Suprathermal plasma flows in current sheets formed in two- and three-dimensional magnetic configurations

Dynamics of the thermal and directed motions of argon plasma ions in current sheets formed in various magnetic configurations was investigated experimentally Measurements in three-dimensional magnetic configurations with an X line were carried out for the first time. The results of these measurements were compared with the data obtained in experiments with two-dimensional magnetic configurations. The ion temperature and the energies and velocities of directed plasma flows within the current sheet were determined by analyzing the shapes of argon ion spectral lines broadened due to the Doppler effect. It is found that, under the given experimental conditions, the axial magnetic field does not affect the ion temperature and plasma acceleration in the sheet.     

Plasma acceleration in current sheets formed in helium in two- and three-dimensional magnetic configurations

The processes of heating and acceleration of plasma in current sheets formed in 2D and 3D magnetic configurations with an X-line in helium plasma have been investigated using spectroscopic methods. It is found that, in 2D magnetic configurations, plasma flows with energies of 400?C1000 eV, which are substantially higher than the ion thermal energy, are generated and propagate along the width (the larger transverse dimension) of the sheet. In 3D configurations, the influence of the longitudinal (directed along the X-line) component of the magnetic field on the plasma parameters in the current sheet has been studied. It is shown that plasma acceleration caused by the Amp??re force can be spatially inhomogeneous in the direction perpendicular to the sheet surface, which should lead to sheared plasma flows in the sheet.     

Engineering of human hepatic tissue with functional vascular networks

Although absolute organ shortage highlights the needs of alternative organ sources for regenerative medicine, the generation of a three-dimensional (3D) and complex vital organ, such as well-vascularized liver, remains a challenge. To this end, tissue engineering holds great promise; however, this approach is significantly limited by the failure of early vascularization in vivo after implantation. Here, we established a stable 3D in vitro pre-vascularization platform to generate human hepatic tissue after implantation in vivo. Human fetal liver cells (hFLCs) were mixed with human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (hMSCs) and were implanted into a collagen/fibronectin matrix composite that was used as a 3-D carrier. After a couple of days, the fluorescent HUVECs developed premature vascular networks in vitro, which were stabilized by hMSCs. The establishment of functional vessels inside the pre-vascularized constructs was proven using dextran infusion studies after implantation under a transparency cranial window. Furthermore, dynamic morphological changes during embryonic liver cell maturation were intravitaly quantified with high-resolution confocal microscope analysis. The engineered human hepatic tissue demonstrated multiple liver-specific features, both structural and functional. Our new techniques discussed here can be implemented in future clinical uses and industrial uses, such as drug testing.     

Engineering of human hepatic tissue with functional vascular networks

Although absolute organ shortage highlights the needs of alternative organ sources for regenerative medicine, the generation of a three-dimensional (3D) and complex vital organ, such as well-vascularized liver, remains a challenge. To this end, tissue engineering holds great promise; however, this approach is significantly limited by the failure of early vascularization in vivo after implantation. Here, we established a stable 3D in vitro pre-vascularization platform to generate human hepatic tissue after implantation in vivo. Human fetal liver cells (hFLCs) were mixed with human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (hMSCs) and were implanted into a collagen/fibronectin matrix composite that was used as a 3-D carrier. After a couple of days, the fluorescent HUVECs developed premature vascular networks in vitro, which were stabilized by hMSCs. The establishment of functional vessels inside the pre-vascularized constructs was proven using dextran infusion studies after implantation under a transparency cranial window. Furthermore, dynamic morphological changes during embryonic liver cell maturation were intravitaly quantified with high-resolution confocal microscope analysis. The engineered human hepatic tissue demonstrated multiple liver-specific features, both structural and functional. Our new techniques discussed here can be implemented in future clinical uses and industrial uses, such as drug testing.     

Evaluation of cooperativity for phase transitions in two- and three-dimensional systems

Use of the so-called "cooperative unit" (readily obtainable from the midpoint slope of phase transition curves) is discussed for the determination of cluster sizes and cooperative interaction energies. This quantity has been commonly employed in a rather empirical way since its correct interpretation is known only for some special cases (linear systems, all-or-none transitions). It is shown in the framework of a lattice model (Ising model) that the cooperative unit may be interpreted in terms of correlation functions and that it defines an average cluster corresponding to the patch size as obtained from scattering experiments. Relations between the cooperative unit and a cooperativity parameter are given for various lattices. Different types of transition curves are discussed using a simple analytical formalism, the quasichemical approximation. Some important nonideality effects are investigated which may lead to a "smearing-out" of first-order transitions.     

Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro

The fabrication of 3D tissues retaining the original functions of tissues/organs in vitro is crucial for optimal tissue engineering and regenerative medicine. The fabrication of 3D tissues also contributes to the establishment of in vitro tissue/organ models for drug screening. Our laboratory has developed a fabrication system for functional 3D tissues by stacking cell sheets of confluent cultured cells detached from a temperature-responsive culture dish. Here we describe the protocols for the fabrication of 3D tissues by cell sheet engineering. Three-dimensional cardiac tissues fabricated by stacking cardiac cell sheets pulsate spontaneously, synchronously and macroscopically. Via this protocol, it is also possible to fabricate other tissues, such as 3D tissue including capillary-like prevascular networks, from endothelial cells sandwiched between layered cell sheets. Cell sheet stacking technology promises to provide in vitro tissue/organ models and more effective therapies for curing tissue/organ failures.     

Software techniques for two- and three-dimensional kinematic measurements of biological and biomimetic systems

Researchers studying aspects of locomotion or movement in biological and biomimetic systems commonly use video or stereo video recordings to quantify the behaviour of the system in question, often with an emphasis on measures of position, velocity and acceleration. However, despite the apparent simplicity of video analysis, it can require substantial investment of time and effort, even when performed with adequate software tools. This paper reviews the underlying principles of video and stereo video analysis as well as its automation and is accompanied by fully functional and freely available software implementation.     

Stereotypic culture systems for liver and bone marrow: evidence for the development of functional tissue in vitro and following implantation in vivo

Stromal cell-associated liver cell and bone marrow (BM) culture on three-dimensiional nylon screen or polyglycolic acid (PGA) felt templates conveys certain functional advantages to the parenchyma of these tissues. Hepatic parenchymal cells (PC) manifest long-term ( approximately 2 month) expression of liver-specific activities including cytochrome P450 enzyme activity and the synthesis of albumin, fibrinogen, transferrin, and other proteins. PC also undergo proliferation in association with stromal cells that were pre-established on these templates. PC mitoses are directly proportional to available space within the template for their expansion indication that geometric or sterotypic parameters influence the growth of these cells in vitro. BM cultured on a similar template exhibits long-term multilineage hematopoietic expression and limited expansion of progenitor cell numbers. Progenitor cell concentration within the cultures can be substantially enhanced if these cells are liberated from co-culture and reseeded onto a template containing fresh stromal cells. BM and liver cel cultures established on felt composed of bioresorbable PGA filaments was grafted into various sites in rats. Liver co-cultures generated sinusoids and other liver-like structures in situ; active hematopoietic blasts were observed at sites of BM co-culture grafts. Biodegradable polymer constructs may prove useful for certain clinical applications as vehicles for the delivery of tissues that were engineered in culture.     

Segmentation of two- and three-dimensional data from electron microscopy using eigenvector analysis

An automatic image segmentation method is used to improve processing and visualization of data obtained by electron microscopy. Exploiting affinity criteria between pixels, e.g., proximity and gray level similarity, in conjunction with an eigenvector analysis, the image is subdivided into areas which correspond to objects or meaningful regions. Extending a proposal by Shi and Malik (1997, Proceedings of the IEEE conference on Computer Vision and Pattern Recognition, pp. 731-737) the approach was adapted to the field of electron microscopy, especially to three-dimensional application as needed by electron tomography. Theory, implementation, parameter setting, and results obtained with a variety of data are presented and discussed. The method turns out to be a powerful tool for visualization with the potential for further improvement by developing and tuning new affinity.     

Recent advances in X-ray imaging of breast tissue: From two- to three-dimensional imaging

Breast cancer is a globally widespread disease whose detection has already been significantly improved by the introduction of screening programs. Nevertheless, mammography suffers from low soft tissue contrast and the superposition of diagnostically relevant anatomical structures as well as from low values for sensitivity and specificity especially for dense breast tissue. In recent years, two techniques for X-ray breast imaging have been developed that bring advances for the early detection of breast cancer. Grating-based phase-contrast mammography is a new imaging technique that is able to provide three image modalities simultaneously (absorption-contrast, phase-contrast and dark-field signal). Thus, an enhanced detection and delineation of cancerous structures in the phase-contrast image and an improved visualization and characterization of microcalcifications in the dark-field image is possible. Furthermore, latest studies about this approach show that dose-compatible imaging with polychromatic X-ray sources is feasible. In order to additionally overcome the limitations of projection-based imaging, efforts were also made towards the development of breast computed tomography (BCT), which recently led to the first clinical installation of an absorption-based BCT system. Further research combining the benefits of both imaging technologies is currently in progress. This review article summarizes the latest advances in phase-contrast imaging for the female breast (projection-based and three-dimensional view) with special focus on possible clinical implementations in the future.     

Spectroscopic measurements of the electron and ion temperatures and effective ion charge in current sheets formed in two- and three-dimensional magnetic configurations

The spatial distributions of the electron temperature and density, the effective and average ion charges, and the thermal and directed ion velocities in current sheets formed in two-dimensional magnetic fields and three-dimensional magnetic configurations with an X line were studied using spectroscopic and interference holographic methods. The main attention was paid to studying the time evolution of the intensities of spectral lines of the working-gas (argon) and impurity ions under different conditions. Using these data, the electron temperature was calculated with the help of an original mathematical code based on a collisional-radiative plasma model incorporating the processes of ionization and excitation, as well as MHD plasma flows generated in the stage of the current-sheet formation. It is shown that the electron temperature depends on the longitudinal magnetic field, whereas the ion temperature is independent of it. The effective ion charge of the current-sheet plasma was determined for the first time.     

Dynamic interaction between breast cancer cells and osteoblastic tissue: comparison of two- and three-dimensional cultures

Breast cancer cell colonization of osteoblast monolayers grown in standard tissue culture (2D) is compared to colonization of a multi-cell-layer osteoblastic tissue (3D) grown in a specialized bioreactor. Colonization of 3D tissue recapitulates events observed in clinical samples including cancer penetration of tissue, growth of microcolonies, and formation of "Single cell file" commonly observed in end-stage pathological bone tissue. By contrast, adherent cancer cell colonies did not penetrate 2D tissue and did not form cell files. Thus, it appears that 3D tissue is a more biologically (clinically) relevant model than 2D monolayers in which to study cancer cell interactions with osteoblastic tissue. This direct comparison of 2D and 3D formats is implemented using MC3T3-E1 murine osteoblasts and MDA-MB-231 human metastatic breast cancer cells, or the metastasis-suppressed line, MDA-MB-231BRMS1, for comparison. When osteoblasts were co-cultured with metastatic cells, production of osteocalcin (a mineralization marker) decreased and secretion of the pro-inflammatory cytokine IL-6 increased in both 2D and 3D formats. Cancer cell penetration of the 3D tissue coincided with a changed osteoblast morphology from cuboidal to spindle-shaped, and with osteoblasts alignment parallel to the cancer cells. Metastasis-suppressed cells did not penetrate 3D tissue, did not cause a change in osteoblast morphology or align in rows. Moreover, they proliferated much less in the 3D culture than in the 2D culture in a manner similar to their growth in bone. In both systems, the cancer cells proliferated to a greater extent with immature osteoblasts compared to more mature osteoblasts.     

Engineering of bone tissue with porcine bone marrow stem cells in three-dimensional trabecular metal: in vitro and in vivo studies

The aim of this study was to investigate capability of cell attachment and ectopic bone formation in pigs after either ex vivo transplantation and expansion of bone marrow stem cells (BMSc) into three-dimensional porous tantalum, or porous tantalum supplemented with BMSc. After 24 hours incubation, cells adhering to the porous tantalum discs were quantified by means of scintillation counting of 3H-thymidine-labeled cells. After 7 days of incubation, the cell-loaded porous tantalum discs were harvested for histological analysis or implanted in the infrasternal muscle; an empty disc and disc implanted immediately after cell loading served as controls. All implants were taken out after 8 weeks of implantation and histological examination was performed. The results of in vitro cell attachment to the porous tantalum discs were not improved significantly with gelatin, collagen or fibronectin coatings. Histological analysis of cell loaded discs in vitro demonstrated viable BMSc within the 3-D tantalum structure. In vivo bone induction was demonstrated when the porous tantalum discs were cultured with BMSc. Our findings indicated that porous tantalum was suitable for cell attachment, and ectopic bone formation in pigs was achieved by means of BMSc cultured with porous tantalum. The present study suggests that cell-mediated hard bone tissue repair technology makes it possible to prefabricate autologous BMSc into three-dimensional trabecular metal in order to engineer bone tissue.     

Vinculin regulates directionality and cell polarity in two- and three-dimensional matrix and three-dimensional microtrack migration

During metastasis, cells can use proteolytic activity to form tube-like “microtracks” within the extracellular matrix (ECM). Using these microtracks, cells can migrate unimpeded through the stroma. To investigate the molecular mechanisms of microtrack migration, we developed an in vitro three-dimensional (3D) micromolded collagen platform. When in microtracks, cells tend to migrate unidirectionally. Because focal adhesions are the primary mechanism by which cells interact with the ECM, we examined the roles of several focal adhesion molecules in driving unidirectional motion. Vinculin knockdown results in the repeated reversal of migration direction compared with control cells. Tracking the position of the Golgi centroid relative to the position of the nucleus centroid reveals that vinculin knockdown disrupts cell polarity in microtracks. Vinculin also directs migration on two-dimensional (2D) substrates and in 3D uniform collagen matrices, as indicated by reduced speed, shorter net displacement, and decreased directionality in vinculin-deficient cells. In addition, vinculin is necessary for focal adhesion kinase (FAK) activation in three dimensions, as vinculin knockdown results in reduced FAK activation in both 3D uniform collagen matrices and microtracks but not on 2D substrates, and, accordingly, FAK inhibition halts cell migration in 3D microtracks. Together these data indicate that vinculin plays a key role in polarization during migration.     

Examination of the distribution of arsenic in hydrated and fresh cowpea roots using two- and three-dimensional techniques

Arsenic (As) is considered to be the environmental contaminant of greatest concern due to its potential accumulation in the food chain and in humans. Using novel synchrotron-based x-ray fluorescence techniques (including sequential computed tomography), short-term solution culture studies were used to examine the spatial distribution of As in hydrated and fresh roots of cowpea (Vigna unguiculata 'Red Caloona') seedlings exposed to 4 or 20 μm arsenate [As(V)] or 4 or 20 μm arsenite. For plants exposed to As(V), the highest concentrations were observed internally at the root apex (meristem), with As also accumulating in the root border cells and at the endodermis. When exposed to arsenite, the endodermis was again a site of accumulation, although no As was observed in border cells. For As(V), subsequent transfer of seedlings to an As-free solution resulted in a decrease in tissue As concentrations, but growth did not improve. These data suggest that, under our experimental conditions, the accumulation of As causes permanent damage to the meristem. In addition, we suggest that root border cells possibly contribute to the plant's ability to tolerate excess As(V) by accumulating high levels of As and limiting its movement into the root.     

Fabrication of two- and three-dimensional model catalyst systems with monodispersed platinum nanoparticles as active metal building blocks

Well-defined Pt monodispersed nanoparticles within the catalytically relevant 1–10 nm size regime were synthesized in solution phase by several synthetic methods which differed in the choice of reducing agent, surface stabilizer, reaction temperature and solvent. Three-dimensional model catalysts were fabricated by incorporating the metal nanoparticles into ordered channels of high surface area mesoporous oxides such as SiO₂, Al₂O₃ and Ta₂O₅ through either sonication or direct synthesis of the oxide support around the particles. Deposition of the same nanocrystals onto silica supports by means of the Langmuir–Schaeffer technique produced two-dimensional model catalysts.     

A three-dimensional microfluidized liver system to assess hepatic drug metabolism and hepatotoxicity

In this study, we propose the design and fabrication of a liver system on a chip. We first chose the most suitable three-dimensional liver-like model between cell spheroids and microtissue precursors, both based on the use of hepatocellular carcinoma cells (HepG2) to provide proof-of-concept data. Spheroids displayed high cell density but low expression of the typical hepatic biomarkers, whereas microtissue precursors showed stable viability and function over the entire culture time. The two liver-like models were compared in terms of cell viability, function, metabolism, and the P-glycoprotein 1 (P-gp) transport-protein expression with the microtissue precursors showing the best performance. Thus, we cultured them into a microfluidic biochip featured with three parallel channels shaped to mimic the hepatic sinusoids. To assess the detoxification potential of the microtissue-loaded biochip we challenged it with a model molecule (ethanol) at different concentrations and time points. Ethanol cytotoxicity was detected by a noninvasive measurement of cell viability based on cell autofluorescence. As expected, a dose-dependent decrease of albumin and urea secretion was observed in the ethanol-treated samples. We believe that the described totally human-derived platform, suitable for integration into a multiorgan microfluidic system, can provide a consistent innovative platform for drug development and toxicity studies.     

Expansion and differentiation of human primary osteoblasts in two- and three-dimensional culture

Abstract

Despite the regenerative capability of bone, treatment of large defects often requires bone grafts. The challenge for bone grafting is to establish rapid and sufficient vascularization. Three-dimensional (3D) multicellular spheroids consisting of the relevant cell types can be used as “mini tissues” to study the complexity of angiogenesis. We investigated two-dimensional (2D) expansion, differentiation and characterization of primary osteoblasts as steps toward the establishment of 3D multicellular spheroids. Supplementation of cell culture medium with vitamin D₃ induces the osteocalcin expression of osteoblasts. An increased osteocalcin concentration of 10.8 ± 0.58 ng/ml could be measured after 19 days in supplemented medium. Vitamin D₃ has no influence on the expression of alkaline phosphatase or the deposition of calcium. Expression of these additional osteogenic markers requires addition of a cocktail of osteogenic factors that, conversely, have no influence on the expression of osteocalcin. Supplementation of the cell culture medium with both vitamin D₃ and a cocktail of osteogenic factors is recommended to produce an osteoblast phenotype that secretes osteocalcin, expresses alkaline phosphatase and deposits calcium. In such a supplemented medium, a mean osteocalcin concentration of 11.63 ± 4.85 ng/ml was secreted by the osteoblasts. Distinguishing osteoblasts and fibroblasts remains a challenge. Neither differentiated nor undifferentiated osteoblasts can be distinguished from fibroblasts by the expression of CD90, ED-A-fibronectin or α-smooth muscle actin; however, these cell types exhibit clear differences in their growth characteristics. Osteoblasts can be arranged as 3D spheroids by coating the bottom of the cell culture device with agarose. The cellular composition of 3D multicellular spheroids can be evaluated quantitatively using vital fluorescence labeling techniques. Spheroids are a promising tool for studying angiogenic and osteogenic phenomena in vivo and in vitro.     

Classification of small molecules by two- and three-dimensional decomposition kernels

MOTIVATION: Several kernel-based methods have been recently introduced for the classification of small molecules. Most available kernels on molecules are based on 2D representations obtained from chemical structures, but far less work has focused so far on the definition of effective kernels that can also exploit 3D information. RESULTS: We introduce new ideas for building kernels on small molecules that can effectively use and combine 2D and 3D information. We tested these kernels in conjunction with support vector machines for binary classification on the 60 NCI cancer screening datasets as well as on the NCI HIV data set. Our results show that 3D information leveraged by these kernels can consistently improve prediction accuracy in all datasets. AVAILABILITY: An implementation of the small molecule classifier is available from http://www.dsi.unifi.it/neural/src/3DDK.