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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Toxicity of tungsten carbide and cobalt-doped tungsten carbide nanoparticles in mammalian cells in vitro.

BACKGROUND: Tungsten carbide nanoparticles are being explored for their use in the manufacture of hard metals. To develop nanoparticles for broad applications, potential risks to human health and the environment should be evaluated and taken into consideration. OBJECTIVE: We aimed to assess the toxicity of well-characterized tungsten carbide (WC) and cobalt-doped tungsten carbide (WC-Co) nanoparticle suspensions in an array of mammalian cells. METHODS: We examined acute toxicity of WC and of WC-Co (10% weight content Co) nanoparticles in different human cell lines (lung, skin, and colon) as well as in rat neuronal and glial cells (i.e., primary neuronal and astroglial cultures and the oligodendrocyte precursor cell line OLN-93). Furthermore, using electron microscopy, we assessed whether nanoparticles can be taken up by living cells. We chose these in vitro systems in order to evaluate for potential toxicity of the nanoparticles in different mammalian organs (i.e., lung, skin, intestine, and brain). RESULTS: Chemical-physical characterization confirmed that WC as well as WC-Co nanoparticles with a mean particle size of 145 nm form stable suspensions in serum-containing cell culture media. WC nanoparticles were not acutely toxic to the studied cell lines. However, cytotoxicity became apparent when particles were doped with Co. The most sensitive were astrocytes and colon epithelial cells. Cytotoxicity of WC-Co nanoparticles was higher than expected based on the ionic Co content of the particles. Analysis by electron microscopy demonstrated presence of WC nanoparticles within mammalian cells. CONCLUSIONS: Our findings demonstrate that doping of WC nanoparticles with Co markedly increases their cytotoxic effect and that the presence of WC-Co in particulate form is essential to elicit this combinatorial effect.

Histologic study of incorporation and resorption of a bone cement-collagen composite: an in vivo study in the minipig.

OBJECTIVE: Calcium phosphates are clinically established as bone defect fillers. They have the capability of osseoconduction and are characterized by a slow resorption process. The present study evaluated the suitability of a newly developed calcium phosphate cement modified with collagen type I. STUDY DESIGN: The modified cement paste was inserted in differently designed defects of 10 minipigs. Further, an alveolar ridge augmentation was performed, applying the cement paste. The cement hardened in situ during the operation, forming a hydroxyapatite collagen composite. Animals were sacrificed after 1, 3, 6, 12, and 18 months. The tissue integration and resorption process was then evaluated using nondecalcified microsections. All animals were evaluated for histology. RESULTS: The implanted material showed osseoconductive characteristics. Resorption started from the edge of the defect zone, and bone substitution followed rapidly. Twelve months after placement of the cement, complete remodeling was observed. CONCLUSION: It can be concluded that the applied hydroxyapatite-collagen cement composite shows good resorption and bone integration.

Mineralised collagen--an artificial, extracellular bone matrix--improves osteogenic differentiation of bone marrow stromal cells.

In the field of bone tissue engineering there is a high demand on bone graft materials which promote bone formation. By combination of collagen type I with nanocrystalline hydroxyapatite (HA) we generated a resorbable material which structure and composition is close to those of the extracellular bone matrix. This nanocomposite material was produced in a biomimetic process in which collagen fibril assembly and mineralisation with hydroxyapatite occur simultaneously. In this study the proliferation and osteogenic differentiation of human bone marrow-derived stromal cells (hBMSC) on membranes of biomimetically mineralised collagen type I was investigated. To this end, we optimised biochemical assays for determination of cell number and alkaline phosphatase activity corresponding to the special properties of this biomaterial. For cell experiments hBMSC were seeded on the mineralised collagen membranes and cultivated over 35 days, both in static and perfusion culture, in the presence and absence of dexamethasone, beta-glycerophosphate and ascorbate. Compared to cells grown on tissue culture polystyrene we found attenuated proliferation rates, but markedly increased activity of alkaline phosphatase on the mineralised collagen indicating its promoting effect on the osteogenic differentiation of hBMSC. Therefore this bone-like material may act as a suitable artificial extracellular matrix for bone tissue engineering. Perfusion of the 2D cell matrix constructs with cell culture medium did not improve proliferation and osteogenic differentiation of the hBMSC.

Electrostatic interactions modulate the conformation of collagen I.

The pH- and electrolyte-dependent charging of collagen I fibrils was analyzed by streaming potential/streaming current experiments using the Microslit Electrokinetic Setup. Differential scanning calorimetry and circular dichroism spectroscopy were applied in similar electrolyte solutions to characterize the influence of electrostatic interactions on the conformational stability of the protein. The acid base behavior of collagen I was found to be strongly influenced by the ionic strength in KCl as well as in CaCl(2) solutions. An increase of the ionic strength with KCl from 10(-4) M to 10(-2) M shifts the isoelectric point (IEP) of the protein from pH 7.5 to 5.3. However, a similar increase of the ionic strength in CaCl(2) solutions shifts the IEP from 7.5 to above pH 9. Enhanced thermal stability with increasing ionic strength was observed by differential scanning calorimetry in both electrolyte systems. In line with this, circular dichroism spectroscopy results show an increase of the helicity with increasing ionic strength. Better screening of charged residues and the formation of salt bridges are assumed to cause the stabilization of collagen I with increasing ionic strength in both electrolyte systems. Preferential adsorption of hydroxide ions onto intrinsically uncharged sites in KCl solutions and calcium binding to negatively charged carboxylic acid moieties in CaCl(2) solutions are concluded to shift the IEP and influence the conformational stability of the protein.

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.

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.