Antibacterial biodegradable Mg-Ag alloys.

The use of magnesium alloys as degradable metals for biomedical applications is a topic of ongoing research and the demand for multifunctional materials is increasing. Hence, binary Mg-Ag alloys were designed as implant materials to combine the favourable properties of magnesium with the well-known antibacterial property of silver. In this study, three Mg-Ag alloys, Mg2Ag, Mg4Ag and Mg6Ag that contain 1.87 %, 3.82 % and 6.00 % silver by weight, respectively, were cast and processed with solution (T4) and aging (T6) heat treatment. The metallurgical analysis and phase identification showed that all alloys contained Mg4Ag as the dominant ß phase. After heat treatment, the mechanical properties of all Mg-Ag alloys were significantly improved and the corrosion rate was also significantly reduced, due to presence of silver. Mg(OH)2 and MgO present the main magnesium corrosion products, while AgCl was found as the corresponding primary silver corrosion product. Immersion tests, under cell culture conditions, demonstrated that the silver content did not significantly shift the pH and magnesium ion release. In vitro tests, with both primary osteoblasts and cell lines (MG63, RAW 264.7), revealed that Mg-Ag alloys show negligible cytotoxicity and sound cytocompatibility. Antibacterial assays, performed in a dynamic bioreactor system, proved that the alloys reduce the viability of two common pathogenic bacteria, Staphylococcus aureus (DSMZ 20231) and Staphylococcus epidermidis (DSMZ 3269), and the results showed that the killing rate of the alloys against tested bacteria exceeded 90%. In summary, biodegradable Mg-Ag alloys are cytocompatible materials with adjustable mechanical and corrosion properties and show promising antibacterial activity, which indicates their potential as antibacterial biodegradable implant materials.

Production, characterisation, and cytocompatibility of porous titanium-based particulate scaffolds.

Despite its non-matching mechanical properties titanium remains the preferred metal implant material in orthopaedics. As a consequence in some cases stress shielding effect occurs, leading to implant loosening, osteopenia, and finally revision surgery. Porous metal scaffolds to allow easier specialised cells ingrowth with mechanical properties closer to the ones of bone can overcome this problem. This should improve healing processes, implant integration, and dynamic strength of implants retaining. Three Ti-6Al-4V materials were metal injection moulded and tailored porosities were effectively achieved. After microstructural and mechanical characterisation, two different primary cells of mesenchymal origin (human umbilical cord perivascular cells and human bone derived cells which revealed to be two pertinent models) as well as one cell line originated from primary osteogenic sarcoma, Saos-2, were bestowed to investigate cell-material interaction on genomic and proteome levels. Biological examinations disclosed that no material has negative impact on early adhesion, proliferation or cell viability. An efficient cell ingrowth into material with an average porosity of 25-50 µm was proved.

Microstructure, mechanical and corrosion properties of Mg-Dy-Gd-Zr alloys for medical applications.

In previous investigations, a Mg-10Dy (wt.%) alloy with a good combination of corrosion resistance and cytocompatibility showed great potential for use as a biodegradable implant material. However, the mechanical properties of Mg-10Dy alloy are not satisfactory. In order to allow the tailoring of mechanical properties required for various medical applications, four Mg-10(Dy+Gd)-0.2Zr (wt.%) alloys were investigated with respect to microstructure, mechanical and corrosion properties. With the increase in Gd content, the number of second-phase particles increased in the as-cast alloys, and the age-hardening response increased at 200°C. The yield strength increased, while the ductility reduced, especially for peak-aged alloys with the addition of Gd. Additionally, with increasing Gd content, the corrosion rate increased in the as-cast condition owing to the galvanic effect, but all the alloys had a similar corrosion rate (~0.5 mm year(-1)) in solution-treated and aged condition.

Magnesium alloys as implant materials--principles of property design for Mg-RE alloys.

Magnesium alloys have attracted increasing interest in the past years due to their potential as implant materials. This interest is based on the fact that magnesium and its alloys are degradable during their time of service in the human body. Moreover magnesium alloys offer a property profile that is very close or even similar to that of human bone. The chemical composition triggers the resulting microstructure and features of degradation. In addition, the entire manufacturing route has an influence on the morphology of the microstructure after processing. Therefore the composition and the manufacturing route have to be chosen carefully with regard to the requirements of an application. This paper discusses the influence of composition and heat treatments on the microstructure, mechanical properties and corrosion behaviour of cast Mg-Gd alloys. Recommendations are given for the design of future degradable magnesium based implant materials.

Comparative structure analysis of non-polar organic ferrofluids stabilized by saturated mono-carboxylic acids.

The structure of ferrofluids (magnetite in decahydronaphtalene) stabilized with saturated mono-carboxylic acids of different chain lengths (lauric, myristic, palmitic and stearic acids) is studied by means of magnetization analysis and small-angle neutron scattering. It is shown that in case of saturated acid surfactants, magnetite nanoparticles are dispersed in the carrier approximately with the same size distribution whose mean value and width are significantly less as compared to the classical stabilization with non-saturated oleic acid. The found thickness of the surfactant shell around magnetite is analyzed with respect to stabilizing properties of mono-carboxylic acids.

[Conformation of the ribosomal protein S1 of Thermus thermophilus in solution under different ionic conditions].

The structure of protein SI of Thermus thermophilus (M = 61 kDa) in solution at low and moderate ionic strengths (0 M and 100 mM NaCl, respectively) has been studied by small-angle X-ray and neutron scattering. It was found that protein S1 has a globular conformation under both ionic conditions. The modelling of different packing of six homologous domains of S1 on the basis of the NMR-resolved structure of one domain showed that the best fit of calculated scattering patterns from such complexes to experimental ones is observed at a compact package of the domains. The calculated value of the radius of gyration of the models is 28-29 angtroms, which is characteristic for globular proteins with a molecular mass of about 60 kDa. It was found that protein S1 has a tendency to form associates, and the type of the associate depends on ionic strength. These associates have, in general, two or three monomers at a moderate ionic strength, while at a low ionic strength the number of monomers exceeds three and they are packed in a compact manner. Strongly elongated associates were observed in neutron experiments at a moderate ionic strength in heavy water. The association of protein molecules was also confirmed by the data of dynamic light scattering. From these data, the translational diffusion coefficient of protein S1 at a moderate ionic strength was calculated to be (D20,w = (2.7 +/- 0.1) x 10(-7)cm2/s). This value is essentially smaller than the expected value (D20,w = (5.8 - 6.0) x 10(-7)cm2/s) for the S1 monomer in the globular conformation, indicating the association of protein molecules under equilibrium conditions.

In-vitro interactions of human chondrocytes and mesenchymal stem cells, and of mouse macrophages with phospholipid-covered metallic implant materials.

Phospholipid-coatings on metallic implant surfaces were evaluated in terms of adhesion, proliferation and matrix production of skeletal cells, and of macrophage stimulation. The working hypothesis is that mimicking a model biomembrane by phospholipids on surfaces to which cells adhere, the surface recognition by surrounding cells is altered. In this study, 1) mirror-like polished Ti-6Al-7Nb and 2) porous Ti-6Al-4V specimens were covered with the phospholipids POPE (palmitoyl-oleoyl phosphatidyl-ethanolamine) and POPC (palmitoyl-oleoyl phosphatidyl-choline), and the interactions of a) human articular chondrocytes (HAC), b) human mesenchymal stem cells (HMSC), and c) mouse macrophages (RAW 264.7Rpar; were tested in vitro. On POPE-covered polished surfaces adherence of HAC (42% of seeded cells after 2 hrs) and metabolic activity (MTT after 3 days) were reduced, while on porous surfaces 99% HAC adhered, and metabolic activity was significantly increased, compared to respective native surfaces. On both POPE-covered surfaces the chondrocyte phenotype was present. After 3 weeks of chondrogenic differentiation, cartilage matrix production (measuring chondroitin sulphate per HAC number) was significantly increased by about 30% on both POPE-covered metallic surfaces. On both POPC-covered surfaces nearly no adhering and surviving HAC were found. HMSC grown on POPE-covered porous substrates showed osteogenic differentiation by improved osteopontin and collagen I expression in RT-PCR, and osteocalcin fluorescence and bone nodule formation was only detectable on POPE-covered porous surfaces. In contrast to POPC and other phospholipids used as positive controls, POPE did not stimulate the NO production in mouse macrophage cultures. We therefore conclude that a phospholipid coating by POPE shows potential as surface modification for metallic implant materials.

Phospholipids as implant coatings.

Bio-interfaces such as bio-membranes are of outmost importance for a variety of live processes. Among them are cell-interactions which take place in, on or through cell membranes. Therefore we propose to cover metallic surfaces with phospholipids to facilitate cell-material interaction. Four lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2- oleoyl-sn-glycero-3-[phospho-L-serine] (POPS) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (POPG), were applied to four metallic growth substrates with different surface structure, roughness and porosity. The interaction of the osteosarcoma cell line MG-63 was investigated in terms of cell adhesion and viability (MTT (methylthiazolyldiphenyl-tetrazolium bromide) assay). While POPS in general had a negative influence, the most suitable combination in terms of viability per adherent MG-63 is the coating of porous Ti6Al4V material with the phospholipids POPE or POPC. The analysis of viability of mouse macrophages RAW 264.7 and their tumor necrosis factor alpha (TNF-alpha) release showed that the adhesion and viability is worst on POPS while the TNF-alpha release was highest. To elucidate the potential of phospholipids to prevent or support bacterial growth, the bacterial number of Gram positive and Gram negative bacteria was investigated. For lipid concentrations higher than 1 mM in solution a growth stimulating effect independent of the lipid type was detected. On a lipid coated surface the number of bacteria was reduced by 81%, 74% and 51% for POPC, POPG and POPE.

Synthesis and mesogenic properties of a Y-shaped glyco-glycero-lipid.

We synthesised a new glyco-glycero-lipid [1,3-di-O-(beta-D-glucopyranosyl)-2-deoxy-2-amino-N-palmitoyl-sn-glycerol] with a Y-shaped structure bearing two sugar head groups and only one fatty acid chain. Instead of an ester linkage between the glycerol and the fatty acid an amido function was introduced. The mesogenic properties were investigated using polarising microscopy and are discussed with respect to similar compounds. The lyotropism was measured using the contact preparation method and small-angle neutron-scattering.

Extended conformation of mammalian translation elongation factor 1A in solution.

The conformation of mammalian elongation factor eEF1A in solution was examined by the small angle neutron scattering and scanning microcalorimetry. We have found that in contrast to the bacterial analogue the eEF1A molecule has no fixed rigid structure in solution. The radius of gyration of the eEF1A molecule (5.2 nm) is much greater than that of prokaryotic EF1A. The specific heat of denaturation is considerably lower for eEF1A than for EF1A, suggesting that the eEF1A conformation is significantly more disordered. Despite its flexible conformation, eEF1A is found to be highly active in different functional tests. According to the neutron scattering data, eEF1A becomes much more compact in the complex with uncharged tRNA. The absence of a rigid structure and the possibility of large conformational change upon interaction with a partner molecule could be important for eEF1A functioning in channeled protein synthesis and/or for the well-known capability of the protein to interact with different ligands besides the translational components.

Mapping proteins of the 50S subunit from Escherichia coli ribosomes.

Mapping of protein positions in the ribosomal subunits was first achieved for the 30S subunit by means of neutron scattering about 15 years ago. Since the 50S subunit is almost twice as large as the 30S subunit and consists of more proteins, it was difficult to apply classical contrast variation techniques for the localisation of the proteins. Polarisation dependent neutron scattering (spin-contrast variation) helped to overcome this restriction. Here a map of 14 proteins within the 50S subunit from Escherichia coli ribosomes is presented including the proteins L17 and L20 that are not present in archeal ribosomes. The results are compared with the recent crystallographic map of the 50S subunit from the archea Haloarcula marismortui.

Localization of the protein L2 in the 50 S subunit and the 70 S E. coli ribosome.

The protein L2 is found in all ribosomes and is one of the best conserved proteins of this mega-dalton complex. The protein was localized within both the isolated 50 S subunit and the 70 S ribosome of the Escherichia coli bacteria with the neutron-scattering technique of spin-contrast variation. L2 is elongated, exposing one end of the protein to the surface of the intersubunit interface of the 50 S subunit. The protein changes its conformation slightly when the 50 S subunit reassociates with the 30 S subunit to form a 70 S ribosome, becoming more elongated and moving approximately 30 A into the 50 S matrix. The results support a recent observation that L2 is essential for the association of the ribosomal subunits and might participate in the binding and translocation of the tRNAs.

Thermotropic and lyotropic properties of long chain alkyl glycopyranosides. Part II. Disaccharide headgroups.

We have investigated the thermotropic and lyotropic properties of some long chain alkyl glycosides with disaccharide headgroups. The thermotropism was measured with polarising microscopy and additionally the lyotropism with the contact preparation method, Fourier-transform Infrared (FTIR) spectroscopy, X-ray diffraction and small angle neutron scattering. A broad thermotropic as well as lyotropic polymorphism was found. The compounds displayed thermotropic S(A) (lamellar) and cubic phases, and the investigation of the lyotropic phase behaviour led to the observation of inverted bicontinuous cubic V(II) phases, lamellar L(alpha) phases, normal bicontinuous cubic V(I) phases, normal columnar H(I) phases, normal discontinuous cubic I(I) phases and lyotropic cholesteric phases. The phases are discussed with respect to the chemical structures that have been varied systematically to derive structure-property relationships.

Structure of a beheaded 30 S ribosomal subunit from Thermus thermophilus.

The 22 S ribonucleoproten particles containing the 5' (body) and the central (platform) domains of the Thermus thermophilus 30 S subunit has been studied by sedimentation, neutron scattering and electron microscopy. The RNP particles have been obtained by oligonucleotide-directed cleavage of 16 S RNA with ribonulease H in the region of the 900th nucleotide of the protein-deficient derivatives of the 30 S subunits. It is shown that these RNP particles are very compact, though their form and dimensions differ slightly from those expected from the electron microscopy model of the 30 S subunit beheaded by computer simulation. The particles are subdivided into two structural domains whose mutual arrangement differs from that of the corresponding morphological parts of the native 30 S subunit. Electron microscopy demonstrates that the mutual arrangement of domains in the RNP particles is not strictly fixed suggesting that interaction with the third domain of the 30 S subunit is a requisite for their correct fitting.

Small angle scattering in ribosomal structure research: localization of the messenger RNA within ribosomal elongation states.

Besides EM and biochemical studies small angle scattering (SAS) examinations have contributed significantly to our current knowledge about the ribosomal structure. SAS does not only allow the validation of competing models but permits independent model building. However, the major contribution of SAS to ribosomal structure research derived from its ability to reveal the spatial distribution of the individual ribosomal components (57 in the E. coli ribosome) within the ribosomal structure. More recently, an improved scattering method (proton-spin contrast variation) made it possible also to address the question of mapping functional ligands in defined ribosomal elongation states. Here, we review the contributions of SAS to the current understanding of the ribosome. Furthermore we present the direct localization of a small mRNA fragment within 70S elongation complexes and describe its movement upon the translocation reaction. The successful mapping of this fragment comprising only about 0.6% of the total mass of the complex proves that proton-spin contrast-variation is a powerful tool in modern ribosome research.

Structure of the elongating ribosome: arrangement of the two tRNAs before and after translocation.

The ribosome uses tRNAs to translate the genetic information into the amino acid sequence of proteins. The mass ratio of a tRNA to the ribosome is in the order of 1:100; because of this unfavorable value it was not possible until now to determine the location of tRNAs within the ribosome by neutron-scattering techniques. However, the new technique of proton-spin contrast-variation improves the signal-to-noise ratio by more than one order of magnitude, thus enabling the direct determination of protonated tRNAs within a deuterated ribosome for the first time. Here we analyze a pair of ribosomal complexes being either in the pre- or post-translocational states that represent the main states of the elongating ribosome. Both complexes were derived from one preparation. The orientation of both tRNAs within the ribosome and their mutual arrangement are determined by using an electron microscopy model for the Escherichia coli ribosome and the tRNA structure. The mass center of gravity of the (tRNA)2mRNA complex moves within the ribosome by 12 +/- 4 A in the course of translocation as previously reported. The main results of the present analysis are that the mutual arrangement of the two tRNAs does not change on translocation and that the angle between them is, depending on the model used, 110 degrees +/- 10 degrees before and after translocation. The translocational movement of the constant tRNA complex within the ribosome can be described as a displacement toward the head of the 30S subunit combined with a rotational movement by about 18 degrees.

Direct localization of the tRNAs within the elongating ribosome by means of neutron scattering (proton-spin contrast-variation).

A new technique for neutron scattering, the proton-spin contrast-variation, improves the signal-to-noise ratio more than one order of magnitude as compared to conventional techniques. The improved signal enables small RNA ligands within a large deuterated ribonucleic acid-protein complex to be measured. We used this technique to determine the positions of the two tRNAs within the elongating ribosome before and after translocation. Using a four-sphere model for each of the L-shaped tRNAs, unequivocal solutions were found for the localization of the mass centre of both tRNAs. The centre of gravity is located in the interface cavity separating the ribosomal subunits near the neck of the 30 S subunit. It moves during translocation by 12(+/-4) A towards the head of the 30 S subunit and slightly towards the L1 protuberance of the 50 S subunit.