Time-lapsed assessment of microcrack initiation and propagation in murine cortical bone at submicrometer resolution.

The strength of bone tissue is not only determined by its mass, but also by other properties usually referred to as bone quality, such as microarchitecture, distribution of bone cells, or microcracks and damage. It has been hypothesized that the bone ultrastructure affects microcrack initiation and propagation. Due to its high resolution, bone assessment by means of synchrotron radiation (SR)-based computed tomography (CT) allows unprecedented three-dimensional (3D) and non-invasive insights into ultrastructural bone phenotypes, such as the canal network and the osteocyte lacunar system. The aims of this study were to describe the initiation and propagation of microcracks and their relation with these ultrastructural phenotypes. To this end, femora from the two genetically distinct inbred mouse strains C3H/He (C3H) and C57BL/6 (B6) were loaded axially under compression, from 0% strain to failure, with 1% strain steps. Between each step, a high-resolution 3D image (700 nm nominal resolution) was acquired at the mid-diaphysis using SR CT for characterization and quantitative analysis of the intracortical porosity, namely the bone canal network, the osteocyte lacunar system and the emerging microcracks. For C3H mice, the canal, lacunar, and microcrack volume densities accounted typically for 1.91%, 2.11%, and 0.27% of the cortical total volume at 2% apparent strain, respectively. Due to its 3D nature, SR CT allowed to visualize and quantify also the volumetric extent of microcracks. At 2% apparent strain, the average microcrack thickness for both mouse strains was 2.0 microm for example. Microcracks initiated at canal and at bone surfaces, whereas osteocyte lacunae provided guidance to the microcracks. Moreover, we observed that microcracks could appear as linear cracks in one plane, but as diffuse cracks in a perpendicular plane. Finally, SR CT images permitted visualization of uncracked ligament bridging, which is thought to be of importance in bone toughening mechanisms. In conclusion, this study showed the power of SR CT for 3D visualization and quantification of the different ultrastructural phases of the intracortical bone porosity. We particularly postulate the necessity of 3D imaging techniques to unravel microcrack initiation and propagation and their effects on bone mechanics. We believe that this new investigation tool will be very useful to further enhance our understanding of bone failure mechanisms.

Batch fabrication of carbon nanotube bearings.

Relative displacements between the atomically smooth, nested shells in multiwalled carbon nanotubes (MWNTs) can be used as a robust nanoscale motion enabling mechanism. Here, we report on a novel method suited for structuring large arrays of MWNTs into such nanobearings in a parallel fashion. By creating MWNT nanostructures with nearly identical electrical circuit resistance and heat transport conditions, uniform Joule heating across the array is used to simultaneously engineer the shell geometry via electric breakdown. The biasing approach used optimizes process metrics such as yield and cycle-time. We also present the parallel and piecewise shell engineering at different segments of a single nanotube to construct multiple, but independent, high density bearings. We anticipate this method for constructing electromechanical building blocks to be a fundamental unit process for manufacturing future nanoelectromechanical systems (NEMS) with sophisticated architectures and to drive several nanoscale transduction applications such as GHz-oscillators, shuttles, memories, syringes and actuators.

Time-lapsed investigation of three-dimensional failure and damage accumulation in trabecular bone using synchrotron light.

Synchrotron radiation micro-computed tomography (SRmicroCT) is a very useful technique when it comes to three-dimensional (3D) imaging of complex internal and external geometries. Being a fully non-destructive technique, SRmicroCT can be combined with other experiments in situ for functional imaging. We are especially interested in the combination of SRmicroCT with mechanical testing in order to gain new insights in the failure mechanism of trabecular bone. This interest is motivated by the immense costs in health care due to patients suffering from osteoporosis, a systemic skeletal disease resulting in decreased bone stability and increased fracture risk. To better investigate the different failure mechanisms on the microlevel, we have developed a novel in situ mechanical compression device, capable of exerting both static and dynamic displacements on experimental samples. The device was calibrated for mechanical testing using solid aluminum and bovine trabecular bone samples. To study different failure mechanisms in trabecular bone, we compared a fatigued and a non-fatigued bovine bone sample with respect to failure initiation and propagation. The fatigued sample failed in a burst-like fashion in contrast to the non-fatigued sample, which exhibited a distinct localized failure band. Moreover, microscopic cracks - microcracks and microfractures - were uncovered in a 3D fashion illustrating the failure process in great detail. The majority of these cracks were connected to a bone surface. The data also showed that the classification of microcracks and -fractures from 2D section can sometimes be ambiguous, which is also true for the distinction of diffuse and distinct microdamage. Detailed investigation of the failure mechanism in these samples illustrated that trabecular bone often fails in delamination, providing a mechanism for energy dissipation while conserving trabecular bone architecture. In the future, this will allow an even better understanding of bone mechanics related to its hierarchical structural organization.

3D morphology of cell cultures: a quantitative approach using micrometer synchrotron light tomography.

Current issues in both tissue engineering and cell biology deal with cell behavior extensively in 3D. Here, we explore synchrotron radiation micro-computed tomography as a tool for morphological characterization of such 3D cellular constructs, providing micrometer resolution in soft and hard tissues. Novel image processing techniques allowed quantification of local and global cell distributions, cell density, adhesive cell culture surface, and scaffold geometry. For proof of concept, we applied this technique to characterize the morphology of two cell cultures of different phenotypes, namely human dermal fibroblasts and mouse calvarial osteoblast-like cells, both seeded on a polymer multifilament yarn. From 3D visualizations in these case studies, we saw that the fibroblasts spanned between the yarn filaments and in this way encapsulated the yarn, whereas the osteoblast-like cells lined the filament surfaces and did not span between them. Differences found in cell distribution as a function of distance to the median yarn axis and the closest filament surface, respectively, quantified these qualitative impressions gained from 3D visualizations. Moreover, the volume-normalized adhesive surface differed by one order of magnitude between the two phenotypes. Our approach allows quantitative correlation of local scaffold geometry and cell morphology. It can be used to investigate the influence of cell phenotype as well as various biochemical agents on tissue engineering constructs and the behavior of cells in culture.

Multiwavelength shearography for quantitative measurements of two-dimensional strain distributions.

We report on the development of a multiwavelength speckle pattern shearing interferometer for the determination of two-dimensional strain distributions. This system is based on simultaneous illumination of the object with three diode lasers that emit at different wavelengths between 800 and 850 nm. Wavelength separation and image acquisition were performed with a special optical arrangement, including narrow-bandpass filters and three black-and-white cameras. The shearographic camera with a variable shearing element, in combination with the appropriate illumination geometry, permitted us to isolate all six displacement derivatives from phase-stepped fringe patterns. The optical system and the measurement procedure were validated with two different experiments. First, the shearographic sensor head was used for the determination of in-plane displacements, and, second, in-plane strain distributions of an aluminum block caused by temperature expansion were measured.

Additive-Subtractive Two-Wavelength ESPI Contouring by Using a Synthetic Wavelength Phase Shift.

The addition correlation of two speckle fields by simultaneousillumination at different wavelengths is used for object contouring ina Twyman-Green-type interferometer. Fringe visibility is enhancedwhen the stochastic speckle background intensity obtained from areference plane modulation is subtracted. We calculate the contourphase map by using a phase-shift algorithm in the syntheticwavelength. A comparison with a sequential illumination, phasedifference method based on a laser wavelength phase shift isgiven. The test setup does not need to be stable on aninterferometric scale, and therefore a method is provided that lendsitself to applications in noisy environments.

Beta-N-acetylhexosaminidase: a target for the design of antifungal agents.

This review provides biochemical, analytical, and biological background information relating to beta-N-acetylhexosaminidase (HexNAc'ase; EC 3.2.1.52) as an emerging target for the design of low-molecular-weight antifungals. The article includes the following: (1) a biochemical description of HexNAc'ase (reaction catalyzed, nomenclature, and mechanism of action) that sets it apart from other, similar enzymes; (2) an overview and a critical evaluation of methods to assay the enzyme, including in crude extracts (photo- and fluorometric procedures with model substrates; HPLC/pulsed amperometric detection of N-acetylglucosamine and chito-oligomers; end-point vs. rate measurements); (3) a summary of some general characteristics of HexNAc'ases from fungi and organisms of other types (Km values, substrate preference, and glycoconjugation); (4) an hypothesis of a specific target function of wall-associated HexNAc'ase (a component of the assembly of surface-located enzymes effecting a continuous turnover and remodelling of the wall fabric through its combined hydrolytic and transglycosylating activities, and a mediator enzyme acting in concert with chitinase and chitin synthase to provide for the controlled lysis and synthesis of chitin during growth); (5) a tabulation of the structural formulae of reaction-based HexNAc'ase inhibitors with Ki values < or = 100 microM (some of them representing transition state mimics that could serve as leads for the development of new antifungals); and (6) an outline of approaches towards the establishment of a three-dimensional model of HexNAc'ase suitable for a truly rational design of antimycotics as well as agricultural fungicides.