with various extracellular matrix proteins was analyzed using a quartz crystal microbalance with dissipation monitoring. The data indicated that integrin was functionally incorporated into the lipid vesicles, thus enabling integrin-specific adhesion of the engineered liposomes to fibrinogen- and fibronectin-functionalized surfaces. Then, we were able to initiate the detachment of integrin liposomes from these surfaces in the presence of the peptide GRGDSP, a process that is even faster with our newly synthesized peptide mimetic SN529, which specifically inhibits the integrin αIIb ß3 .

Self-Assembled Fibrinogen Scaffolds Support Cocultivation of Human Dermal Fibroblasts and HaCaT Keratinocytes.

Self-assembled fibrinogen scaffolds are highly attractive biomaterials to mimic native blood clots. To explore their potential for wound healing, we studied the interaction of cocultures of human dermal fibroblasts (HDFs) and HaCaT keratinocytes with nanofibrous, planar, and physisorbed fibrinogen. Cell viability analysis indicated that the growth of HDFs and HaCaTs was supported by all fibrinogen topographies until 14 days, either in mono- or coculture. Using scanning electron microscopy and cytoskeletal staining, we observed that the native morphology of both cell types was preserved on all topographies. Expression of the marker proteins vimentin and cytokeratin-14 showed that the native phenotype of fibroblasts and undifferentiated keratinocytes, respectively, was maintained. HDFs displayed their characteristic wound healing phenotype, characterized by expression of fibronectin. Finally, to mimic the multilayered microenvironment of skin, we established successive cocultures of both cells, for which we found consistently high metabolic activities. SEM analysis revealed that HaCaTs arranged into a confluent top layer after 14 days, while fluorescent labeling confirmed the presence of both cells in the layered structure after 6 days. In conclusion, all fibrinogen topographies successfully supported the cocultivation of fibroblasts and keratinocytes, with fibrinogen nanofibers being particularly attractive for skin regeneration due to their biomimetic porous architecture and the technical possibility to be detached from an underlying substrate.

Assembly of Rolled-Up Collagen Constructs on Porous Alumina Textiles.

Developing new techniques to prepare free-standing tubular scaffolds has always been a challenge in the field of regenerative medicine. Here, we report a new and simple way to prepare free-standing collagen constructs with rolled-up architecture by self-assembling nanofibers on porous alumina (Al2O3) textiles modified with different silanes, carbon or gold. Following self-assembly and cross-linking with glutaraldehyde, collagen nanofibers spontaneously rolled up on the modified Al2O3 textiles and detached. The resulting collagen constructs had an inner diameter of approximately 2 to 4 mm in a rolled-up state and could be easily detached from the underlying textiles. Mechanical testing of wet collagen scaffolds following detachment yielded mean values of 3.5 ± 1.9 MPa for the tensile strength, 41.0 ± 20.8 MPa for the Young's modulus and 8.1 ± 3.7% for the elongation at break. No roll-up was observed on Al2O3 textiles without any modification, where collagen did not assemble into fibers, either. Blends of collagen and chitosan were also found to roll into fibrous constructs on silanized Al2O3 textiles, while fibrinogen nanofibers or blends of collagen and elastin did not yield such structures. Based on these differences, we hypothesize that textile surface charge and protein charge, in combination with the porous architecture of protein nanofibers and differences in mechanical strain, are key factors in inducing a scaffold roll-up. Further studies are required to develop the observed roll-up effect into a reproducible biofabrication process that can enable the controlled production of free-standing collagen-based tubes for soft tissue engineering.

A nanoporous alumina microelectrode array for functional cell-chip coupling.

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.

Nanofiber Topographies Enhance Platelet-Fibrinogen Scaffold Interactions.

The initial contact with blood and its components, including plasma proteins and platelets, directs the body's response to foreign materials. Natural scaffolds of extracellular matrix or fibrin contain fibrils with nanoscale dimensions, but how platelets specifically respond to the topography and architecture of fibrous materials is still incompletely understood. Here, planar and nanofiber scaffolds are fabricated from native fibrinogen to characterize the morphology of adherent platelets and activation markers for phosphatidylserine exposure and α-granule secretion by confocal fluorescence microscopy and scanning electron microscopy. Different fibrinogen topographies equally support the spreading and α-granule secretion of washed platelets. In contrast, preincubation of the scaffolds with plasma diminishes platelet spreading on planar fibrinogen surfaces but not on nanofibers. The data show that the enhanced interactions of platelets with nanofibers result from a higher locally accessible surface area, effectively increasing the ligand density for integrin-mediated responses. Overall, fibrinogen nanofibers direct platelets toward robust adhesion formation and α-granule secretion while minimizing their procoagulant activity. Similar results on fibrinogen-coated polydimethylsiloxane substrates with micrometer-sized 3D features suggest that surface topography could be used more generally to steer blood-materials interactions on different length scales for enhancing the initial wound healing steps.

Large-scale patterning of gold nanopillars in a porous anodic alumina template by replicating gold structures on a titanium barrier.

Gold nanopillars are grown in patterns inside a porous anodic alumina template. On selected positions, defined by a gold "seed" pattern, gold is electroplated into the pores, while a barrier layer underneath the porous template blocks the deposition on the rest of the surface. Large-scale arrays of free-standing nanopillar islands are obtained after selective etching of the alumina template.

Effect of Collagen Nanofibers and Silanization on the Interaction of HaCaT Keratinocytes and 3T3 Fibroblasts with Alumina Nanopores.

During wound healing, a complex cascade of cellular and molecular events occurs, which is governed by topographical and biochemical cues. Therefore, optimal tissue repair requires scaffold materials with versatile structural and biochemical features. Nanoporous anodic aluminum oxide (AAO) membranes exhibit good biocompatibility along with customizable nanotopography and antimicrobial properties, which has brought them into the focus of wound treatment. However, despite their good permeability, such bioinert ceramic nanopores cannot actively promote cell growth as they lack biochemical cues to support specific ligand-receptor interactions. Therefore, we modified AAO nanopores with the biochemical features of collagen nanofibers or amino groups provided by silanization with (3-aminopropyl)triethoxysilane (APTES) to design a permeable scaffold material that can additionally promote cell adhesion. Viability assays revealed that the metabolic activity of both 3T3 fibroblasts and HaCaT keratinocytes on bare and silanized AAO pores was comparable to glass controls until 72 h. Interestingly, both cell types showed a reduced proliferation on AAO with collagen nanofibers. Nevertheless, scanning electron and fluorescence microscopy revealed that 3T3 fibroblasts exhibited a well-spread morphology with filopodia attached to the nanoporous surface of the underlying AAO membranes or nanofibrous collagen networks, thus indicating a close interaction with the composites. Keratinocytes, although growing in clusters on bare and APTES-modified AAO, also adhered well on collagen-modified AAO membranes. When in contact with Escherichia coli suspensions for 20 h, the AAO membranes successfully prevented bacteria penetration irrespective of the biochemical functionalization. In summary, both functionalization strategies have high potential to specifically control molecular signaling and cell migration to further develop alumina nanopores for wound healing.

Principles of Fibrinogen Fiber Assembly In Vitro.

Fibrinogen nanofibers hold great potential for applications in wound healing and personalized regenerative medicine due to their ability to mimic the native blood clot architecture. Although versatile strategies exist to induce fibrillogenesis of fibrinogen in vitro, little is known about the underlying mechanisms and the associated length scales. Therefore, in this manuscript the current state of research on fibrinogen fibrillogenesis in vitro is reviewed. For the first time, the manifold factors leading to the assembly of fibrinogen molecules into fibers are categorized considering three main groups: substrate interactions, denaturing and non-denaturing buffer conditions. Based on the meta-analysis in the review it is concluded that the assembly of fibrinogen is driven by several mechanisms across different length scales. In these processes, certain buffer conditions, in particular the presence of salts, play a predominant role during fibrinogen self-assembly compared to the surface chemistry of the substrate material. Yet, to tailor fibrous fibrinogen scaffolds with defined structure-function-relationships for future tissue engineering applications, it still needs to be understood which particular role each of these factors plays during fiber assembly. Therefore, the future combination of experimental and simulation studies is proposed to understand the intermolecular interactions of fibrinogen, which induce the assembly of soluble fibrinogen into solid fibers.

Self-assembled fibrinogen nanofibers support fibroblast adhesion and prevent E. coli infiltration.

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.

Spatial patterning of nanofibrous collagen scaffolds modulates fibroblast morphology.

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.

Wet-spinning of magneto-responsive helical chitosan microfibers.

Helical structures can be found in nature at various length scales ranging from the molecular level to the macroscale. Due to their ability to store mechanical energy and to optimize the accessible surface area, helical shapes contribute particularly to motion-driven processes and structural reinforcement. Due to these special features, helical fibers have become highly attractive for biotechnological and tissue engineering applications. However, there are only a few methods available for the production of biocompatible helical microfibers. Given that, we present here a simple technique for the fabrication of helical chitosan microfibers with embedded magnetic nanoparticles. Composite fibers were prepared by wet-spinning and coagulation in an ethanol bath. Thereby, no toxic components were introduced into the wet-spun chitosan fibers. After drying, the helical fibers had a diameter of approximately 130 µm. Scanning electron microscopy analysis of wet-spun helices revealed that the magnetic nanoparticles agglomerated into clusters inside the fiber matrix. The helical constructs exhibited a diameter of approximately 500 µm with one to two windings per millimeter. Due to their ferromagnetic properties they are easily attracted to a permanent magnet. The results from the tensile testing show that the helical chitosan microfibers exhibited an average Young's modulus of 14 MPa. By taking advantage of the magnetic properties of the feedstock solution, the production of the helical fibers could be automated. The fabrication of the helical fibers was achieved by utilizing the magnetic properties of the feedstock solution and winding the emerging fiber around a rotating magnetic collector needle upon coagulation. In summary, our helical chitosan microfibers are very attractive for future use in magnetic tissue engineering or for the development of biocompatible actuator systems.

Fabrication of 3D-nanofibrous fibrinogen scaffolds using salt-induced self assembly.

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.

Single-molecule mechanics of protein-labelled DNA handles.

DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA-protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot-streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein-DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition in time-critical molecular motor studies.

Template-assisted extrusion of biopolymer nanofibers under physiological conditions.

Biomedical applications ranging from tissue engineering to drug delivery systems require versatile biomaterials based on the scalable and tunable production of biopolymer nanofibers under physiological conditions. These requirements can be successfully met by a novel extrusion process through nanoporous aluminum oxide templates, which is presented in this study. With this simple method we are able to control the nanofiber diameter by chosing the size of the nanopores and the concentration of the biopolymer feed solution. Nanofiber assembly into different hierarchical fiber arrangements can be achieved with a wide variety of different proteins ranging from the intracellular proteins actin, α-actinin and myosin to the extracellular matrix components collagen, fibronectin, fibrinogen, elastin and laminin. The extrusion of nanofibers can even be applied to the polysaccharides hyaluronan, chitosan and chondroitin sulphate. Moreover, blends of different proteins or proteins and polysaccharides can be extruded into composite nanofibers. With these features our template-assisted extrusion process will lead to new avenues in the development of nanofibrous biomaterials.

Model systems for studying cell adhesion and biomimetic actin networks.

Many cellular processes, such as migration, proliferation, wound healing and tumor progression are based on cell adhesion. Amongst different cell adhesion molecules, the integrin receptors play a very significant role. Over the past decades the function and signalling of various such integrins have been studied by incorporating the proteins into lipid membranes. These proteolipid structures lay the foundation for the development of artificial cells, which are able to adhere to substrates. To build biomimetic models for studying cell shape and spreading, actin networks can be incorporated into lipid vesicles, too. We here review the mechanisms of integrin-mediated cell adhesion and recent advances in the field of minimal cells towards synthetic adhesion. We focus on reconstituting integrins into lipid structures for mimicking cell adhesion and on the incorporation of actin networks and talin into model cells.

Force measurements of the disruption of the nascent polypeptide chain from the ribosome by optical tweezers.

We show that optical tweezers are a valuable tool to study the co-translational folding of a nascent polypeptide chain at the ribosome in real-time. The aim of this study was to demonstrate that a stable and intact population of ribosomes can be tethered to polystyrene beads and that specific hook-ups to the nascent polypeptide chain by dsDNA handles, immobilized on a second bead, can be detected. A rupture force of the nascent chain in the range of 10-50 pN was measured, which demonstrates that the system is anchored to the surface in a stable and specific way. This will allow in numerous future applications to follow protein folding using much lower forces.