Evaluating 3D bone tissue engineered constructs with different seeding densities using the alamarBlue™ assay and the effect on in vivo bone formation

Bone tissue engineering using patient derived cells seeded onto porous scaffolds has gained much attention in recent years. Evaluating the viability of these 3D constructs is an essential step in optimizing the process. The alamarBlue (aB) assay was evaluated for its potential to follow in vitro cell proliferation on architecturally standardized hydroxyapatite scaffolds. The impact of the aB assayed and seeding density on subsequent in vivo bone formation was investigated. Twelve scaffolds were seeded with various densities from 250 to 2.5×10⁶ cells/scaffold and assay by aB at 5 time points during the 7-day culture period. Twelve additional scaffolds were seeded with 2.5×10⁵ cells/scaffold. Two control and 2 aB treated scaffolds were subcutaneously implanted into each of 6 nude mice for 6 weeks. Four observers ranked bone formation using a pair wise comparison of histological sections form each mouse. The aB assay successfully followed cell proliferation, however, the diffusion kinetics of the 3D constructs must be considered. The influence of in vitro aB treatment on subsequent in vivo bone formation cannot be ruled out but was not shown to be significant in the current study. The aB assay appears to be quite promising for evaluating a maximum or end-point viability of 3D tissue engineered constructs. Finally, higher seeding densities resulted in more observed bone formation.     

Wave-front sensing from defocused images by use of wave-front slopes

We describe a novel technique for deriving wave-front aberrations from two defocused intensity measurements. The intensity defines a probability density function, and the method is based on the evolution of the cumulative density function of the intensity with light propagation. In one dimension, the problem is easily solved with a histogram specification procedure, with a linear relationship between the wave-front slope and the difference in the abscissas of the histograms. In two dimensions, the method requires use of a Radon transform. Simulation results demonstrate that good reconstructions can be attained down to 100 photons in each detector. In addition, the method is insensitive to scintillation at the aperture.     

The principle cause for lower plate numbers in capillary zone electrophoresis with most organic solvents.

With longitudinal diffusion as an unavoidable source of peak broadening, the peak efficiency (expressed by the plate number, N) in capillary zone electrophoresis depends on the ratio of electrophoretic mobility, mu, and tracer- or self-diffusion coefficient, D. Both parameters are functions of the ionic strength of the electrolyte solution. According to theory, the mobility is decreased with increasing ionic strength by the relaxation effect (depending on the relative permittivity) and the electrophoretic effect (depending on the relative permittivity and the viscosity of the solvent), whereas the diffusion coefficient is decreased only by the relaxation effect. This allows the theoretical predictions that the plate number, which is proportional to the ratio mu/D, decreases with increasing ionic strength and that the magnitude of this reduction depends on the solvent. Taking the values for relative permittivity and viscosity allows forecasting that, in general, water as a solvent exhibits the smallest lowering of the plate number, as compared to organic solvents. The theoretical predictions are confirmed by the data for the ratio calculated from measured mobilities and diffusion coefficients for iodide as the analyte ion in water, methanol, and acetonitrile with ionic strength of the background electrolyte varying between 0.005 and 0.080 mol L(-1). Whereas the experimentally observed plate number per volt is reduced from its "ultimate value" of about 20 (analyte charge number z = 1, zero ionic strength) in water by only 10%, the decrease at the same ionic strength in methanol and acetonitrile reaches 25 to 30%. Thus, the maximum plate number should read Nmax approximately equals 13 zU (with U being the effective voltage) for these solvents with ionic strengths normally applied in capillary electrophoresis. This reduction is not stemming from inappropriate experimental conditions, but has fundamental physicochemical causes.     

Integrated microfluidic system enabling (bio)chemical reactions with on-line MALDI-TOF mass spectrometry

A continuous flow micro total analysis system (micro-TAS) consisting of an on-chip microfluidic device connected to a matrix assisted laser desorption ionization [MALDI] time-of-flight [TOF] mass spectrometer (MS) as an analytical screening system is presented. Reaction microchannels and inlet/outlet reservoirs were fabricated by powderblasting on glass wafers that were then bonded to silicon substrates. The novel lab-on-a-chip was realized by integrating the microdevice with a MALDI-TOFMS standard sample plate used as carrier to get the microfluidic device in the MALDI instrument. A novel pressure-driven pumping mechanism using the vacuum of the instrument as a driving force induces flow in the reaction microchannel in a self-activating way. Organic syntheses as well as biochemical reactions are carried out entirely inside the MALDI-MS ionization vacuum chamber and analyzed on-line by MALDI-TOFMS in real time. The effectiveness of the micro-TAS system has been successfully demonstrated with several examples of (bio)chemical reactions.     

Simultaneous measurement of foveal spectral reflectance and cone-photoreceptor directionality

An instrument for simultaneous measurement of foveal spectral reflectance and cone-photoreceptor directionality is described. The key element is an imaging spectrograph (spectral range of 420-790 nm) with its entrance slit conjugate to the pupil plane of a human eye. A 1.9-deg spot on the retina is sampled in 1 s. Video observation of the retina and the pupil facilitates proper alignment. Measurements were performed on 21 healthy subjects. Model analysis of the spectra provided densities of photostable ocular absorbers. As an example, macular pigment and melanin are discussed in more detail. Spatial profiles exhibited the optical Stiles-Crawford effect, reflecting cone-photoreceptor directionality.     

Benchmarking international scientific excellence: Are highly cited research papers an appropriate frame of reference?

This paper introduces a citation-based "systems approach" for analyzing the various institutional and cognitive dimensions of scientific excellence within national research systems. The methodology, covering several aggregate levels, focuses on the most highly cited research papers in the international journal literature. The distribution of these papers across institutions and disciplines enables objective comparisons their (possible) international-level scientific excellence. By way of example, we present key results from a recent series of analyses of the research system in the Netherlands in the mid 1990s, focussing on the performance of the universities across the various major scientific disciplines within the context of the entire system"s scientific performance. Special attention is paid to the contribution in the world"s top 1% and top 10% most highly cited research papers. The findings indicate that these high performance papers provide a useful analytical framework - both in terms of transparency, cognitive and institutional differentiation, as well as its scope for domestic and international comparisons - providing new indicators for identifying "world class" scientific excellence at the aggregate level. The average citation scores of these academic "Centres of Scientific Excellence" appear to be an inadequate predictor of their production of highly cited papers. However, further critical reflection and in-depth validation studies are needed to establish the true potential of this approach for science policy analyses and evaluation of research performance. This revised version was published online in August 2006 with corrections to the Cover Date.     

Liouville Invariance in Quantum and Classical Mechanics

The density-matrix and Heisenberg formulations of quantum mechanics follow—for unitary evolution—directly from the Schrödinger equation. Nevertheless, the symmetries of the corresponding evolution operator, the Liouvillian L = i[, H], need not be limited to those of the Hamiltonian H. This is due to L only involving eigenenergy differences, which can be degenerate even if the energies themselves are not. Remarkably, this possibility has rarely been mentioned in the literature, and never pursued more generally. We consider an example involving mesoscopic Josephson devices, but the analysis only assumes familiarity with basic quantum mechanics. Subsequently, such L-symmetries are shown to occur more widely, in particular also in classical mechanics. The symmetry's relevance to dissipative systems and quantum-information processing is briefly discussed. PACS: 03.65.-w, 03.67.-a, 45.20.Jj, 74.50.+r     

Branching rates and growth functions in the outgrowth of dendritic branching patterns

The outgrowth of dendritic branching patterns proceeds by neurite elongation and branching. These actions are supported by growth cones, specialized dynamic structures at the tips of outgrowing neurites, in response to a multitude of intracellular and extracellular signals and mechanisms. Branching rates of growth cones and their temporal patterns thus reflect the extent and changes in these responses. The present study outlines a model framework to relate branching rates of individual growth cones with the growth rate of the entire dendritic tree. The branching rate of an individual growth cone is assumed to depend on the total number of growth cones at any given moment (representing competition between growth cones), on its position along the dendrite, and on a baseline component representing all other factors. Four different strategies are discussed for determining quantitatively these components from experimental data. The methods are applied in the analysis of dendritic trees of Wistar rat multipolar non-pyramidal neurons, quantitatively reconstructed at several developmental stages (Parnavelas J G and Uylings H B M 1980 Brain Res. 193 373-82, Uylings H B M, Parnavelas J G, Walg H and Veltman W A M 1980 Mikroskopie 37 220-4). It is shown that the baseline branching rate is a rapidly decreasing function of time, indicating the largest baseline drive for branching in the early days of outgrowth.     

A shape analysis framework for neuromorphometry

This paper addresses in an integrated and systematic fashion the relatively overlooked but increasingly important issue of measuring and characterizing the geometrical properties of nerve cells and structures, an area often called neuromorphology. After discussing the main motivation for such an endeavour, a comprehensive mathematical framework for characterizing neural shapes, capable of expressing variations over time, is presented and used to underline the main issues in neuromorphology. Three particularly powerful and versatile families of neuromorphological approaches, including differential measures, symmetry axes/skeletons, and complexity, are presented and their respective potentials for applications in neuroscience are identified. Examples of applications of such measures are provided based on experimental investigations related to automated dendrogram extraction, mental retardation characterization, and axon growth analysis.