Evaluating the morphology of the degradation layer of pure magnesium via 3D imaging at resolutions below 40 nm.

Magnesium is attractive for the application as a temporary bone implant due to its inherent biodegradability, non-toxicity and suitable mechanical properties. The degradation process of magnesium in physiological environments is complex and is thought to be a diffusion-limited transport problem. We use a multi-scale imaging approach using micro computed tomography and transmission X-ray microscopy (TXM) at resolutions below 40 nm. Thus, we are able to evaluate the nanoporosity of the degradation layer and infer its impact on the degradation process of pure magnesium in two physiological solutions. Magnesium samples were degraded in simulated body fluid (SBF) or Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) for one to four weeks. TXM reveals the three-dimensional interconnected pore network within the degradation layer for both solutions. The pore network morphology and degradation layer composition are similar for all samples. By contrast, the degradation layer thickness in samples degraded in SBF was significantly higher and more inhomogeneous than in DMEM+10%FBS. Distinct features could be observed within the degradation layer of samples degraded in SBF, suggesting the formation of microgalvanic cells, which are not present in samples degraded in DMEM+10%FBS. The results suggest that the nanoporosity of the degradation layer and the resulting ion diffusion processes therein have a limited influence on the overall degradation process. This indicates that the influence of organic components on the dampening of the degradation rate by the suppression of microgalvanic degradation is much greater in the present study.

Three-dimensional imaging of xylem at cell wall level through near field nano holotomography.

Detailed imaging of the three-dimensionally complex architecture of xylary plants is important for studying biological and mechanical functions of woody plants. Apart from common two-dimensional microscopy, X-ray micro-computed tomography has been established as a three-dimensional (3D) imaging method for studying the hydraulic function of wooden plants. However, this X-ray imaging method can barely reach the resolution needed to see the minute structures (e.g. pit membrane). To complement the xylem structure with 3D views at the nanoscale level, X-ray near-field nano-holotomography (NFH) was applied to analyze the wood species Pinus sylvestris and Fagus sylvatica. The demanded small specimens required focused ion beam (FIB) application. The FIB milling, however, influenced the image quality through gallium implantation on the cell-wall surfaces. The measurements indicated that NFH is appropriate for imaging wood at nanometric resolution. With a 26 nm voxel pitch, the structure of the cell-wall surface in Pinus sylvestris could be visualized in genuine detail. In wood of Fagus sylvatica, the structure of a pit pair, including the pit membrane, between two neighboring fibrous cells could be traced tomographically.

Label-free highly sensitive probe detection with novel hierarchical SERS substrates fabricated by nanoindentation and chemical reaction methods.

Nanostructures have been widely employed in surface-enhanced Raman scattering (SERS) substrates. Recently, in order to obtain a higher enhancement factor at a lower detection limit, hierarchical structures, including nanostructures and nanoparticles, appear to be viable SERS substrate candidates. Here we describe a novel method integrating the nanoindentation process and chemical redox reaction to machine a hierarchical SERS substrate. The micro/nanostructures are first formed on a Cu(110) plane and then Ag nanoparticles are generated on the structured copper surface. The effect of the indentation process parameters and the corrosion time in the AgNO3 solution on the Raman intensities of the SERS substrate with hierarchical structures are experimentally studied. The intensity and distribution of the electric field of single and multiple Ag nanoparticles on the surface of a plane and with multiple micro/nanostructures are studied with COMSOL software. The feasibility of the hierarchical SERS substrate is verified using R6G molecules. Finally, the enhancement factor using malachite green molecules was found to reach 5.089 × 109, which demonstrates that the production method is a simple, reproducible and low-cost method for machining a highly sensitive, hierarchical SERS substrate.

Chondrogenic differentiation of ATDC5-cells under the influence of Mg and Mg alloy degradation.

Biodegradable magnesium (Mg)-based materials are a potential alternative to permanent implants for application in children. Nevertheless effects of those materials on growth plate cartilage and chondrogenesis have not been previously evaluated. In vitro differentiation of ATDC5 cells was evaluated under the influence of pure Mg (PMg), Mg with 10wt% of gadolinium (Mg-10Gd) and Mg with 2wt% of silver (Mg-2Ag) degradation products (extracts) and direct cell culture on the materials. Gene expression showed an inhibitory effect on ATDC5 mineralization with the three extracts and a chondrogenic potential of Mg-10Gd. Cells cultured in Mg-10Gd and Mg-2Ag extracts showed the same proliferation and morphology than cells cultured in growth conditions. Mg-10Gd induced an increase in production of ECM and a bigger cell size, similar to the effects found with differentiation conditions. An increased metabolic activity was observed in cells cultured under the influence of Mg-10Gd extracts, indicated by an acidic pH during most of the culture period. After 7days of culture on the materials, ATDC5 growth, distribution and ECM synthesis were higher on Mg-10Gd samples, followed by Mg-2Ag and PMg, which was influenced by the homogeneity and composition of the degradation layer. This study confirmed the tolerance of ATDC5 cells to Mg-based materials and a chondrogenic effect of Mg-10Gd. Further studies in vitro and in vivo are necessary to evaluate cell reactions to those materials, as well as the effects on bone growth and the biocompatibility of the alloying system in the body.

Determination of the packing fraction in photonic glass using synchrotron radiation nanotomography.

Photonic glass is a material class that can be used as photonic broadband reflectors, for example in the infrared regime as thermal barrier coating films. Photonic properties such as the reflectivity depend on the ordering and material packing fraction over the complete film thickness of up to 100 µm. Nanotomography allows acquiring these key parameters throughout the sample volume at the required resolution in a non-destructive way. By performing a nanotomography measurement at the PETRA III beamline P05 on a photonic glass film, the packing fraction throughout the complete sample thickness was analyzed. The results showed a packing fraction significantly smaller than the expected random close packing giving important information for improving the fabrication and processing methods of photonic glass material in the future.

The Degradation Interface of Magnesium Based Alloys in Direct Contact with Human Primary Osteoblast Cells.

Magnesium alloys have been identified as a new generation material of orthopaedic implants. In vitro setups mimicking physiological conditions are promising for material / degradation analysis prior to in vivo studies however the direct influence of cell on the degradation mechanism has never been investigated. For the first time, the direct, active, influence of human primary osteoblasts on magnesium-based materials (pure magnesium, Mg-2Ag and Mg-10Gd alloys) is studied for up to 14 days. Several parameters such as composition of the degradation interface (directly beneath the cells) are analysed with a scanning electron microscope equipped with energy dispersive X-ray and focused ion beam. Furthermore, influence of the materials on cell metabolism is examined via different parameters like active mineralisation process. The results are highlighting the influences of the selected alloying element on the initial cells metabolic activity.

Neutron study of phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine spray coating on titanium implants.

Permanent implants made from titanium are widely used and successfully implemented in medicine to address problems related to orthopedic and oral disorders. However, implants that interact in all cases optimally and durably with bone tissue have yet to be developed. Here, the authors suggest a phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine (POPE) lipid coating to partially mimic the biological cell membrane. To improve the homogeneity of the POPE distribution on the metal surface, the lipids are applied by spray coating. It is shown that the spray coating leads to two types of multilamellar POPE structures. Our experimental results demonstrate that these coatings are stable in a liquid environment in the range of physiological temperatures due to the unique interbilayer interaction of POPE lipids. Additionally, the interaction of the POPE multilayer structure with human serum albumin is considered. A simultaneous analysis of the specular and off-specular data provides structural information necessary to assess the quality of the coating for future applications.

On radiation damage in FIB-prepared softwood samples measured by scanning X-ray diffraction.

The high flux density encountered in scanning X-ray nanodiffraction experiments can lead to severe radiation damage to biological samples. However, this technique is a suitable tool for investigating samples to high spatial resolution. The layered cell wall structure of softwood tracheids is an interesting system which has been extensively studied using this method. The tracheid cell has a complex geometry, which requires the sample to be prepared by cutting it perpendicularly to the cell wall axis. Focused ion beam (FIB) milling in combination with scanning electron microscopy allows precise alignment and cutting without splintering. Here, results of a scanning X-ray diffraction experiment performed on a biological sample prepared with a focused ion beam of gallium atoms are reported for the first time. It is shown that samples prepared and measured in this way suffer from the incorporation of gallium atoms up to a surprisingly large depth of 1 µm.

A Porous TiAl6V4 Implant Material for Medical Application.

Increased durability of permanent TiAl6V4 implants still remains a requirement for the patient's well-being. One way to achieve a better bone-material connection is to enable bone "ingrowth" into the implant. Therefore, a new porous TiAl6V4 material was produced via metal injection moulding (MIM). Specimens with four different porosities were produced using gas-atomised spherical TiAl6V4 with different powder particle diameters, namely, "Small" (<45 µm), "Medium" (45-63 µm), "Mix" (90% 125-180 µm + 10% <45 µm), and "Large" (125-180 µm). Tensile tests, compression tests, and resonant ultrasound spectroscopy (RUS) were used to analyse mechanical properties. These tests revealed an increasing Young's modulus with decreasing porosity; that is, "Large" and "Mix" exhibit mechanical properties closer to bone than to bulk material. By applying X-ray tomography (3D volume) and optical metallographic methods (2D volume and dimensions) the pores were dissected. The pore analysis of the "Mix" and "Large" samples showed pore volumes between 29% and 34%, respectively, with pore diameters ranging up to 175 µm and even above 200 µm for "Large." Material cytotoxicity on bone cell lines (SaOs-2 and MG-63) and primary cells (human bone-derived cells, HBDC) was studied by MTT assays and highlighted an increasing viability with higher porosity.

Element distribution in the corrosion layer and cytotoxicity of alloy Mg-10Dy during in vitro biodegradation.

The present work investigates the corrosion behaviour, the element distribution in the corrosion layer and the cytocompatibility of alloy Mg-10Dy. The corrosion experiments were performed in a cell culture medium (CCM) under cell culture conditions close to the in vivo environment. The element distribution on the surface as well as in cross-sections of the corrosion layer was investigated using scanning electron microscopy, energy-dispersive X-ray analysis, X-ray photoelectron spectroscopy and X-ray diffraction. The cytocompatibility of alloy Mg-10Dy with primary human osteoblasts was evaluated by MTT, cell adhesion and live/dead staining tests. The results show that the corrosion layer was enriched in Dy, while the P and Ca content gradually decreased from the surface to the bottom of the corrosion layer. In addition, large amounts of MgCO3·3H2O formed in the corrosion layer after 28 days immersion. Both extracts and the Dy-enriched corrosion layer of alloy Mg-10Dy showed no cytotoxicity to primary human osteoblasts.

Ion release from magnesium materials in physiological solutions under different oxygen tensions.

Although magnesium as degradable biomaterial already showed clinical proof of concepts, the design of new alloys requires predictive in vitro methods, which are still lacking. Incubation under cell culture conditions to obtain "physiological" corrosion may be a solution. The aim of this study was to analyse the influence of different solutions, addition of proteins and of oxygen availability on the corrosion of different magnesium materials (pure Mg, WE43, and E11) with different surface finishing. Oxygen content in solution, pH, osmolality and ion release were determined. Corrosion led to a reduction of oxygen in solution. The influence of oxygen on pH was enhanced by proteins, while osmolality was not influenced. Magnesium ion release was solution-dependent and enhanced in the initial phase by proteins with delayed release of alloying elements. The main corrosion product formed was magnesium carbonate. Therefore, cell culture conditions are proposed as first step toward physiological corrosion.