Image-plate sensitivity to x rays at 2 to 60 keV.

The sensitivity of Fuji SR and MS image plates (IPs) used in x-ray spectrometers on OMEGA and the National Ignition Facility has been measured using two techniques. A set of radioisotopes has been used to constrain image-plate sensitivity between 6 and 60 keV, while a Manson source has been used to expose image plates to x rays at energies between 1.5 and 8 keV. These data have shown variation in sensitivity on the order of 5% for a given IP type and scanner settings. The radioisotope technique has also been used to assess IP fading properties for MS-type plates over long times. IP sensitivity as a function of scanner settings and pixel size has been systematically examined, showing variations of up to a factor of 2 depending on the IP type. Cross-calibration of IP scanners at different facilities is necessary to produce a consistent absolute sensitivity curve spanning the energy range of 2-60 keV.

Measuring and predicting the diffraction properties of cylindrically bent potassium acid phthalate, KAP(001), crystals.

This report presents the results from measuring the X-ray diffraction properties of a curved potassium acid phthalate (KAP(001)) spectrometer crystal using two measurement methods. One method used a diode type X-ray source and a dual goniometer analysis system, utilizing a flat, perfect KAP(001) crystal as the monochromator. The second method used a synchrotron source and dual crystal Si(111) monochromator. Bent crystals are used in X-ray spectrometers as dispersion elements. These crystals are bent into a circular cylinder section, and this bending can alter the rocking curve properties. The crystal rocking curves were measured for spectral energies ranging from 1250 to 4500 eV. A multi-lamellar model was compared to the measurements and showed good quantitative agreement. This provides a valuable tool for predicting the changes to the KAP (001) for any radius of curvature when the crystal is bent into a cylindrical section.

A technique for measuring the quality of an elliptically bent pentaerythritol [PET(002)] crystal.

We present a technique for determining the X-ray spectral quality from each region of an elliptically curved PET(002) crystal. The investigative technique utilizes the shape of the crystal rocking curve which changes significantly as the radius of curvature changes. This unique quality information enables the spectroscopist to verify where in the spectral range that the spectrometer performance is satisfactory and where there are regions that would show spectral distortion. A collection of rocking curve measurements for elliptically curved PET(002) has been built up in our X-ray laboratory. The multi-lamellar model from the XOP software has been used as a guide and corrections were applied to the model based upon measurements. But, the measurement of RI at small radius of curvature shows an anomalous behavior; the multi-lamellar model fails to show this behavior. The effect of this anomalous RI behavior on an X-ray spectrometer calibration is calculated. It is compared to the multi-lamellar model calculation which is completely inadequate for predicting RI for this range of curvature and spectral energies.

A multi-wavelength, high-contrast contact radiography system for the study of low-density aerogel foams.

A multi-wavelength, high contrast contact radiography system has been developed to characterize density variations in ultra-low density aerogel foams. These foams are used to generate a ramped pressure drive in materials strength experiments at the National Ignition Facility and require precision characterization in order to reduce errors in measurements. The system was used to characterize density variations in carbon and silicon based aerogels to ∼10.3% accuracy with ∼30 µm spatial resolution. The system description, performance, and measurement results collected using a 17.8 mg/cc carbon based JX-6 (C20H30) aerogel are discussed in this manuscript.

Primary cilia expression in bone marrow in response to mechanical stimulation in explant bioreactor culture.

Bone marrow contains a multitude of mechanically sensitive cells that may participate in mechanotransduction. Primary cilia are sensory organelles expressed on mesenchymal stem cells (MSCs), osteoblasts, osteocytes, and other cell types that sense fluid flow in monolayer culture. In marrow, cilia could similarly facilitate the sensation of relative motion between adjacent cells or interstitial fluid. The goal of this study was to determine the response of cilia to mechanical stimulation of the marrow. Bioreactors were used to supply trabecular bone explants with low magnitude mechanical stimulation (LMMS) of 0.3 ×g at 30 Hz for 1 h/d, 5 d/week, inducing shear stresses in the marrow. Four groups were studied: unstimulated (UNSTIM), stimulated (LMMS), and with and without chloral hydrate (UNSTIM+CH and LMMS+CH, respectively), which was used to disrupt cilia. After 19 days of culture, immunohistochemistry for acetylated α-tubulin revealed that more cells expressed cilia in culture compared to in vivo controls. Stimulation decreased the number of cells expressing cilia in untreated explants, but not in CH-treated explants. MSCs represented a greater fraction of marrow cells in the untreated explants than CH-treated explants. MSCs harvested from the stimulated groups were more proliferative than in the unstimulated explants, but this effect was absent from CH treated explants. In contrast to the marrow, neither LMMS nor CH treatment affected bone formation as measured by mineralising surface. Computational models indicated that LMMS does not induce bone strain, and the reported effects were thus attributed to shear stress in the marrow. From a clinical perspective, genetic or pharmaceutical alterations of cilia expression may affect marrow health and function.

Measuring the diffraction properties of an imaging quartz(211) crystal.

A dual goniometer X-ray system was used to measure the reflectivity curve for a spherically bent quartz(211) crystal. An analysis of the dual goniometer instrument response function for the rocking curve width measurement was developed and tested against the actual measurements. The rocking curve was measured at 4510.8 eV using the Ti Kα1 characteristic spectral line. The crystal is the dispersion element for a high resolution spectrometer used for plasma studies. It was expected to have a very narrow rocking curve width. The analysis showed that we could measure the upper bound for the rocking curve width of the Qz(211) crystal. The upper bound was 58 µrad giving a lower bound for the instrument resolving power E/ΔE = 34 000. Greatly improved insight into the dual goniometer operation and its limitations was achieved.

Osteocyte differentiation and the formation of an interconnected cellular network in vitro.

Extracellular matrix (ECM) stiffness and cell density can regulate osteoblast differentiation in two dimensional environments. However, it is not yet known how osteoblast-osteocyte differentiation is regulated within a 3D ECM environment, akin to that existing in vivo. In this study we test the hypothesis that osteocyte differentiation is regulated by a 3D cell environment, ECM stiffness and cell density. We encapsulated MC3T3-E1 pre-osteoblastic cells at varied cell densities (0.25, 1 and 2 × 106 cells/mL) within microbial transglutaminase (mtgase) gelatin hydrogels of low (0.58 kPa) and high (1.47 kPa) matrix stiffnesses. Cellular morphology was characterised from phalloidin-FITC and 4',6-diamidino-2-phenylindole (DAPI) dilactate staining. In particular, the expression of cell dendrites, which are phenotypic of osteocyte differentiation, were identified. Immunofluorescent staining for the osteocytes specific protein DMP-1 was conducted. Biochemical analyses were performed to determine cell number, alkaline phosphatase activity and mineralisation at 2.5 hours, 3, 21 and 56 days. We found that osteocyte differentiation and the formation of an interconnected network between dendritic cells was significantly increased within low stiffness 3D matrices, compared to cells within high stiffness matrices, at high cell densities. Moreover we saw that this network was interconnected, expressed DMP-1 and also connected with osteoblast-like cells at the matrix surface. This study shows for the first time the role of the 3D physical nature of the ECM and cell density for regulating osteocyte differentiation and the formation of the osteocyte network in vitro. Future studies could apply this method to develop 3D tissue engineered constructs with an osteocyte network in place.

Investigation of the optimal timing for chondrogenic priming of MSCs to enhance osteogenic differentiation in vitro as a bone tissue engineering strategy.

Recent in vitro tissue engineering approaches have shown that chondrogenic priming of human bone marrow mesenchymal stem cells (MSCs) can have a positive effect on osteogenesis in vivo. However, whether chondrogenic priming is an effective in vitro bone regeneration strategy is not yet known. In particular, the appropriate timing for chondrogenic priming in vitro is unknown albeit that in vivo cartilage formation persists for a specific period before bone formation. The objective of this study is to determine the optimum time for chondrogenic priming of MSCs to enhance osteogenic differentiation by MSCs in vitro. Pellets derived from murine and human MSCs were cultured in six different media groups: two control groups (chondrogenic and osteogenic) and four chondrogenic priming groups (10, 14, 21 and 28 days priming). Biochemical analyses (Hoechst, sulfate glycosaminoglycan (sGAG), Alkaline Phosphate (ALP), calcium), histology (Alcian Blue, Alizarin Red) and immunohistochemistry (collagen types I, II and X) were performed on the samples at specific times. Our results show that after 49 days the highest amount of sGAG production occurred in MSCs chondrogenically primed for 21 days and 28 days. Moreover we found that chondrogenic priming of MSCs in vitro for specific amounts of time (14 days, 21 days) can have optimum influence on their mineralization capacity and can produce a construct that is mineralized throughout the core. Determining the optimum time for chondrogenic priming to enhance osteogenic differentiation in vitro provides information that might lead to a novel regenerative treatment for large bone defects, as well as addressing the major limitation of core degradation and construct failure.

High Miller-index germanium crystals for high-energy x-ray imaging applications.

Near-normal-incidence bent crystals are widely used for x-ray imaging applications. Advantages include high collection solid angle and potentially high efficiency for narrow-band sources, while disadvantages include relatively large (several Å) interatomic spacings and a limited number of suitable matches between a crystal 2d value and an integral multiple of useful emission line wavelengths. The disadvantages become more significant at x-ray energies >10 keV. The former disadvantage can be mitigated by using high-order reflections from crystal planes having low Miller indices, but both disadvantages can be mitigated by using low-order reflections from crystal planes having high Miller indices. We report here on integrated reflectivity measurements we performed of Ge (15,7,7) (2d=0.6296 Å), a candidate for imaging Ru He-α (θ(B)=87°). We find good agreement with calculations, and the data show a multitude of closely spaced reflections with slightly different Bragg angles including a fifth-order reflection of Ge (3,1,1) that has comparable reflectivity. This demonstrates that arbitrary choices of Miller indices in Ge crystals can be used to fine-tune Bragg angles for near-normal-incidence x-ray imaging at tens of kiloelectron volt x-ray energies with minimal lower-energy contamination from lower-order reflections, and that existing calculational tools can be used to reliably estimate integrated reflectivity.

Measuring the x-ray resolving power of bent potassium acid phthalate diffraction crystals.

This report presents the results from measuring the X-ray resolving power of a curved potassium acid phthalate (KAP(001)) spectrometer crystal using two independent methods. It is part of a continuing effort to measure the fundamental diffraction properties of bent crystals that are used to study various characteristics of high temperature plasmas. Bent crystals like KAP(001) do not usually have the same diffraction properties as corresponding flat crystals. Models that do exist to calculate the effect of bending the crystal on the diffraction properties have simplifying assumptions and their accuracy limits have not been adequately determined. The type of crystals that we measured is being used in a spectrometer on the Z machine at Sandia National Laboratories in Albuquerque, New Mexico. The first technique for measuring the crystal resolving power measures the X-ray spectral line width of the characteristic lines from several metal anodes. The second method uses a diode X-ray source and a double crystal diffractometer arrangement to measure the reflectivity curve of the KAP(001) crystal. The width of that curve is inversely proportional to the crystal resolving power. The measurement results are analyzed and discussed.

Estrogen withdrawal from osteoblasts and osteocytes causes increased mineralization and apoptosis.

Recent studies have demonstrated increased bone mineral heterogeneity following estrogen withdrawal in vivo. Such changes likely contribute to fracture risk during post-menopausal osteoporosis since tissue mineralization is correlated with bone strength and stiffness. However, the cellular mechanisms responsible for increased mineral variability have not yet been distinguished. The objective of this study is to elucidate how alterations in mineral distribution are initiated during estrogen depletion. Specifically, we tested two separate hypotheses; (1) estrogen deficiency directly alters osteoblast mineralization and (2) estrogen deficiency increases bone cell apoptosis. Osteoblast-like cells (MC3T3-E1) and osteocyte-like cells (MLO-Y4) were pretreated with or without estrogen (17ß-estradiol) for 14 days. Estrogen deficiency was subsequently induced by either withdrawing estrogen from cells or blocking estrogen receptors using an estrogen antagonist, fulvestrant (ICI 182,780). Cell number (Hoechst DNA), alkaline phosphatase activity (p-NPP), mineralization (alizarin red) and apoptosis (Caspase 3/7) were evaluated. Whether estrogen withdrawal altered apoptosis rates in the presence of an apoptosis promoting agent (etoposide) was also determined. Interestingly, estrogen withdrawal from cells accustomed to estrogen exposure caused significantly increased osteoblast mineralization and osteocyte apoptosis compared with continued estrogen treatment. In contrast, blocking estrogen receptors with fulvestrant abrogated the mineralization induced by estrogen treatment. When apoptosis was induced using etoposide, cells undergoing estrogen withdrawal increased apoptosis compared to cells with continued estrogen treatment. Recognizing the underlying mechanisms regulating bone cell mineralization and apoptosis during estrogen deficiency and their consequences is necessary to further our knowledge of osteoporosis.

Osteocyte differentiation is regulated by extracellular matrix stiffness and intercellular separation.

Osteocytes are terminally differentiated bone cells, derived from osteoblasts, which are vital for the regulation of bone formation and resorption. ECM stiffness and cell seeding density have been shown to regulate osteoblast differentiation, but the precise cues that initiate osteoblast-osteocyte differentiation are not yet understood. In this study, we cultured MC3T3-E1 cells on (A) substrates of different chemical compositions and stiffnesses, as well as, (B) substrates of identical chemical composition but different stiffnesses. The effect of cell separation was investigated by seeding cells at different densities on each substrate. Cells were evaluated for morphology, alkaline phosphatase (ALP), matrix mineralisation, osteoblast specific genes (Type 1 collagen, Osteoblast specific factor (OSF-2)), and osteocyte specific proteins (dentin matrix protein 1 (DMP-1), sclerostin (Sost)). We found that osteocyte differentiation (confirmed by dendritic morphology, mineralisation, reduced ALP, Col type 1 and OSF-2 and increased DMP-1 and Sost expression) was significantly increased on soft collagen based substrates, at low seeding densities compared to cells on stiffer substrates or those plated at high seeding density. We propose that the physical nature of the ECM and the necessity for cells to establish a communication network contribute substantially to a concerted shift toward an osteocyte-like phenotype by osteoblasts in vitro.

A fluid-structure interaction model to characterize bone cell stimulation in parallel-plate flow chamber systems.

Bone continuously adapts its internal structure to accommodate the functional demands of its mechanical environment and strain-induced flow of interstitial fluid is believed to be the primary mediator of mechanical stimuli to bone cells in vivo. In vitro investigations have shown that bone cells produce important biochemical signals in response to fluid flow applied using parallel-plate flow chamber (PPFC) systems. However, the exact mechanical stimulus experienced by the cells within these systems remains unclear. To fully understand this behaviour represents a most challenging multi-physics problem involving the interaction between deformable cellular structures and adjacent fluid flows. In this study, we use a fluid-structure interaction computational approach to investigate the nature of the mechanical stimulus being applied to a single osteoblast cell under fluid flow within a PPFC system. The analysis decouples the contribution of pressure and shear stress on cellular deformation and for the first time highlights that cell strain under flow is dominated by the pressure in the PPFC system rather than the applied shear stress. Furthermore, it was found that strains imparted on the cell membrane were relatively low whereas significant strain amplification occurred at the cell-substrate interface. These results suggest that strain transfer through focal attachments at the base of the cell are the primary mediators of mechanical signals to the cell under flow in a PPFC system. Such information is vital in order to correctly interpret biological responses of bone cells under in vitro stimulation and elucidate the mechanisms associated with mechanotransduction in vivo.

Integrated x-ray reflectivity measurements of elliptically curved pentaerythritol crystals.

The elliptically curved pentaerythritol (PET) crystals used in the Supersnout 2 x-ray spectrometer on the National Ignition Facility at Lawrence Livermore National Laboratory have been calibrated photometrically in the range of 5.5-16 keV. The elliptical geometry provides broad spectral coverage and minimizes the degradation of spectral resolution due to the finite source size. The reflectivity curve of the crystals was measured using a x-ray line source. The integrated reflectivity (R(I)) and width of its curve (ΔΘ) were the measurements of major interest. The former gives the spectrometer throughput, and the latter gives the spectrometer resolving power. Both parameters are found to vary considerably with the radius of curvature of the crystal and with spectral energy. The results are attributed to an enhanced mosaic effect due to the increase in curvature. There are also contributions from the crystal cleaving and gluing processes.

Soft x-ray images of the laser entrance hole of ignition hohlraums.

Hohlraums are employed at the national ignition facility to convert laser energy into a thermal x-radiation drive, which implodes a fusion capsule, thus compressing the fuel. The x-radiation drive is measured with a low spectral resolution, time-resolved x-ray spectrometer, which views the region around the hohlraum's laser entrance hole. This measurement has no spatial resolution. To convert this to the drive inside the hohlraum, the size of the hohlraum's opening ("clear aperture") and fraction of the measured x-radiation, which comes from this opening, must be known. The size of the clear aperture is measured with the time integrated static x-ray imager (SXI). A soft x-ray imaging channel has been added to the SXI to measure the fraction of x-radiation emitted from inside the clear aperture. A multilayer mirror plus filter selects an x-ray band centered at 870 eV, near the peak of the x-ray spectrum of a 300 eV blackbody. Results from this channel and corrections to the x-radiation drive are discussed.

Heat-shock-induced cellular responses to temperature elevations occurring during orthopaedic cutting.

Severe heat-shock to bone cells caused during orthopaedic procedures can result in thermal damage, leading to cell death and initiating bone resorption. By contrast, mild heat-shock has been proposed to induce bone regeneration. In this study, bone cells are exposed to heat-shock for short durations occurring during surgical cutting. Cellular viability, necrosis and apoptosis are investigated immediately after heat-shock and following recovery of 12, 24 h and 4 days, in osteocyte-like MLO-Y4 and osteoblast-like MC3T3-E1 cells, using flow cytometry. The regeneration capacity of heat-shocked Balb/c mesenchymal stem cells (MSCs) and MC3T3-E1s has been investigated following 7 and 14 day's recovery, by quantifying proliferation, differentiation and mineralization. An immediate necrotic response to heat-shock was shown in cells exposed to elevated temperatures (45°C, 47°C and most severe at 60°C). A longer-term apoptotic response is induced in MLO-Y4s and, to a lesser extent, in MC3T3-E1s. Heat-shock-induced differentiation and mineralization by MSCs. These findings indicate that heat-shock is more likely to induce apoptosis in osteocytes than osteoblasts, which might reflect their role as sensors detecting and communicating damage within bone. Furthermore, it is shown for the first time that mild heat-shock (less than equal to 47°C) for durations occurring during surgical cutting can positively enhance osseointegration by osteoprogenitors.

High performance imaging streak camera for the National Ignition Facility.

An x-ray streak camera platform has been characterized and implemented for use at the National Ignition Facility. The camera has been modified to meet the experiment requirements of the National Ignition Campaign and to perform reliably in conditions that produce high electromagnetic interference. A train of temporal ultra-violet timing markers has been added to the diagnostic in order to calibrate the temporal axis of the instrument and the detector efficiency of the streak camera was improved by using a CsI photocathode. The performance of the streak camera has been characterized and is summarized in this paper. The detector efficiency and cathode measurements are also presented.

High-energy x-ray backlighter spectrum measurements using calibrated image plates.

The x-ray spectrum between 18 and 88 keV generated by a petawatt laser driven x-ray backlighter target was measured using a 12-channel differential filter pair spectrometer. The spectrometer consists of a series of filter pairs on a Ta mask coupled with an x-ray sensitive image plate. A calibration of Fuji™ MS and SR image plates was conducted using a tungsten anode x-ray source and the resulting calibration applied to the design of the Ross pair spectrometer. Additionally, the fade rate and resolution of the image plate system were measured for quantitative radiographic applications. The conversion efficiency of laser energy into silver Kα x rays from a petawatt laser target was measured using the differential filter pair spectrometer and compared to measurements using a single photon counting charge coupled device.

Images of the laser entrance hole from the static x-ray imager at NIF.

The static x-ray imager at the National Ignition Facility is a pinhole camera using a CCD detector to obtain images of Hohlraum wall x-ray drive illumination patterns seen through the laser entrance hole (LEH). Carefully chosen filters, combined with the CCD response, allow recording images in the x-ray range of 3-5 keV with 60 µm spatial resolution. The routines used to obtain the apparent size of the backlit LEH and the location and intensity of beam spots are discussed and compared to predictions. A new soft x-ray channel centered at 870 eV (near the x-ray peak of a 300 eV temperature ignition Hohlraum) is discussed.