Search

In this work we describe the manufacturing of cellulosic, cell compatible scaffolds with an inherent 3D origami crease pattern for applications in cardiac tissue engineering. Different cellulosic materials were studied, among them cotton linters, fibers obtained from eucalyptus, pine, spruce and lyocell. Formed sheets made of cotton linters were chosen for further study due to the highest biocompatibility and mechanical properties best suited for cardiomyocytes in wet and dry conditions: E - modulus of 0.8 GPa, tensile strength of 4.7 MPa and tensile strength in wet environment of 2.28 MPa. Cell alignment is desired to achieve directional contraction of the cardiac tissue, and several options were investigated to achieve fiber alignment, e.g. a dynamic sheet former and Rapid Köthen sheet former. Although the orientation was minimal, cells cultured on the cellulose fibers grew and aligned along the fibers. Origami inspired crease patterns were applied to the cellulose scaffolds in order to introduce directional flexibility beneficial for cardiac contraction. The transfer of a Miura crease pattern was successfully applied in two ways: folding of the dried sheet between PET foils pre-formed in a 3D printed mold, and in situ wet fiber molding on a 3D-patterned mesh mounted in the sheet former’s sieve section. The latter approach enables upscaling for potential mass production.

Phosphorus (P) is a limited natural resource and essential for global food supply, particularly given our ever-growing world population. However, natural P deposits are restricted to just a few countries and the quality of exploited primary sources is declining. Phosphorus recovery from sewage sludge or sewage sludge ash leached by acids is hence gaining importance. During P recovery the removal of impurities is a challenge that can be addressed by nanofiltration retaining e. g. multivalent metals and allowing phosphoric acid to pass. However, currently available membranes show low permeability and limit this process option economically. Layer-by-layer (LbL) membrane synthesis is a technology that allows membrane performance to be tailored to individual filtration tasks. Little is known for such membranes with respect to acid resistance, acid permeability and impurity rejection. We show that LBL membranes based on PDADMAC/PSS show the desired passage of phosphoric acid with retentions values below 10%, aluminum retention was always above 95%. Permeabilities up to 4 L/(m2 h bar) were reached even up to a phosphorous recovery of 75%. Overall permeabilities were 16 times higher than a commercial benchmark membrane. Initial stability tests and upscaling into a larger module show the viability of the proposed modification approach.

We report an advanced treatment method for phosphoric acid recovery from leached sewage sludge ash. Layer-by-layer (LbL) polyelectrolyte deposition has been used as a tool to modify and convert a hollow ultrafiltration membrane into a nanofiltration (NF) LbL membrane for H3PO4 recovery. To build the LbL membrane, poly(styrenesulfonate) PSS was chosen as polyanion, while three different polycations were used: a permanently charged polyelectrolyte, poly(diallyldimethylammonium chloride), PDADMAC; a pH-dependent charged polyelectrolyte poly(allylamine hydrochloride), PAH; and a PAH modified with guanidinium groups (PAH-Gu). Based on detailed surface characterizations (AFM, XPS and Zeta-potential) it was concluded that both charge density and pH-responsiveness of the polycations are key parameters to control the final membrane surface structure and transport properties. The surface properties of LbL-coated membranes were correlated with the membrane filtration performance, when exposed to the real leached sewage sludge ash solution. The highest permeability was recorded for (PDADMAC/PSS)6, a result that was rationalized on its loose, and possibly less interpenetrated, structure, followed by (PAH-Gu/PSS)6 characterized by a more dense, compact layer. H3PO4 recovery was the highest in the case of (PDADMAC/PSS)6, but the retention of multivalent metals (Fe3+ and Mg2+) was low, leading to a more contaminated permeate. The opposite trend was observed for (PAH-Gu/PSS)6, resulting in a less metal-contaminated, but also a less H3PO4-concentrated permeate. Our LbL-modified membranes were found to improve the permeability and H3PO4 recovery compared to a commercially available acid-resistant NF membrane.

Surface roughness is a vital factor for medical implants since the cells of the surrounding tissue interact with the underlying substrate on the micro- and nanometer scales. In order to improve the surface morphology of implants, appropriate large-area micro- and nanostructuring techniques have to be identifi ed being applicable to irregularly shaped structures. We demonstrate that plasma treatments of polyetheretherketone (PEEK) thin fi lms produce nanostructured surfaces in a reproducible manner. They are easily tailored by varying plasma intensity using oxygen and ammonia as process gases. It was observed that roughness and nanostructure density linearly depend on plasma intensity. Oxygen plasma turned out to exhibit a stronger effect compared to ammonia plasma at the same processing conditions. For cell interaction studies, the mean size of the nanostructures was intentionally varied between 10 nm and 100 nm. In vitro experiments revealed that human mesenchymal stem cells (hMSC) adhere inhomogenously on untreated PEEK fi lms, but the plasma treatment with oxygen or ammonia allows the hMSC to adhere and proliferate. Fluorescence microscopy of the cells on the PEEK fi lms turned out to be diffi cult because of the strong auto-fl uorescence of the PEEK substrate. Stains including the whole cell vital stain Calcein-AM allowed cell morphology studies on plasma-treated PEEK fi lms. In the case of the analysis of cell compartments such as the actin cytoskeleton, confocal laser scanning microscopy (CLSM) was successfully applied.

Several properties of bio-inspired surfaces like chemical composition, surface topography, surface hydrophilicity and even surface charge could influence bacterial adhesion to implant materials. Therefore, a nanostructured surface is being investigated to avoid bacterial colonization by their physico-mechanical and chemical aspects. Both smooth and rough-surfaced titanium (PT, SLA) and zirconia (M and ZLA) surfaces were used as controls. Titanium SLA was modified by two-step-etching to create nanostructured surface. Antibacterial properties of the materials were tested by adhesion of Porphyromonas gingivalis (ATCC 33277). The vitality of bacteria was assessed by Live/Dead BacLight™ Bacterial Viability Kit or by conventional culturing on Columbia blood agar. Conventional culturing revealed reduction of bacteria on nanostructured titanium (5.27±0.8 x 104 CFU/mm2) in comparison to rough-surfaced control materials (ZLA 6.16±4.86 x 104 and SLA 1.53±0.75 x 105 CFU/mm2). However, smooth-surfaced control materials (M 2.25±0.84 x 104 and PT 6.63±5.77 x 103 CFU/mm2) showed similar results to the nanostructured material. Live/dead staining demonstrated the antimicrobial efficacy of the nanostructured material revealing reduction of vital bacteria population up to 70%. This effect was not observed on the control materials (bacterial vitality ≥95%). In conclusion, nanostructured titanium surface shows a reduction of vital bacteria. Therefore, bio-inspired nanostructures can modify the bacteria–titanium interaction.

Selected mono- and bicationic quats were compounded with polyethylene. The physicochemical surface properties, leaching behavior, and antibacterial activity of such modified samples were investigated. Contact angle measurements and fluorescein binding assays showed the presence of quaternary ammonium groups at the surface. After storing the samples in 50°C warm water for 30 days, several were still antimicrobially active. No correlation between the number of exposed N+ head groups after leaching and the antibacterial activity was observed. There is however a qualitative correlation of the antibacterial activity with the contact angles and surface concentrations of N+ before leaching/storing in warm water.

The nuclear pore complex regulates cargo transport between the cytoplasm and the nucleus. We set out to correlate the governing biochemical interactions to the nanoscopic responses of the phenylalanineglycine (FG)–rich nucleoporin domains, which are involved in attenuating or promoting cargo translocation. We found that binding interactions with the transport receptor karyopherin-β1 caused the FG domains of the human nucleoporin Nup153 to collapse into compact molecular conformations. This effect was reversed by the action of Ran guanosine triphosphate, which returned the FG domains into a polymer brush-like, entropic barrier conformation. Similar effects were observed in Xenopus oocyte nuclei in situ. Thus, the reversible collapse of the FG domains may play an important role in regulating nucleocytoplasmic transport.

Nuclear pore complexes (NPCs) facilitate macromolecular exchange between the nucleus and cytoplasm of eukaryotic cells. The vertebrate NPC is composed of approximately 30 different proteins (nucleoporins), of which around one third contain phenylalanine-glycine (FG)-repeat domains that are thought to mediate the main interaction between the NPC and soluble transport receptors. We have recently shown that the FG-repeat domain of Nup153 is flexible within the NPC, although this nucleoporin is anchored to the nuclear side of the NPC. By using domain-specific antibodies, we have now mapped the domain topology of Nup214 in Xenopus oocytes and in human somatic cells by immuno-EM. We have found that whereas Nup214 is anchored to the cytoplasmic side of the NPC via its N-terminal and central domain, its FG-repeat domain appears flexible, residing on both sides of the NPC. Moreover, the spatial distribution of the FG-repeat domains of both Nup153 and Nup214 shifts in a transport-dependent manner, suggesting that the location of FG-repeat domains within the NPC correlates with cargo/receptor interactions and that they concomitantly move with cargo through the central pore of the NPC.

The nuclear pore complex (NPC) is the only gateway between the cell nucleus and the cytoplasm, and it mediates all movement between these two compartments. The past few years have witnessed much progress on its structure and molecular architecture, its interaction with soluble transport receptors, and how nucleocytoplasmic transport is regulated by the small GTPase Ran. However, the exact molecular mechanisms underlying nucleocytoplasmic transport and in particular cargo translocation through the NPC are understood only poorly. Recent findings on the interaction between the NPC and transport factors might provide insights into how efficient NPC translocation is accomplished. Moreover, in addition to mediating nucleocytoplasmic transport, the NPC seems to be involved, either directly or indirectly, in many other cellular processes such as chromosome segregation, gene expression, the secretory pathway and apoptosis. Here we review recent findings on these unexpected roles of the NPC.

Amplitude-modulation atomic force microscopy (AM-AFM) is used to determine the retention properties of CaF2 nanoparticles adsorbed on mica and on tooth enamel in liquid. From the phase-lag of the forced cantilever oscillation the local energy dissipation at the detachment point of the nanoparticle was determined. This enabled us to compare different as-synthesized CaF2 nanoparticles that vary in shape, size and surface structure. CaF2 nanoparticles are candidates for additives in dental care products as they could serve as fluorine-releasing containers preventing caries during a cariogenic acid attack on the teeth. We show that the adherence of the nanoparticles is increased on the enamel substrate compared to mica, independently of the substrate roughness, morphology and size of the particles.

The nuclear envelope (NE) is a double membrane physical barrier, which separates the nucleus from the cytoplasm. Underlying the NE are the nuclear lamins, which in combination with inner nuclear membrane proteins form the lamina. The lamina is crucial for maintaining the structural integrity of the nucleus and for positioning of nuclear pore complexes (NPCs) within the NE. The nucleoporin Nup153 has previously been reported to bind to B-type lamins. However, the specificity of this interaction is not well established. Here we show that Nup153 exhibits multiple binding sites for A- and B-type lamins. Using GST-pull down assays, we found that both the N-terminal domain of Nup153 and its C terminus associate with the Ig-fold domain of A- and B-type lamins. By employing purified Nup153 and lamin proteins in blot overlay assays we revealed that both the N-terminal and the C-terminal domain of Nup153 are directly interacting with the lamins. Moreover, we provide evidence that mutations in the lamin A Ig-fold domain selectively affect Nup153-binding, suggesting that Nup153 may play a role in lamin-associated diseases, known as laminopathies. Together our results indicate a far more intricate interplay between Nup153 and nuclear lamins than previously accepted.

Adhesive cells including fibroblasts produce contractile forces to the underlying substrate for their locomotion. Such forces are not only high enough to deform compliant membranes such as silicone but also bend rigid micro-cantilevers. The cell-induced bending of silicon micro-cantilevers was determined using laser deflection during trypsin treatment. The observed cantilever relaxation corresponds to a contractile cell force of (16 ±7) µN per rat-2 fibroblast. The cantilever bending approach represents a unique method for the determination of contractile cell forces on any kind of rigid substrate in desired (physiological) environment. Hence, the fundamental technique allows building cell-based biosensors or should provide a quantitative parameter for characterising the cyto-compatibility of load bearing implant surfaces.

Deep insight on The nuclear pore complex becomes alive: new insights into its dynamics and involvement in different cellular processes.

A new preparation procedure was developed for the stable adsorption of either the cytoplasmic or the nuclear face of native (i.e. in physiological buffer without detergent extraction and in the absence of chemical fixatives) Xenopus oocyte nuclear envelopes (NEs) onto silicon (Si) surfaces. This yields optimal structural preservation of the nuclear pore complexes (NPCs) without compromising their functional properties. The functional viability of thus prepared NPCs was documented by time-lapse atomic force microscopy (AFM) of the reversible calcium-mediated opening (i.e. +Ca(2+)) and closing (i.e. -Ca(2+)) of the iris diaphragm-like distal ring topping the NPCs' nuclear baskets. Moreover, site-specific single colloidal gold particle detection was documented by AFM imaging one and the same NPC before and after immuno-gold labeling the sample with a nucleoporin-specific antibody. With this new preparation protocol at hand, we should eventually be able to follow by time-lapse AFM transport of single gold-conjugated cargos across individual NPCs.

Phenylalanine-glycine (FG)-repeat nucleoporins (Nups) form the major components of the selective gating mechanism in the nuclear pore complex (NPC). Hence, a primary requirement is to understand how they vacillate between preventing the access of passively diffusing molecules and promoting the translocation of receptor-bound cargo into the NPC. To shed light on such behavior, we have studied the nanomechanical properties of a cysteine-modified FG-rich C-terminal domain of hNup153 (i.e., cNup153) and its interactions with importin-beta. This is carried out using single molecule force spectroscopy (SMFS) with the atomic force microscope (AFM). In the absence of importin-beta, cNup153 is highly flexible and can be reversibly stretched and relaxed without any change to its intrinsic entropic elasticity, indicating a lack of intra-FG interactions, i.e., natively unfolded. Importin-beta-modified AFM tips reveal complex binding topologies with cNup153, and provide evidence for binding promiscuity in FG-receptor interactions. These differences suggest that cooperativity between FG-domains arises from FG-receptor interactions instead of FG-FG interactions. On a technical note, this work highlights an improved SMFS technique which involves pre-passivating the underlying substrate surface with polyethylene glycol to reduce undesirable AFM tip-surface effects. A high yield of acceptable data is subsequently obtained from the low surface coverage of target molecules by implementing SMFS measurements in force-volume (FV) mode.

Natively unfolded phenylalanine-glycine (FG)-repeat domains are alleged to form the physical constituents of the selective barrier-gate in nuclear pore complexes during nucleocytoplasmic transport. Presently, the biophysical mechanism behind the selective gate remains speculative because of a lack of information regarding the nanomechanical properties of the FG domains. In this work, we have applied the atomic force microscope to measure the mechanical response of individual and clusters of FG molecules. Single-molecule force spectroscopy reveals that FG molecules are unfolded and highly flexible. To provide insight into the selective gating mechanism, an experimental platform has been constructed to study the collective behavior of surface-tethered FG molecules at the nanoscale. Measurements indicate that the collective behavior of such FG molecules gives rise to an exponentially decaying long-range steric repulsive force. This finding indicates that the molecules are thermally mobile in an extended polymer brush-like conformation. This assertion is confirmed by observing that the brush-like conformation undergoes a reversible collapse transition in less polar solvent conditions. These findings reveal how FG-repeat domains may simultaneously function as an entropic barrier and a selective trap in the near-field interaction zone of nuclear pore complexes; i.e., selective gate.

Nup153, one of the best characterized nuclear pore complex proteins (nucleoporins), plays a critical role in the import of proteins into the nucleus as well as in the export of RNAs and proteins from the nucleus. Initially an epitope of Nup153 was found to reside at the distal ring of the NPC, whereas more recently another epitope was localized to the nuclear ring moiety of the NPC. In an effort to more definitively determine the location of Nup153 within the 3-D architecture of the NPC we have generated domain-specific antibodies against distinct domains of Xenopus Nup153. With this approach we have found that the N-terminal domain is exposed at the nuclear ring of the NPC, whereas the zinc-finger domain of Nup153 is exposed at the distal ring of the NPC. In contrast, the C-terminal domain of Nup153 is not restricted to one particular subdomain of the NPC but rather appears to be highly flexible. Exogenous epitope-tagged hNup153 incorporated into Xenopus oocyte NPCs further underscored these findings. Our data illustrate that multiple domain-specific antibodies are essential to understanding the topology of a nucleoporin within the context of the NPC. Moreover, this approach has revealed new clues to the mechanisms by which Nup153 may contribute to nucleocytoplasmic transport.

Using variothermal polymer micro-injection molding, disposable arrays of eight polymer micro-cantilevers each 500 μm long, 100 μm wide and 25 μm thick were fabricated. The present study took advantage of an easy flow grade polypropylene. After gold coating for optical read-out and asymmetrical sensitization, the arrays were introduced into the Cantisens(®) Research system to perform mechanical and functional testing. We demonstrate that polypropylene cantilevers can be used as biosensors for medical purposes in the same manner as the established silicon ones to detect single-stranded DNA sequences and metal ions in real-time. A differential signal of 7 nm was detected for the hybridization of 1 μM complementary DNA sequences. For 100 nM copper ions the differential signal was found to be (36 ± 5) nm. Nano-mechanical sensing of medically relevant, nanometer-size species is essential for fast and efficient diagnosis.