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International audience; The Total Focusing Method (TFM) was generalized about ten years ago to form images with complex ultrasoundpaths involving reflections and mode conversions at the interfaces of a testing sample. The resulting multi-mode imaging allows to fully image the face of a crack-type defect. More recently, the Plane Wave Imaging (PWI) was revisited to perform multi-mode reconstructions with a small number of transmissions compared to the TFM. In order to further accelerate the imaging process, we propose here to combine plane wave emissions with a fast reconstruction algorithm in the Fourier domain. The method was developed in medical imaging with the pioneeringworks of J.- Y. Lu, and was studied in a recent paper to form images in solids with direct reconstruction modes. In the present paper, the theory is extended to various reconstruction modes (half-skip and one-skip modes with/without mode conversions), and the imaging method is evaluated with experimental results in a sample featuring different planar defects. We show that the method provides multi-mode images equivalent to those computed with the time-domain PWI, while reducing the computation times by a factor up to 13.

In this paper, defects in a high density polyethylene pipe are imaged with the total focusing method. The viscoelastic attenuation of this material greatly reduces the signal level and leads to a poor signal-to-noise ratio (SNR) due to electronic noise. To improve the image quality, the decomposition of the time reversal operator method is combined with the spatial Hadamard coded transmissions before calculating images in the time domain. Because the Hadamard coding is not compatible with conventional imaging systems, this paper proposes two modified coding methods based on sparse Hadamard matrices with +1/0 coefficients. The SNRs expected with the different spatial codes are demonstrated, and then validated on both simulated and experimental data. Experiments are performed with a transducer array in contact with the base material of a polyethylene pipe. In order to improve the noise filtering procedure, the singular values associated with electronic noise are expressed on the basis of the random matrix theory. This model of noise singular values allows a better identification of the defect response in noisy experimental data. Finally, the imaging method is evaluated in a more industrial inspection configuration, where an immersion array probe is used to image defects in a butt fusion weld with a complex geometry.

Category: Basic Sciences/Biologics Introduction/Purpose: Charcot foot is a serious complication of diabetes present in up to 13% carrying with high morbidity (dislocations, fractures, deformities) and up to 14% mortality. In Charcot, osteoclasts are found in increased numbers and with increased activity3,4 while C-telopeptide of type I collagen (CTX) is upregulated as opposed to ALP suggesting this is indeed an osteoclast and not an osteoblast disease. Methods: The scientific literature was reviewed to identify relevant osteoclastic activators and their role in the hyper activated state. We will also show videos of our current micromanipulation research techniques used to study osteoclast inhibitors in the lab. Results: The blood of Charcot patients is different at the nano level. Micro-nano-particles are extracellular vesicles derived from cells into plasma. Microparticles are found in increased quantity in Charcot;which contain more cytokines; known osteoclast activators. MicroRNAs – Epigenetic changes also are in Charcot; 16 different circulating microRNA known osteoclast activators are associated with Charcot. Advanced glycation end products (AGE). The hyperglycemic state in Charcot is a driving force in pathogenesis. AGEs are normal proteins turned dud after hyperglycemic induced glycation, a post translational modification which is irreversible affects both intracellular and extracellular proteins. AGEs may bind receptor RAGE, increasing osteoclast activity . RANKL is the main osteoclast differentiator and activator. RANKL has been the target of osteoclast control almost since it was found in 1998 . Conclusion: Charcot mediated bony destruction and remodeling; both of which are mediated by the osteoclast has traditionally had a tremendous cytokine focus. Our review suggests the mechanism for modifying a Charcot response on a local and systemic level. It is interesting to note that osteoclast inhibitors Denosumab, Bisphosphonates, Calcitonin and osteoblcast activators rPTH are not completely effective in switching off the Charcot response, implying there is a far more complex interaction between the inflammatory response, osteoclastic stimulators and osteoblastic inhibitors.

Category: Diabetes Introduction/Purpose: Diabetic ulcers carry with it extensive morbidity. The microbiome of diabetic foot infections have previously been shown to be polymicrobial. We hypothesized that ulcer treatment would normalize the foot microbiome. Methods: Microbiome profiles of the fore, mid and hind diabetic foot. Diabetic foot (with ulcer) were taken pre and post treatment with a combination of surgical debridement, local and systemic antibiotics. 10 subjects were studied. Microbiome graphs and images pre and post treatment of feet are shown. Results: The microbiome profile of the diabetic foot almost completely normalized to that of normal flora. Before treatment, the foot microbiome was associated with polymicrobial pathogenic bacteria mostly away from the ulcer. The ulcer itself only contained minimal species, albeit pathogenic bacteria. The differences in profiles between the pre and post treatment were significantly different p < 0.01. Conclusion: Chicken or egg, the foot microbiome almost completely reverts to normal flora with simple ulcer debridement and local/systemic antibiotics. Our data point possibly suggest ulcers could be prevented by reducing pathogenic microbiome load (chlorhexidine washes) or microbiome transplants. We next move to controls as a proof of concept.

International audience; Time-domain plane-wave imaging (PWI) has recently emerged in medical imaging and is now taking to nondestructive testing (NDT) due to its ability to provide images of good resolution and contrast with only a few steered plane waves. Insonifying a medium with plane waves is a particularly interesting approach in 3-D imaging with matrix arrays because it allows to tremendously reduce the volume of data to be stored and processed as well as the acquisition time. However, even if the data volume is reduced with plane wave emissions, the image reconstruction in the time domain with a delay-and-sum algorithm is not sufficient to achieve low computation times in 3-D due to the number of voxels. Other reconstruction algorithms take place in the wavenumber-frequency (f-k) domain and have been shown to accelerate computation times in seismic imaging and in synthetic aperture radar. In this paper, we start from time-domain PWI in 2-D and compare it to two algorithms in the f-k domain, coming from the Stolt migration in seismic imaging and the Lu theory of limited diffraction beams in medical imaging. We then extend them to immersion testing configurations where a linear array is facing a plane water-steel interface. Finally, the reconstruction algorithms are generalized to 3-D imaging with matrix arrays. A comparison dwelling on image quality and algorithmic complexities is provided, as well as a theoretical analysis of the image amplitudes and the limits of each method. We show that the reconstruction schemes in the f-k domain improve the lateral resolution and offer a theoretical and numerical computation gain of up to 36 in 3-D imaging in a realistic NDT configuration. Index Terms-2-D and 3-D imaging, nondestructive testing (NDT), transducer arrays, ultrasounds, wavenumber-frequency (f-k) migration.

National audience; Ce travail vise à améliorer l'imagerie ultrasonore multi-éléments dans des aciers anisotropes issus du domaine nucléaire (soudures et revêtements en acier inoxydable). En général, les paramètres gouvernant la propagation des ultrasons dans ces aciers ne sont pas connus en cours d'inspection. Ceci peut donner lieu à des images très dégradées lorsque le modèle de propagation est trop éloigné du milieu physique. Pour améliorer la qualité des images, une méthode d'optimisation est étudiée. Elle consiste à faire varier les paramètres du modèle de propagation jusqu'à ce que l'image reconstruite satisfasse un critère d'optimalité choisi. Cette méthode associe un modèle de substitution pour contourner le modèle direct de propagation, qui est coûteux en temps de calcul, et un algorithme de montée de gradient pour maximiser une fonction coût multivariée. L'optimisation avec l'algorithme de montée du gradient est comparée à un algorithme d'optimisation global de type Essaim Particulaire. Enfin, les paramètres du modèle (tenseur d'élasticité, orientation des grains…) issus de l'optimisation sont comparés aux données mesurées par d'autres techniques de laboratoire.

International audience; An optimization method is studied to enhance the reliability of TFM (Total Focusing Method) images in anisotropic nuclear materials. The method is able to adapt to a given anisotropic structure (weld, cladded steel) when the parameters governing the wave propagation are uncertain. The optimization scheme combines a surrogate model to bypass the extensive computation times of the propagation forward model, and a gradient descent algorithm to minimize a multivariate cost function. The gradient-based local optimization is compared with a global optimization tool, the Particle Swarm algorithm. Finally, the parameters (stiffness constant, grain orientation, cladding thickness…) corresponding to the optimal TFM image are compared with those measured by other characterization techniques.

ObjectivesLevel of education and genetic risk are key predictors of cardiovascular disease (CVD). While several studies have explored the causal mechanisms of education effects, it remains uncertain to what extent genetic risk is mediated by established CVD risk factors. This study sought to investigate this and explored the mediation of education and genetic effects on CVD by established cardiovascular risk factors in the Framingham Heart Study (FHS).DesignProspective observational cohort study.Participants7017 participants from the FHS.SettingCommunity-based cohort of adults in Framingham, Massachusetts, USA.Primary outcome measureIncident CVD. The total effects of education and genetic predisposition using a 63-variant genetic risk score (GRS) on CVD, as well as those mediated by established CVD risk factors, were assessed via mediation analysis based on the counterfactual framework using Cox proportional hazards regression models.ResultsOver a median follow-up time of 12.0 years, 1091 participants experienced a CVD event. Education and GRS displayed significant associations with CVD after adjustment for age and sex and the established risk factors smoking, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), body mass index, systolic blood pressure (SBP) and diabetes. For education effects, smoking, HDL-C and SBP were estimated to mediate 18.8% (95% CI 9.5% to 43%), 11.5% (95% CI 5.7% to 29.0%) and 4.5% (95% CI 1.6% to 13.3%) of the total effect of graduate degree, respectively, with the collective of all risk factors combined mediating 38.5% (95% 24.1% to 64.9%). A much smaller proportion of the effects of GRS were mediated by established risk factors combined (17.6%, 95% CI 2.4% to 35.7%), with HDL-C and TC mediating 11.5% (95% CI 6.2% to 21.5%) and 3.1% (95% CI 0.2% to 8.3%), respectively.ConclusionsUnlike education inequalities, established risk factors mediated only a fraction of GRS effects on CVD. Further research is required to elucidate the underlying causal mechanisms of genetic contributions to CVD.

International audience; In the present work, the Synthetic Transmit Aperture (STA) imaging is combined with the Decomposition of the Time Reversal Operator (DORT) method to image a coarse grained austenitic-ferritic steel using a contact transducer array. The highly heterogeneous structure of this material produces a strong scattering noise in ultrasound images. Furthermore, the surface waves guided along the array interfere with the bulk waves backscattered by defects. In order to overcome these problems, the DORT method is applied before calculating images with the STA algorithm. The method consists in analyzing in the frequency domain the singular values and singular vectors of the full array transfer matrix. This paper first presents an analysis of the singular values of different waves contained in the data acquisition, which facilitates the identification of the subspace associated with the surface guided waves for filtering operations. Then, a filtered matrix is defined where the contribution of structural noise and guided waves are reduced. Finally, in the time domain, the STA algorithm is applied to this matrix in order to calculate an image with reduced structural noise. Experiments demonstrate that this filtering improves the signal-to-noise ratio by more than 12 dB in comparison with the STA image before filtering.

Conference of ICU International Congress on Ultrasonics, ICU 2015 ; Conference Date: 11 May 2015 Through 14 May 2015; International audience; In the present work, the Synthetic Transmit Aperture (STA) imaging is applied on coarse grained steels using a contact phased-array probe. In order to reduce the noise introduced by the heterogeneous structure, as well as artifacts due to surface guided waves, the Decomposition of the Time Reversal Operator method is performed before calculating STA images.

Investigations into the pharmacology of different types of cys-loop GABA receptor have relied for years on the chemical modification of GABA-like compounds. The GABA metabolite GABOB is an attractive molecule to modify due to its convenient chemical structure. In the process of developing new GABA-mimic compounds from GABOB as a starting compound three small molecule GABA derivatives were synthesized using a variety of chemical transformations. Amongst these, a new and reliable method to synthesize TACA (trans-4-aminocrotonic acid) is reported.

Expansion of pluripotent stem cells in defined media devoid of animal-derived feeder cells to generate multilayered three-dimensional (3D) bulk preparations or spheroids, rather than two-dimensional (2D) monolayers, is advantageous for many regenerative, biological or disease-modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder-free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty-like' spheroid structures in defined xeno-free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly, we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial-scale expansion and intricate organ–tissue engineering applications with human pluripotent stem cells, where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation.

Bone remodeling requires osteoclasts to generate and maintain an acidified resorption compartment between the apical membrane and the bone surface to solubilize hydroxyapatite crystals within the bone matrix. This acidification process requires ( i ) apical proton secretion by a vacuolar H + -ATPase, ( ii ) actin cytoskeleton reorganization into a podosome belt that forms a gasket to restrict lacunar acid leakage, and ( iii ) basolateral chloride uptake and bicarbonate extrusion by an anion exchanger to provide Cl − permissive for apical acid secretion while preventing cytoplasmic alkalinization. Here we show that osteoclast-targeted deletion in mice of solute carrier family 4 anion exchanger member 2 ( Slc4a2 ) results in osteopetrosis. We further demonstrate a previously unrecognized consequence of SLC4A2 loss of function in the osteoclast: dysregulation of calpain-dependent podosome disassembly, leading to abnormal actin belt formation, cell spreading, and migration. Rescue of SLC4A2-deficient osteoclasts with functionally defined mutants of SLC4A2 indicates regulation of actin cytoskeletal reorganization by anion-exchange activity and intracellular pH, independent of SLC4A2’s long N-terminal cytoplasmic domain. These data suggest that maintenance of intracellular pH in osteoclasts through anion exchange regulates the actin superstructures required for bone resorption.

Conference of 2016 IEEE International Ultrasonics Symposium, IUS 2016 ; Conference Date: 18 September 2016 Through 21 September 2016; Conference Code:124585; International audience; In this paper, we present two frequency-domain algorithms for 2D imaging in solids, namely Stolt's method and Lu's method, the latter being extended to the 3D imaging with matrix arrays. First, the performances of both frequency-domain methods are evaluated in 2D imaging and compared with the time-domain plane wave imaging (PWI). Then, Lu's method and PWI are applied to 3D to image a distribution of small porosities in a steel block. 2D and 3D images calculated with the f-k methods are comparable to the PWI images in terms of contrast and spatial resolution while achieving computation times more than 25 times shorter.

Conference of 43rd Annual Review of Progress in Quantitative Nondestructive Evaluation, QNDE 2016 ; Conference Date: 17 July 2016 Through 22 July 2016; Conference Code:126650; International audience; In the present work, the Total Focusing Method (TFM) is used to image defects in a High Density Polyethylene (HDPE) pipe. The viscoelastic attenuation of this material corrupts the images with a high electronic noise. In order to improve the image quality, the Decomposition of the Time Reversal Operator (DORT) filtering is combined with spatial Walsh-Hadamard coded transmissions before calculating the images. Experiments on a complex HDPE joint demonstrate that this method improves the signal-to-noise ratio by more than 40 dB in comparison with the conventional TFM.

Conference of 2016 IEEE International Ultrasonics Symposium, IUS 2016 ; Conference Date: 18 September 2016 Through 21 September 2016; Conference Code:124585; International audience; In this work, the total focusing method is used to image defects in a high density polyethylene pipe. The viscoelastic attenuation of this material corrupts the images with a high electronic noise. To improve the image quality, the decomposition of the time reversal operator method is combined with spatial Walsh-Hadamard coded transmissions before calculating the images.

Pre-print; International audience; This paper describes a new ultrasonic array imaging method for Non-Destructive Testing (NDT) which is derived from the medical Plane Wave Imaging (PWI) technique. The objective is to perform fast ultrasound imaging with high image quality. The approach is to transmit plane waves at several angles and to record the back-scattered signals with all the array elements. Focusing in receive is then achieved by coherent summations of the signals in every point of a region of interest. The medical PWI is generalized to immersion setups where water acts as a coupling medium and to multimodal (direct, half-skip modes) imaging in order to detect different types of defects (inclusions, porosities, cracks). This method is compared to the Total Focusing Method (TFM) which is the reference imaging technique in NDT. First, the two post-processing algorithms are described. Then experimental results with the array probe either in contact or in immersion are presented. A good agreement between the TFM and the PWI is observed, with three to ten times less transmissions required for the PWI.

Investigations into the pharmacology of different types of cys-loop GABA receptor have relied for years on the chemical modification of GABA-like compounds. The GABA metabolite GABOB is an attractive molecule to modify due to its convenient chemical structure. In the process of developing new GABA-mimic compounds from GABOB as a starting compound three small molecule GABA derivatives were synthesized using a variety of chemical transformations. Amongst these, a new and reliable method to synthesize TACA (trans-4-aminocrotonic acid) is reported.

Twersky's theory is generalized to multiple scattering by a uniform random distribution of cylinders in a poro-elastic medium. The high-frequency regime only, where no dispersion effects occur in the absence of scatterers, is investigated in the frame of Biot's theory. The scatterers lie within a slab of the host medium, and an incident wave gives rise to a fast longitudinal coherent wave, a slow longitudinal one, as well as a shear one in the slab. The dispersion equations of those three coherent waves are derived. The shear coherent wave propagates independently of the other two, while the longitudinal coherent waves obey a coupled dispersion equation involving conversion terms. Numerically speaking, coupling effects are significant only when forward scattering by a single cylinder of the fast wave into the slow one (or the slow wave into the fast) is larger than forward scattering with no conversion.

Conference of 42nd Annual Review of Progress in Quantitative Nondestructive Evaluation, QNDE 2015, Incorporating the 6th European-American Workshop on Reliability of NDE ; Conference Date: 26 July 2015 Through 31 July 2015; Conference Code:119404; International audience; In the present work, the Total Focusing Method (TFM) is used to image flaws in coarse-grained steels with a contact phased-array probe. In order to reduce the noise introduced by the heterogeneous structure, as well as artifacts due to surface guided waves, the Decomposition of the Time Reversal Operator method is performed before calculating TFM images.

Conference of 42nd Annual Review of Progress in Quantitative Nondestructive Evaluation, QNDE 2015, Incorporating the 6th European-American Workshop on Reliability of NDE ; Conference Date: 26 July 2015 Through 31 July 2015; Conference Code:119404; International audience; Ultrasonic imaging with high frame rates is of great interest to perform fast inspections and to reduce NDE costs. In this communication, we propose a new fast imaging method which is derived from the medical Plane Wave Imaging (PWI). The PWI method is applied to different immersion-testing configurations (specimens with plane or complex surfaces) and to different imaging modes (imaging with direct or half-skip wave paths) according to the type of defects (point-like or extended crack-types defects).

Immersion ultrasonic testing of structures with complex geometries may be significantly improved by using phased-arrays and specific adaptive algorithms that allow to image flaws under a complex and unknown interface. In this context, this paper presents a comparative study of different Surface Estimation Methods (SEM) available in the CIVA software and used for adaptive imaging. These methods are based either on time-of-flight measurements or on image processing. We also introduce a generalized adaptive method where flaws may be fully imaged with half-skip modes. In this method, both the surface and the back-wall of a complex structure are estimated before imaging flaws.

The scattering of elastic waves by a finite number of close cylindrical cavities embedded in an elastic matrix is considered. Depending on whether the cavities are empty or fluid-filled, the regimes of interaction between the scatterers, resonant and interferential, are identified and compared with the ones already investigated in the case of elastic scatterers immersed in a fluid. To this end, the general formalism of the elastic scattering by N inclusions has been introduced. Numerical results are given for one, two and three identical inclusions. The related scattering S -matrix is next defined. Its unitarity allows indeed to express the energy conservation and consequently to check the numerical results. For empty cavities, the computations show that the interferential interaction is predominant even for very close cavities. In the case of fluid-filled cavities, a strong resonant coupling occurs with the emergence of a large number of resonances, while the interferential phenomenon appears only for large distances between scatterers. The resonant interaction examination shows new and unknown resonant interaction mechanisms. Indeed, except for a few narrow resonances, the single fluid-filled cavity resonances do not obey anymore the splitting law existing for immersed elastic scatterers: they split into a number of interaction resonances which is not in relation with the number of scatterers.

41st Annual Review of Progress in Quantitative Nondestructive Evaluation (QNDE), Boise, ID, JUL 20-25, 2014; International audience; In this paper, we propose an ultrasonic adaptive imaging method based on the phased-array technology and the synthetic focusing algorithm Total Focusing Method (TFM). The general principle is to image the surface by applying the TFM algorithm in a semi-infinite water medium, Then, the reconstructed surface is taken into account to make a second TFM image inside the component. In the surface reconstruction step, the TFM algorithm has been optimized to decrease computation time and to limit noise in water. In the second step, the ultrasonic paths through the reconstructed surface are calculated by the Fermat's principle and an iterative algorithm, and the classical TFM is applied to obtain an image inside the component. This paper presents several results of TFM imaging in components of different geometries, and a result obtained with a new technology of probes equipped with a flexible wedge filled with water (manufactured by Imasonic).

In osteoclasts (OCs) podosomes are organized in a belt, a feature critical for bone resorption. Although microtubules (MTs) promote the formation and stability of the belt, the MT and/or podosome molecules that mediate the interaction of the two systems are not identified. Because the growing "plus" ends of MTs point toward the podosome belt, plus-end tracking proteins (+TIPs) might regulate podosome patterning. Among the +TIPs, EB1 increased as OCs matured and was enriched in the podosome belt, and EB1-positive MTs targeted podosomes. Suppression of MT dynamic instability, displacement of EB1 from MT ends, or EB1 depletion resulted in the loss of the podosome belt. We identified cortactin as an Src-dependent interacting partner of EB1. Cortactin-deficient OCs presented a defective MT targeting to, and patterning of, podosomes and reduced bone resorption. Suppression of MT dynamic instability or EB1 depletion increased cortactin phosphorylation, decreasing its acetylation and affecting its interaction with EB1. Thus, dynamic MTs and podosomes interact to control bone resorption.

From the SETAC Pellston Workshop on Pesticide Risk Assessment for Pollinators January 15-21, 2011 Pensacola, Florida, USA; International audience

Purpose Artificial materials such as dental implants are at risk of bacterial contamination in the oral cavity. Human beta defensins (HBDs), small cationic antimicrobial peptides that exert a broad-spectrum antibacterial function at epithelial surfaces and within some mesenchymal tissues, could probably help to reduce such contamination. HBDs also have protective immunomodulatory effects and have been reported to promote bone remodeling. The aim of this study, therefore, was to investigate the influence of recombinant HBD-2 on the proliferation and survival of cells in culture. Materials and methods Human mesenchymal stem cells (hMSCs), human osteoblasts, human keratinocytes (control), and the HeLa cancer cell line (control) were incubated with recombinant HBD-2 (1, 5, 10, or 20 μg/mL). Cell proliferation and cytotoxicity were evaluated via a water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase assays, respectively. Results HBD-2 was not toxic in any tested concentration to hMSCs, osteoblasts, keratinocytes, or HeLa cells. Furthermore, proliferation of hMSCs and osteoblasts increased after treatment with HBD-2 at all tested concentrations, and keratinocyte proliferation increased when treated at 20 μg/mL. In contrast, HeLa cancer cells were not affected by HBD-2 as tested. Conclusions HBD-2 is not only biocompatible but also promotes proliferation of hMSCs, osteoblasts, and keratinocytes in culture. Further investigation of HBD-2 functional surface coating of artificial materials is recommended.

A short-cut review was carried out to establish whether propofol can improve recovery and reduce recurrence in patients presenting to the emergency department with acute migraine. Three studies were directly relevant to the question. The author, date and country of publication, patient group studied, study type, relevant outcomes, results and study weaknesses of these papers are shown in table 1. The clinical bottom line is that propofol might be a safe and effective therapy in the treatment of migraine, but more well-designed trials are needed to compare with standard therapy before widespread use is considered.

Transplanted retinal pigment epithelium (RPE) cells hold promise for treatment of age-related macular degeneration (AMD) and Stargardt disease (SD), but it is conceivable that the degenerated host Bruch's membrane (BM) as a natural substrate for RPE might not optimally support transplanted cell survival with correct cellular organization. We fabricated novel ultrathin three-dimensional (3-D) nanofibrous membranes from collagen type I and poly(lactic-co-glycolic acid) (PLGA) by an advanced clinical-grade needle-free electrospinning process. The nanofibrillar 3-D networks closely mimicked the fibrillar architecture of the native inner collagenous layer of human BM. Human RPE cells grown on our nanofibrous membranes bore a striking resemblance to native human RPE. They exhibited a correctly orientated monolayer with a polygonal cell shape and abundant sheet-like microvilli on their apical surfaces. RPE cells built tight junctions and expressed RPE65 protein. Flat 2-D PLGA film and cover glass as controls delivered inferior RPE layers. Our nanofibrous membranes may imitate the natural BM to such extent that they allow for the engineering of an in vivo-like human RPE monolayer that maintains the natural biofunctional characteristics. Such ultrathin membranes may provide a promising vehicle for a functional RPE cell monolayer implantation in the subretinal space in patients with AMD or SD.

Conference of 11th Anglo-French Physical Acoustics Conference, AFPAC 2012 ; Conference Date: 18 January 2012 Through 20 January 2012; Conference Code:98821; International audience; The Total Focusing Method (TFM) is sometimes described in the literature as the "gold standard" compared to conventional imaging techniques. TFM is an algorithm that usually post-processes the full matrix of data, also called Full Matrix Capture (FMC). Real-time piloting of such an algorithm is heavy due to the large number of firings (N for a N-element array) and the large number of signals (N×N) to process that tend to decrease the frame rate and, consequently, the inspection speed. This problem can be overcome to some extent if only a few elements are activated which is equivalent to using a sparse array in transmit. The PSF (Point Spread Function) provides information about important images parameters: lateral resolution and contrast. An algorithm based on PSF optimization is proposed to obtain both the number of transmit pulses and the location of the active elements. However, reducing the number of emissions induces a loss in transmitted energy. To compensate it, each transmit pulse is carried out by multiple transmit elements that emulate a single "virtual" element. The method is evaluated on experimental data in a realistic NDT configuration by comparison of images obtained with FMC and SMC (Sparse Matrix Capture) acquisitions.

Biomaterials that mimic the nanofibrous architecture of the natural extracellular matrix (ECM) are in the focus for stem cell hosting or delivery in tissue engineering of multilayered soft tissues such as skin, mucosa, or retina. Synthetic nanofibers for such ECM are usually produced by single-syringe electrospinning with only one needle-jet at very low production rates of 0.005-0.008 g·min⁻¹. The aim of this study was to utilize a novel industrial needle-free multijet electrospinning device with the potential for mass production of nanofibrous ECM (NF-ECM) exhibiting a controlled three-dimensional (3D) morphology for large-scale applications such as large area skin regeneration in patients with burns.The novel NanoSpider™ NS200, an industrial apparatus originally designed for electrospinning of nanofibrous textile meshes, was used to fabricate 3D NF-ECMs of the following synthetic and natural biopolymers: collagen, gelatin, poly(caprolactone) (PCL), and poly(L-lactide-co-glycolide) (PLGA). Different concentrations of Gelatin polymer solution were electrospun under varying processing conditions, namely speed of spinning electrode rotation (u) and electric field intensity (E) by altering applied voltage (v) or the distance between electrodes (h) to achieve homogeneous desirable 3D morphology. Nanofiber diameters were assessed by scanning electron microscopy (SEM). Biocompatibility was tested by WST-1 (water-soluble tetrazolium salt) proliferation assay of seeded human mesenchymal stem cells (HMSCs). Biological performance of HMSCs on 3D PLGA NF-ECM was compared to two-dimensional (2D) PLGA film controls via SEM and confocal microscopy. Western blotting addressed the expression of surface adhesion proteins; focal adhesion kinase (FAK), phosphorylated FAK (pY397), α-tubulin, paxillin, vinculin. and integrin subunits; α5, αv, and β1 proteins.Large-scale mass production of NF-ECM membranes with a highly homogenous nanofiber morphology and 3D architecture could be produced with an extremely high production rate of 0.394±0.013 g·min⁻¹·m⁻¹ when compared to standard procedures. This was achieved by electrospinning a 20% (wt)/v gelatin solution, in an electric field intensity of 0.381 kV·mm⁻¹. The nanofibers possessed diameters of around 180±40 nm with 28% deviation. HSMCs proliferation was significantly improved on NF-ECMs derived from collagen, gelatin, and PLGA when compared to PCL or flat coverglass controls (p0.01). PLGA NF-ECM in 3D nanofibrous architecture possessed significantly superior biocompatibility when compared to flat 2D PLGA film (p0.05). Furthermore, on 3D PLGA NF-ECMs, HSMCs expressed a higher amount of α-tubulin and paxillin compared to the HMSCs cultured on a 2D PLGA film (p0.05). HMSCs exhibited a complex multifaceted morphology on all NF-ECMs, where cells appeared to be integrated into the 3D NF-ECMs niches with complex cell filopodia extending into to all directions. In contrast, HMSCs on flat 2D films of the same materials or on coverglass displayed a simple flattened, monolayered structure.Needle-free multijet electrospinning can be used to mass produce artificial ECMs with intrinsic biocompatibility and desirable integration of stem cells for large-scale applications.

Novel clinical grade electrospinning methods could provide three-dimensional (3D) nanostructured biomaterials comprising of synthetic or natural biopolymer nanofibers. Such advanced materials could potentially mimic the natural extracellular matrix (ECM) accurately and may provide superior niche-like spaces on the subcellular scale for optimal stem-cell attachment and individual cell homing in regenerative therapies. The goal of this study was to design several novel "nanofibrous extracellular matrices" (NF-ECMs) with a natural mesh-like 3D architecture through a unique needle-free multi-jet electrospinning method in highly controlled manner to comply with good manufacturing practices (GMP) for the production of advanced healthcare materials for regenerative medicine, and to test cellular behavior of human mesenchymal stem cells (HMSCs) on these. Biopolymers manufactured as 3D NF-ECM meshes under clinical grade GMP-like conditions show higher intrinsic cytobiocompatibility with superior cell integration and proliferation if compared to their 2D counterparts or a clinically-approved collagen membrane.

In aeronautical industry, composite structures under testing often have complex and variable geometries. In this case, an optimal use of ultrasonic transducer arrays requires specific algorithms in electronic systems in order to achieve rapid and reliable inspections. To fulfil such requirements, a new real time and adaptive technique is presented. This technique is based on an iterative algorithm that does not require a prior knowledge of the geometrical and acoustical properties of the component undergoing inspection. All different parts of one given component (with flat, concave, convex parts…) can be controlled using a same transducer array, such as a conventional linear array with a flat shape. In this paper, the adaptive processing is demonstrated and illustrated through acquisitions performed with different typical aircraft composite structures.

The transplantation of human stem cells seeded on biomaterials holds promise for many clinical applications in cranio-maxillo-facial tissue engineering and regenerative medicine. However, stem cell propagation necessary to produce sufficient cell numbers currently utilizes fetal calf serum (FCS) as a growth supplement which may subsequently transmit animal pathogens. Human platelet lysate (HPL) could potentially be utilized to produce clinical-grade stem cell-loaded biomaterials as an appropriate FCS substitute that is in line with clinically-applicable practice. The goal of this study was to investigate whether HPL can be successfully used to propagate human mesenchymal stem cells (HMSCs) seeded on clinically-approved collagen materials under clinically-applicable conditions using FCS as a control. HMSCs were isolated from bone marrow and cultured in the presence of 10% FCS or 10% HPL. Characterization of HMSCs was performed by flow cytometry and through osteogenic and adipogenic differentiation assays. Proliferative capacity of HMSCs on both matrices was investigated by mitochondrial dehydrogenase assays (WST) and tissue coverage scanning electron microscopy (SEM). The isolated HMSC differentiated into osteogenic and adipogenic cells authenticating the multipotentiality of the HMSCs. WST tests and the SEM images demonstrated that HPL was generally superior to FCS in promoting growth of seeded HMSCs. For all other tests HPL supported HMSCs at least equal to FCS. In conclusion, HPL is an effective growth factor to allow expansion of clinical-grade HMSCs on clinically-approved biomaterials for maxillofacial and oral implantology applications.

Background Real-time quantitative RT-PCR (qPCR) is a powerful technique capable of accurately quantitating mRNA expression levels over a large dynamic range. This makes qPCR the most widely used method for studying quantitative gene expression. An important aspect of qPCR is selecting appropriate controls or normalization factors to account for any differences in starting cDNA quantities between samples during expression studies. Here, we report on the selection of a concise set of housekeeper genes for the accurate normalization of quantitative gene expression data in differentiating osteoblasts, osteoclasts and macrophages. We implemented the use of geNorm, an algorithm that determines the suitability of genes to function as housekeepers by assessing expression stabilities. We evaluated the expression stabilities of 18S, ACTB, B2M, GAPDH, HMBS and HPRT1 genes. Findings Our analyses revealed that 18S and GAPDH were regulated during osteoblast differentiation and are not suitable for use as reference genes. The most stably expressed genes in osteoblasts were ACTB, HMBS and HPRT1 and their geometric average constitutes a suitable normalization factor upon which gene expression data can be normalized. In macrophages, 18S and GAPDH were the most variable genes while HMBS and B2M were the most stably expressed genes. The geometric average of HMBS and B2M expression levels forms a suitable normalization factor to account for potential differences in starting cDNA quantities during gene expression analysis in macrophages. The expression stabilities of the six candidate reference genes in osteoclasts were, on average, more variable than that observed in macrophages but slightly less variable than those seen in osteoblasts. The two most stably expressed genes in osteoclasts were HMBS and B2M and the genes displaying the greatest levels of variability were 18S and GAPDH. Notably, 18S and GAPDH were the two most variably expressed control genes in all three cell types. The geometric average of HMBS, B2M and ACTB creates an appropriate normalization factor for gene expression studies in osteoclasts. Conclusion We have identified concise sets of genes suitable to use as normalization factors for quantitative real-time RT-PCR gene expression studies in osteoblasts, osteoclasts and macrophages.

Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem cell-mediated therapies for fracture and other orthopedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of stimulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase activity and extracellular matrix mineralization. Furthermore, similar DMSO-mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1, we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among the numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical, and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased alkaline phosphatase activity, and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. A flow on knockdown of bone-specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c, suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts.

This paper deals with multiple scattering by a random arrangement of parallel circular elastic cylinders immersed in a fluid. The cylinders are distributed in a region called “slab” that is located between two parallel planes orthogonal to a given x -direction. The disorder inside the slab depends on the x -variable. The goal is to calculate the reflection and transmission coefficients by this space-varying slab. For low concentrations of cylinders, two methods are developed from Twersky’s theory on the propagation of coherent waves in an effective medium. The first method is based upon the discretization of the properties of the space-varying slab. The second one is based on the WKB method. They are successfully compared in the case of a smooth space-varying slab in which the random distribution of cylinders varies slowly along the x -direction. An effective mass density is defined, which allows the derivation of the mean acoustic displacement from the mean pressure field. The continuity of both pressure and normal displacement is thus shown at the interface between two different effective media as well as at the interface between the space-varying slab and a homogeneous fluid.

Burkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis, a spectrum of potentially fatal diseases endemic in Northern Australia and South-East Asia. We demonstrate that B. pseudomallei rapidly modifies infected macrophage-like cells in a manner analagous to osteoclastogenesis. These alterations include multinucleation and the expression by infected cells of mRNA for factors required for osteoclastogenesis: the chemokines monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 gamma (MIP-1gamma), 'regulated on activation normal T cell expressed and secreted' (RANTES) and the transcription factor 'nuclear factor of activated T-cells cytoplasmic 1' (NFATc1). An increase in expression of these factors was also observed after infection with Burkholderia thailandensis. Expression of genes for the osteoclast markers calcitonin receptor (CTR), cathepsin K (CTSK) and tartrate-resistant acid phosphatase (TRAP) was also increased by B. pseudomallei-infected, but not by B. thailandensis-infected cells. The expression by B. pseudomallei-infected cells of these chemokine and osteoclast marker genes was remarkably similar to cells treated with RANKL, a stimulator of osteoclastogenesis. Analysis of dentine resorption by B. pseudomallei-induced osteoclast-like cells revealed that demineralization may occur but that authentic excavation does not take place under the tested conditions. Furthermore, we identified and characterized lfpA (for lactonase family protein A) in B. pseudomallei, which shares significant sequence similarity with the eukaryotic protein 'regucalcin', also known as 'senescence marker protein-30' (SMP-30). LfpA orthologues are widespread in prokaryotes and are well conserved, but are phylogenetically distinct from eukaryotic regucalcin orthologues. We demonstrate that lfpA mRNA expression is dramatically increased in association with macrophage-like cells. Mutation of lfpA significantly reduced expression of the tested host genes, relative to the response to wild-type B. pseudomallei. We also show that lfpA is required for optimal virulence in vivo.

RhoGTPases regulate actin cytoskeleton dynamics, a key element in osteoclast biology. We identified three novel genes induced during RANKL-stimulated osteoclastogenesis among RhoGTPases and their exchange factors that are essential in osteoclast biology. Introduction: During the process of differentiation, adhesion to the bone matrix or osteolysis, the actin cytoskeleton of osteoclasts undergoes profound reorganization. RhoGTPases are key regulators of actin dynamics. They control cell adhesion, migration, and morphology through their action on actin cytoskeleton. In mice, there are 18 low molecular weight RhoGTPases. They are activated by guanine nucleotide exchange factors: the RhoGEFs. There are 76 RhoGEFs in mice: 65 belong to the Dbl family and 11 to the CZH family. To identify novel genes among RhoGTPases and RhoGEFs important in osteoclasts, we established the expression profiles of the complete families of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis. Materials and Methods: The RAW264.7 cell line, mouse bone marrow macrophages, and hematopoietic stem cells were used as precursors for RANKL-induced osteoclastogenesis. Gene arrays and real-time quantitative PCR analyses were performed to establish the transcription profiles of RhoGTPase and RhoGEF genes during differentiation. Small hairpin RNA was used to knock down genes of interest. Results: Of the 18 RhoGTPases and 76 RhoGEFs, the expression of three genes was upregulated by RANKL: the RhoGTPase RhoU/Wrch1, the Dbl family exchange factor Arhgef8/Net1, and the CZH family exchange factor Dock5. The inductions were observed in gene array and real-time quantitative PCR experiments performed in RAW264.7 cells. They were further confirmed in bone marrow macrophages and hematopoietic stem cells. Silencing of Wrch1 and Arhgef8 expression severely inhibited differentiation and affected osteoclast morphology. Dock5 suppression was lethal in osteoclast precursors while having no effect in fibroblasts. Conclusions: We identified three genes among RhoGTPase signaling pathways that are upregulated during RANKL-induced osteoclastogenesis. These genes are novel essential actors in osteoclasts, most likely through the control of actin cytoskeleton dynamics.

MCP-1 (monocyte chemotactic protein-1) is a CC chemokine that is induced by receptor activator of NFkappaB ligand (RANKL) in human osteoclasts. In the absence of RANKL, treatment of human peripheral blood mononuclear cells with macrophage colony-stimulating factor and MCP-1 resulted in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells that are positive for calcitonin receptor (CTR) and a number of other osteoclast markers, including nuclear factor of activated t cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). Although NFATc1 was strongly induced by MCP-1 and was observed in the nucleus, MCP-1 did not permit the formation of bone-resorbing osteoclasts, although these cells had the typical TRAP(+)/CTR(+) multinuclear phenotype of osteoclasts. Despite a similar appearance to osteoclasts, RANKL treatment was required in order for TRAP(+)/CTR(+) multinuclear cells to develop bone resorption activity. The lack of bone resorption was correlated with a deficiency in expression of certain genes related to bone resorption, such as cathepsin K and MMP9. Furthermore, calcitonin blocked the MCP-1-induced formation of TRAP(+)/CTR(+) multinuclear cells as well as blocking osteoclast bone resorption activity, indicating that calcitonin acts at two stages of osteoclast differentiation. Ablation of NFATc1 in mature osteoclasts did not prevent bone resorption activity, suggesting NFATc1 is involved in cell fusion events and not bone resorption. We propose that the MCP-1-induced TRAP(+)/CTR(+) multinuclear cells represent an arrested stage in osteoclast differentiation, after NFATc1 induction and cellular fusion but prior to the development of bone resorption activity.

Treatment of adherent peripheral blood mononuclear cells (PBMCs) with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) stimulates the formation of multinucleate osteoclast-like cells. Treatment with M-CSF alone results in the formation of macrophage-like cells. Through the use of Atlas human cDNA expression arrays, genes regulated by RANKL were identified. Genes include numerous cytokines and cytokine receptors (RANTES and CSF2R proportional, variant ), transcription factors (nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and GA binding protein transcription factor alpha (GABPalpha)), and ribosomal proteins (60S L17 and 40S S20). Real-time PCR analysis showed significant correlation (R2 of 0.98 P < 0.01) with array data for all genes tested. Time courses showed differential activation patterns of transcription factors with early induction of FUSE binding protein 1 (FBP) and c-Jun, and later steady upregulation of NFATc1 and GABP by RANKL. Treatment with cyclosporin A, a known NFATc1 inhibitor, resulted in a blockade of osteoclast formation. The mononuclear cells resulting from high cyclosporin treatment (1,000 ng/ml) were cathepsin K (CTSK) and tartrate-resistant acid phosphatase (TRAP) positive but expression of calcitonin receptor (CTR) was downregulated by more than 30-fold. Constant exposure of M-CSF- and RANKL-treated cells to GM-CSF resulted in inhibition of osteoclast formation and the downregulation of CTSK and TRAP implicating the upregulation of CSF2R in a possible feedback inhibition of osteoclastogenesis.