Bivalent kinetic binding model to surface plasmon resonance studies of antigen-antibody displacement reactions.

Molecular and functional analysis of small molecule binding to protein can provoke insights into cellular signaling and regulatory systems as well as facilitate pharmaceutical drug discovery. In label free small molecule detection the displacement assay format can be applied. This is beneficial because displacement of high molecular weight receptors is detected instead of low molecular weight ligand as in classical binding analysis. Thus, detection limit is potentially lowered. Using the influenza haemagglutinin (HA) peptide binding to mono or bivalent anti-haemagglutinin peptide antibody displacement assay formats could be established. The exact time resolved analysis of binding and dissolution of ligand HA and anti-Haemagglutinin peptide antibody was achieved with surface plasmon resonance (SPR) spectroscopy. Mathematical models could be developed from kinetic equations of ligand binding to mono or bivalent antibodies. With this, an accurate simulation of the SPR results was reached. The simulation plot had to be exactly adjusted to the SPR results to determine all kinetic rate constants defining ligand and receptor binding kinetics. Large variations in receptor concentration gave almost identical rate constants in binding. It became obvious that rebinding is in any case not necessary to understand the binding kinetics of our model system HA/anti-HA. Maximum decline of SPR response could be used to determine ligand concentrations in analyte.

Biological activity and mechanical stability of sol-gel-based biofilters using the freeze-gelation technique for immobilization of Rhodococcus ruber.

Biofilters with long lifetime and high storage stability are very important for bioremediation processes to ensure the readiness at the occurrence of sudden contaminations. By using the freeze-gelation technique, living cells can be immobilized within a mechanically and chemically stable ceramic-like matrix. Due to a freeze-drying step, the embedded microorganisms are converted into a preserved form. In that way, they can be stored under dry conditions, which comply better with storage, transport, and handling requirements. Thus, in contrast to other immobilization techniques, there is no need for storage in liquid or under humid atmosphere. The biological activity, mechanical strength, and the structure of the biologically active ceramic-like composites (biocers) produced by freeze gelation have been investigated by using the phenol-degrading bacteria Rhodococcus ruber as model organism. Samples of freeze-gelation biocers have been investigated after defined storage periods, demonstrating nearly unchanged mechanical strength of the immobilization matrix as well as good storage stability of the activity of the immobilized cells over several months of storage at 4 °C. Repeated-batch tests demonstrated further that the freeze-gelation biocers can be repeatedly used over a period of more than 12 months without losing its bioactivity. Thus, these results show that freeze-gelation biocers have high potential of being scaled up from laboratory test systems to applications in real environment because of their long bioactivity as well as mechanical stability.

Nested Formation of Calcium Carbonate Polymorphs in a Bacterial Surface Membrane with a Graded Nanoconfinement: An Evolutionary Strategy to Ensure Bacterial Survival.

It is the intention of this study to elucidate the nested formation of calcium carbonate polymorphs or polyamorphs in the different nanosized compartments. With these observations, it can be concluded how the bacteria can survive in a harsh environment with high calcium carbonate supersaturation. The mechanisms of calcium carbonate precipitation at the surface membrane and at the underlying cell wall membrane of the thermophilic soil bacterium Geobacillus stearothermophilus DSM 13240 have been revealed by high-resolution transmission electron microscopy and atomic force microscopy. In this Gram-positive bacterium, nanopores in the surface layer (S-layer) and in the supporting cell wall polymers are nucleation sites for metastable calcium carbonate polymorphs and polyamorphs. In order to observe the different metastable forms, various reaction times and a low reaction temperature (4 °C) have been chosen. Calcium carbonate polymorphs nucleate in the confinement of nanosized pores (⌀ 3-5 nm) of the S-layer. The hydrous crystalline calcium carbonate (ikaite) is formed initially with [110] as the favored growth direction. It transforms into the anhydrous metastable vaterite by a solid-state transition. In a following reaction step, calcite is precipitated, caused by dissolution of vaterite in the aqueous solution. In the larger pores of the cell wall (⌀ 20-50 nm), hydrated amorphous calcium carbonate is grown, which transforms into metastable monohydrocalcite, aragonite, or calcite. Due to the sequence of reaction steps via various metastable phases, the bacteria gain time for chipping the partially mineralized S-layer, and forming a fresh S-layer (characteristic growth time about 20 min). Thus, the bacteria can survive in solutions with high calcium carbonate supersaturation under the conditions of forced biomineralization.

Friction-controlled traction force in cell adhesion.

The force balance between the extracellular microenvironment and the intracellular cytoskeleton controls the cell fate. We report a new (to our knowledge) mechanism of receptor force control in cell adhesion originating from friction between cell adhesion ligands and the supporting substrate. Adherent human endothelial cells have been studied experimentally on polymer substrates noncovalently coated with fluorescent-labeled fibronectin (FN). The cellular traction force correlated with the mobility of FN during cell-driven FN fibrillogenesis. The experimental findings have been explained within a mechanistic two-dimensional model of the load transfer at focal adhesion sites. Myosin motor activity in conjunction with sliding of FN ligands noncovalently coupled to the surface of the polymer substrates is shown to result in a controlled traction force of adherent cells. We conclude that the friction of adhesion ligands on the supporting substrate is important for mechanotransduction and cell development of adherent cells in vitro and in vivo.

Formation of tubes during self-assembly of bacterial surface layers.

Based on experimental studies on tube formation during self-assembly of bacterial surface (S)-layers, a mechanistic model for describing the underlying basic mechanisms is proposed and the effect of process parameters on growth velocity and tube radius is investigated. The S-layer is modeled as a curved sheet with discrete binding sites for the association of monomers distributed along the S-layer edges. Reported changes of the tube radius owing to genetic protein modifications are explained within the framework of continuum mechanics. S-layer growth velocity and shape development are analyzed by Monte Carlo simulation in their dependence on the attachment and detachment frequencies of monomers at the S-layer. For curved S-layer patches, a criterion for the formation of S-layer tubes is derived. Accordingly, tubes can form only within a certain range of the initial monomer concentration. Furthermore, the effect of calcium ion concentration on tube formation is discussed, including recent experimental findings on the calcium effect.

pH- and salt-dependent molecular combing of DNA: experiments and phenomenological model.

λ-DNA as well as plasmids can be successfully deposited by molecular combing on hydrophobic surfaces, for pH values ranging from 4 to 10. On polydimethylsiloxane (PDMS) substrates, the deposited DNA molecules are overstretched by about 60-100%. There is a significant influence of sodium ions (NaCl) on the surface density of the deposited DNA, with a maximum near to 100 mM NaCl for a DNA solution (28 ng µl(-1)) at pH 8. The combing process can be described by a micromechanical model including: (i) the adsorption of free moving coiled DNA at the substrate; (ii) the stretching of the coiled DNA by the preceding meniscus; (iii) the relaxation of the deposited DNA to the final length. The sticky ends of λ-DNA cause an adhesion force in the range of about 400 pN which allows a stable overstretching of the DNA by the preceding meniscus. The exposing of hidden hydrophobic bonds of the overstretched DNA leads to a stable deposition on the hydrophobic substrate. The pH-dependent density of deposited DNA as well as the observed influence of sodium ions can be explained by their screening of the negatively charged DNA backbone and sticky ends, respectively. The final DNA length can be derived from a balance of the stored elastic energy of the overstretched molecules and the energy of adhesion.

Podosome-Driven Defect Development in Lamellar Bone under the Conditions of Senile Osteoporosis Observed at the Nanometer Scale.

The degradation mechanism of human trabecular bone harvested from the central part of the femoral head of a patient with a fragility fracture of the femoral neck under conditions of senile osteoporosis was investigated by high-resolution electron microscopy. As evidenced by light microscopy, there is a disturbance of bone metabolism leading to severe and irreparable damages to the bone structure. These defects are evoked by osteoclasts and thus podosome activity. Podosomes create typical pit marks and holes of about 300-400 nm in diameter on the bone surface. Detailed analysis of the stress field caused by the podosomes in the extracellular bone matrix was performed. The calculations yielded maximum stress in the range of few megapascals resulting in formation of microcracks around the podosomes. Disintegration of hydroxyapatite and free lying collagen fibrils were observed at the edges of the plywood structure of the bone lamella. At the ultimate state, the disintegration of the mineralized collagen fibrils to a gelatinous matrix comes along with a delamination of the apatite nanoplatelets resulting in a brittle, porous bone structure. The nanoplatelets aggregate to big hydroxyapatite plates with a size of up to 10 x 20 µm2. The enhanced plate growth can be explained by the interaction of two mechanisms in the ruffled border zone: the accumulation of delaminated hydroxyapatite nanoplatelets near clusters of podosomes and the accelerated nucleation and random growth of HAP nanoplatelets due to a nonsufficient concentration of process-directing carboxylated osteocalcin cOC.

Dielectrophoretic growth of metallic nanowires and microwires: theory and experiments.

Dielectrophoresis-assisted growth of metallic nanowires from an aqueous salt solution has been previously reported, but so far there has been no clear understanding of the process leading to such a bottom-up assembly. The present work, based on a series of experiments to grow metallic nano- and microwires by dielectrophoresis, provides a general theoretical description of the growth of such wires from an aqueous salt solution. Palladium nanowires and silver microwires have been grown between gold electrodes from their aqueous salt solution via dielectrophoresis. Silver microwire growth has been observed in situ using light microscopy. From these experiments, a basic model of dielectrophoresis-driven wire growth is developed. This model explains the dependence of the growth on the frequency and the local field enhancement at the electrode asperities. Such a process proves instrumental in the growth of metallic nanowires with controlled morphology and site specificity between the electrodes.

First evidence of octacalcium phosphate@osteocalcin nanocomplex as skeletal bone component directing collagen triple-helix nanofibril mineralization.

Tibia trabeculae and vertebrae of rats as well as human femur were investigated by high-resolution TEM at the atomic scale in order to reveal snapshots of the morphogenetic processes of local bone ultrastructure formation. By taking into account reflections of hydroxyapatite for Fourier filtering the appearance of individual alpha-chains within the triple-helix clearly shows that bone bears the feature of an intergrowth composite structure extending from the atomic to the nanoscale, thus representing a molecular composite of collagen and apatite. Careful Fourier analysis reveals that the non-collagenous protein osteocalcin is present directly combined with octacalcium phosphate. Besides single spherical specimen of about 2 nm in diameter, osteocalcin is spread between and over collagen fibrils and is often observed as pearl necklace strings. In high-resolution TEM, the three binding sites of the γ-carboxylated glutamic acid groups of the mineralized osteocalcin were successfully imaged, which provide the chemical binding to octacalcium phosphate. Osteocalcin is attached to the collagen structure and interacts with the Ca-sites on the (100) dominated hydroxyapatite platelets with Ca-Ca distances of about 9.5 Å. Thus, osteocalcin takes on the functions of Ca-ion transport and suppression of hydroxyapatite expansion.

Effect of modification of hydroxyapatite/collagen composites with sodium citrate, phosphoserine, phosphoserine/RGD-peptide and calcium carbonate on bone remodelling.

This study describes the early interface reaction of cancellous bone to a nanocrystalline hydroxyapatite cement containing type I collagen (HA/Coll) and its modifications with sodium citrate (CI), calcium carbonate (CA), phosphoserine (P) and phosphoserine plus RGD-peptide (RGD). Cylindrical implants of HA/Coll and its modifications were inserted into the tibia of Wistar rats. We analysed 6 specimens per group at days 2, 4, 7, 14 and 28. CI, P and RGD modifications showed improved material properties (finer microstructure and higher compressive strength) compared to CA and HA/Coll implants. The powder X-ray diffraction (XRD) showed that the addition of P and CI led to an increase of alpha-TCP peaks while the diffraction patterns of the non-modified cement (HA/Coll) were quite similar with that of natural bone. All of the implants healed without adverse reactions. A significantly higher number of TRAP-positive osteoclasts were observed around CI, RGD and P on day 7 compared to CA and HA/Coll. Around CI, P and RGD a significantly delayed increase of ED1-positive mononuclear cells was detected. The amount of direct bone contact after 28 days was significantly higher around CI, P and RGD compared to CA and HA/Coll implants. The addition of CI, P and RGD appears to enhance bone remodelling at the early stages of bone healing, leading to increased bone formation around HA/Coll composite cements.

Parallel manipulation of bifunctional DNA molecules on structured surfaces using kinesin-driven microtubules.

We have developed a technique to manipulate bifunctional DNA molecules: One end is thiolated to bind to a patterned gold surface and the other end is biotinylated to bind to a microtubule gliding over a kinesin-coated surface. We found that DNA molecules can be stretched and overstretched between the gold pads and the motile microtubules, and that they can form dynamic networks. This serves as a proof-of-principle that biological machineries can be used in vitro to accomplish the parallel formation of structured DNA templates that will have applications in biophysics and nanoelectronics.

In vitro ossification and remodeling of mineralized collagen I scaffolds.

A promising strategy of bone tissue engineering is to repair bone defects by implanting biodegradable scaffolds that can undergo remodeling and be replaced completely by autologous bone tissue. For this purpose, it is necessary to create scaffolds that can be degraded by osteoclasts and enable osteoblasts to build new mineralized bone matrix. In order to achieve this goal a new porous material has been developed using biomimetically mineralized collagen I. These scaffolds were co-cultured with osteoclast-like cells and osteoblasts in order to characterize the capacity of these cells to remodel the material in vitro. It was possible to show the development of biologically active osteoclast- like cells that were able to invade and degrade the scaffold. They degraded the scaffold by internalizing it as intracellular vesicles, thereby making room for osteoblasts to invade and build new bone matrix. In addition, it could be shown that osteoblasts proliferated, differentiated, and produced new mineralized extracellular matrix. Hence, it could be shown that co-culture of osteoclastlike cells and osteoblasts on biomimetically mineralized collagen I is a promising approach for bone tissue engineering. In addition, it can be applied to study the process of bone remodeling in vitro.

Catalytic oxidation activity of Pt3O4 surfaces and thin films.

The catalytic oxidation activity of platinum particles in automobile catalysts is thought to originate from the presence of highly reactive superficial oxide phases which form under oxygen-rich reaction conditions. Here we study the thermodynamic stability of platinum oxide surfaces and thin films and their reactivities toward oxidation of carbon compounds by means of first-principles atomistic thermodynamics calculations and molecular dynamics simulations based on density functional theory. On the Pt(111) surface the most stable superficial oxide phase is found to be a thin layer of alpha-PtO2, which appears not to be reactive toward either methane dissociation or carbon monoxide oxidation. A PtO-like structure is most stable on the Pt(100) surface at oxygen coverages of one monolayer, while the formation of a coherent and stress-free Pt3O4 film is favored at higher coverages. Bulk Pt3O4 is found to be thermodynamically stable in a region around 900 K at atmospheric pressure. The computed net driving force for the dissociation of methane on the Pt3O4(100) surface is much larger than that on all other metallic and oxide surfaces investigated. Moreover, the enthalpy barrier for the adsorption of CO molecules on oxygen atoms of this surface is as low as 0.34 eV, and desorption of CO2 is observed to occur without any appreciable energy barrier in molecular dynamics simulations. These results, combined, indicate a high catalytic oxidation activity of Pt3O4 phases that can be relevant in the contexts of Pt-based automobile catalysts and gas sensors.

Osteocalcin enhances bone remodeling around hydroxyapatite/collagen composites.

The effect of osteocalcin (OC), an extracellular bone matrix protein, on bone healing around hydroxyapatite/collagen composites was investigated. Cylindrical nanocrystalline hydroxyapatite implants of 2.5-mm diameter containing 2.5% biomimetically mineralized collagen type I were inserted press-fit into the tibial head of adult Wistar rats. To one implant group, 10 mug/g OC was added. Six specimens per group were analyzed at 2, 7, 14, 28, and 56 days. After 14 days, newly formed woven bone had reached the implant surface of the OC implants whereas a broad fibrous interface could still be observed around controls. Woven bone was formed directly around both implant groups after 28 days and had been replaced partially by lamellar bone around the OC implants only. No significant differences in total bone contact were seen between both groups after 56 days. The higher number of phagocytosing cells and osteoclasts characterized immunohistochemically with ED1, cathepsin D, and tartate-resistant alkaline phosphatase around the OC implants at the early stages of bone healing suggests an earlier onset of bone remodeling. The earlier and increased expression of bone-specific matrix proteins and multifunctional adhesion proteins (osteopontin, bone sialoprotein, CD44) at the interface around the OC implants indicates that OC may accelerate bone formation and regeneration. This study supports the observations from in vitro studies that OC activates both osteoclasts and osteoblasts during early bone formation.

Biomimetic porous scaffolds with high elasticity made from mineralized collagen--an animal study.

Histological investigations of a new hydroxyapatite-collagen composite material were carried out to evaluate its possible suitability as a bone substitute. The three-dimensional scaffolds made from biomimetically mineralized collagen exhibit an interconnecting pore structure and elastic mechanical properties. They were implanted into the subcutaneous tissue and bone defects made in the femur of rats and harvested with the surrounding tissue at 1, 2, 4, 8, and 12 weeks after surgery. The materials implanted in the subcutaneous tissue were covered by fibrous connective tissue with a slight inflammatory response, and many foreign-body giant cells were observed on the surface of the scaffolds. Most of the material implanted in the subcutaneous tissue was resorbed at 8 weeks by phagocytosis. In the bone defects, new bone formation was observed on the surface of the material at 1 week. New bone increased with time, and osteoclasts were seen on the surface of the scaffolds at 2 weeks. Resorption and replacement by new bone of many parts of the materials implanted in the femur were observed by 12 weeks. These responses occurred faster than those of other hydroxyapatite-collagen composites. The results suggested that the new biomimetically mineralized collagen scaffolds were suitable as an implant material for bone-tissue reconstruction.

Colorimetric As (V) detection based on S-layer functionalized gold nanoparticles.

Herein, we present simple and rapid colorimetric and UV/VIS spectroscopic methods for detecting anionic arsenic (V) complexes in aqueous media. The methods exploit the aggregation of S-layer-functionalized spherical gold nanoparticles of sizes between 20 and 50 nm in the presence of arsenic species. The gold nanoparticles were functionalized with oligomers of the S-layer protein of Lysinibacillus sphaericus JG-A12. The aggregation of the nanoparticles results in a color change from burgundy-red for widely dispersed nanoparticles to blue for aggregated nanoparticles. A detailed signal analysis was achieved by measuring the shift of the particle plasmon resonance signal with UV/VIS spectroscopy. To further improve signal sensitivity, the influence of larger nanoparticles was tested. In the case of 50 nm gold nanoparticles, a concentration of the anionic arsenic (V) complex lower than 24 ppb was detectable.

Octacalcium phosphate - a metastable mineral phase controls the evolution of scaffold forming proteins.

The molecular structure of collagen type 1 can be understood as the result of evolutionary selection in the process of formation of calcium phosphate based biocomposites acting as load bearing components in living organisms. The evolutionary selection fulfills the principle of 'survival of the fittest' in a particular biological environment. Disk-like post-nucleation complexes of Ca2(HPO4)3 2- organized in ribbon-like assemblies in the metastable octacalcium phosphate (OCP) phase, and Ca3 triangles in the stable HAP phase had formed the crystallographic motifs in this selection process. The rotational as well as the translational symmetry of the major tropocollagen (TC) helix agree nearly perfectly with the corresponding symmetries of the OCP structure. The sequence of (Gly-X-Y) motifs of the three α chains constituting the TC molecule enables an optimized structural fit for the nucleation of Ca3 triangles, the directed growth of nanostructured OCP, and the subsequent formation of hydroxyapatite (HAP) in collagen macrofibrils by a topotaxial transition. The known connection between genetic defects of collagen type 1 and Osteogenesis imperfecta should motivate the search for similar dependences of other bone diseases on a disturbed molecular structure of collagen on the genetic scale.

Single-walled carbon nanotube diameter.

Two different single-walled carbon nanotube (SWNT) growth modes (cap growth mode and circumference growth mode) are shown to exist. General SWNT diameter windows are derivable from catalyst particle size considerations. In addition, an almost complete picture of nanotube diameter dependencies for the cap growth mode is drawn from experiment. The nanotube diameter always scales linear with temperature, but the degree of dependence varies with the catalyst element. The nanotube diameter scales logarithmically with the gas pressure and catalyst composition. Very few or exactly one atom of a catalyst additive is sufficient to induce SWNT diameter changes. The experimental data allow the conclusion that the observed nanotube diameter is based on materials properties of sp2-bonded carbon/graphene sheets, on individual properties of the catalyst elements, and on additional kinetic components from temperature and pressure changes. Indications are found for a specific and maybe decisive role of adsorbate atoms at the surface of a catalyst particle on the nanotube diameter and therefore on the process of nanotube nucleation and growth.

Novel Textile Scaffolds Generated by Flock Technology for Tissue Engineering of Bone and Cartilage.

Textile scaffolds can be found in a variety of application areas in regenerative medicine and tissue engineering. In the present study we used electrostatic flocking-a well-known textile technology-to produce scaffolds for tissue engineering of bone. Flock scaffolds stand out due to their unique structure: parallel arranged fibers that are aligned perpendicularly to a substrate, resulting in mechanically stable structures with a high porosity. In compression tests we demonstrated good mechanical properties of such scaffolds and in cell culture experiments we showed that flock scaffolds allow attachment and proliferation of human mesenchymal stem cells and support their osteogenic differentiation. These matrices represent promising scaffolds for tissue engineering.

Selective targeting of green fluorescent nanodiamond conjugates to mitochondria in HeLa cells.

Fluorescent cellular biomarkers play a prominent role in biosciences. Most of the available biomarkers have some drawbacks due to either physical and optical or cytotoxic properties. In view of this, we investigated the potential of green fluorescent nanodiamonds as biomarkers in living cells. Nanodiamonds were functionalized by attaching antibodies that target intracellular structures such as actin filaments and mitochondria. Then, the nanodiamond conjugates were transfected into HeLa cells. Transfections were mediated by 4(th)-generation dendrimers, cationic liposomes and protamine sulfate. Using fluorescence microscopy, we confirmed successful transfections of the nanodiamonds into HeLa cells. Nanodiamond fluorescence could be easily differentiated from cellular autofluorescence. Furthermore, nanodiamonds could be targeted selectively to intracellular structures. Therefore, nanodiamonds are a promising tool for intracellular assays.