Complex Three-dimensional Environment Research Articles

A scaffold-free multicellular three-dimensional in vitro model of osteogenesis.

In vitro models of osteogenesis are essential for investigating bone biology and the effects of pharmaceutical, chemical, and physical cues on bone formation. Osteogenesis takes place in a complex three-dimensional (3D) environment with cells from both mesenchymal and hematopoietic origins. Existing in vitro models of osteogenesis include two-dimensional (2D) single type cell monolayers and 3D cultures. However, an in vitro scaffold-free multicellular 3D model of osteogenesis is missing. We hypothesized that the self-inductive ossification capacity of bone marrow tissue can be harnessed in vitro and employed as a scaffold-free multicellular 3D model of osteogenesis. Therefore, rat bone marrow tissue was cultured for 28 days in three settings: 2D monolayer, 3D homogenized pellet, and 3D organotypic explant. The ossification potential of marrow in each condition was quantified by micro-computed tomography. The 3D organotypic marrow explant culture resulted in the greatest level of ossification with plate-like bone formations (up to 5 mm in diameter and 0.24 mm in thickness). To evaluate the mimicry of the organotypic marrow explants to newly forming native bone tissue, detailed compositional and morphological analyses were performed, including characterization of the ossified matrix by histochemistry, immunohistochemistry, Raman microspectroscopy, energy dispersive X-ray spectroscopy, backscattered electron microscopy, and micromechanical tests. The results indicated that the 3D organotypic marrow explant culture model mimics newly forming native bone tissue in terms of the characteristics studied. Therefore, this platform holds significant potential to be used as a model of osteogenesis, offering an alternative to in vitro monolayer cultures and in vivo animal models.

Quantitative In vivo Imaging of the Effects of Inhibiting Integrin Signaling via Src and FAK on Cancer Cell Movement: Effects on E-cadherin Dynamics

Most cancer-related deaths are due to the development of metastatic disease, and several new molecularly targeted agents in clinical development have the potential to prevent disease progression. However, it remains difficult to assess the efficacy of antimetastatic agents in the clinical setting, and an increased understanding of how such agents work at different stages of the metastatic cascade is important in guiding their clinical use. We used optical window chambers combined with photobleaching, photoactivation, and photoswitching to quantitatively measure (a) tumor cell movement and proliferation by tracking small groups of cells in the context of the whole tumor, and (b) E-cadherin molecular dynamics in vivo following perturbation of integrin signaling by inhibiting focal adhesion kinase (FAK) and Src. We show that inhibition of Src and FAK suppresses E-cadherin-dependent collective cell movement in a complex three-dimensional tumor environment, and modulates cell-cell adhesion strength and endocytosis in vitro. This shows a novel role for integrin signaling in the regulation of E-cadherin internalization, which is linked to regulation of collective cancer cell movement. This work highlights the power of fluorescent, direct, in vivo imaging approaches in the preclinical evaluation of chemotherapeutic agents, and shows that inhibition of the Src/FAK signaling axis may provide a strategy to prevent tumor cell spread by deregulating E-cadherin-mediated cell-cell adhesions.

Waalbot II: Adhesion Recovery and Improved Performance of a Climbing Robot using Fibrillar Adhesives

This paper presents the design and optimization of a wall-climbing robot along with the incorporation of autonomous adhesion recovery and a motion planning implementation. The result is Waalbot II, an untethered 85 g robot able to climb on smooth vertical surfaces with up to a 100 g payload (117% body mass) or, when unburdened, on planar surfaces of any orientation at speeds up to 5 cm/s. Bio-inspired climbing mechanisms, such as Waalbot II’s gecko-like fibrillar adhesives, passive peeling, and force sensing, improve the overall climbing capabilities compared with initial versions, resulting in the ability to climb on non-smooth surfaces as well as on inverted smooth surfaces. Robot length scale optimization reveals and quantifies trends in the theoretical factor of safety and payload carrying capabilities. Autonomous adhesion recovery behavior provides additional climbing robustness without additional mechanical complexity to mitigate degradation and contamination. An implementation of a motion planner, designed to take into account Waalbot II’s kinematic constraints, results in the ability to navigate to a goal in complex three-dimensional environments while properly planning plane-to-plane transitions and avoiding obstacles. Experiments verified the improved climbing capabilities of Waalbot II as well as its novel semi-autonomous adhesion recovery behavior and motion planning.

Enhanced Fluorescence Imaging of Live Cells by Effective Cytosolic Delivery of Probes

BackgroundMicroscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery.Principal FindingsWe show the delivery within several types of mammalian cells using nanometre-sized biomimetic polymer vesicles (a.k.a. polymersomes) that offer both highly efficient cellular uptake and endolysomal escape capability without any effect on the cellular metabolic activity. Such biocompatible polymersomes can encapsulate various types of probes including cell membrane probes and nucleic acid probes as well as labelled nucleic acids, antibodies and quantum dots.SignificanceWe show the delivery of sufficient quantities of probes to the cytosol, allowing sustained functional imaging of live cells over time periods of days to weeks. Finally the combination of such effective staining with three-dimensional imaging by confocal laser scanning microscopy allows cell imaging in complex three-dimensional environments under both mono-culture and co-culture conditions. Thus cell migration and proliferation can be studied in models that are much closer to the in vivo situation.

Everyday memory deficits in very mild Alzheimer's disease

Memory complaints of patients sometimes are not verified via standard cognitive testing. Acquisition of information in everyday life requires memorization in complex three-dimensional environments. The authors mimicked this with a photorealistic virtual environment (VE). Memory for verbal material and spatial scenery was tested in healthy controls (HC) and patients with mild Alzheimer's disease (AD); mini-mental state evaluation (MMSE) 25.7 ± 1.8 (mean ± standard deviation). The number of memorized items increased to 90% in both classical list learning and for items memorized in VE in HC. In contrast, only 40% of items were recalled in list learning and 20% in VE in AD patients. Unlike the gender difference favoring female HC on list learning, performance was alike for both genders in VE. We conclude that verbal learning abilities in healthy elderly subjects are alike in standard settings and under virtual reality conditions. In AD patients memory deficits that are relevant to everyday life yet not detectable with list learning are unmasked in virtual reality. In future, this may aid objective appraisal of interventions with regard to their everyday relevance.

Fully Coupled Multi-Hybrid FEM-PO/PTD-UTD Method for the Analysis of Radiation Problems

A multi-hybrid method combining the finite element method (FEM) and several high frequency techniques (HFTs) for the efficient analysis of the radiation and scattering of electromagnetic waves on complex three-dimensional environments is presented. It makes use of FEM for the regions with small and complex features and one or several HFTs for the analysis of the electrically large objects of the structure. The hybridization is done in a fully-coupled way taking into account mutual interactions between the FEM domains and the objects analyzed with HFTs. Two main HFTs are used: physical optics (PO) and the uniform theory of diffraction (UTD) in order to take advantage of the use of distributed and point sources. Physical theory of diffraction (PTD) is also used when needed in order to improve the accuracy of PO

A Neuromorphic VLSI Model of Bat Interaural Level Difference Processing for Azimuthal Echolocation

Bats use the unusual sensory modality of echolocation to fly in complete darkness with speed and agility through complex three-dimensional environments. Their small head size and the use of high-frequency sound make interaural level differences (ILDs) their primary cue for azimuthal echolocation. In this paper, we present a neuromorphic VLSI-based system that emulates the ILD processing in the bat brainstem and midbrain. By selecting simple neural units, we propose a circuit model that is mathematically tractable and captures the essential elements of bat ILD computation. The chip includes a three-layer network of spiking neurons with 32 neurons on each layer, and the address-event representation for external interface. Emphasizing the neural spike timing and population behavior, we hope this study will contribute to the bat research community in particular as well as neuroscience in general by providing a real-time, fine-grained, neuromorphic bat echolocation simulator that will be used to address system-level performance of low-level neural algorithms. By developing functional models of the bat echolocation system, we hope to emulate the efficient implementation demonstrated by nature

Equibiaxial strain stimulates fibroblastic phenotype shift in smooth muscle cells in an engineered tissue model of the aortic wall

Many cells in the body reside in a complex three-dimensional (3D) environment stimulated by mechanical force. In vitro bioreactor systems have greatly improved our understanding of the mechanisms behind cell mechanotransduction. Current systems to impose strain in vitro are limited either by the lack of uniform strain profile or inability to strain 3D engineered tissues. In this study, we present a system capable of generating cyclic equibiaxial strain to an engineered vascular wall model. Type I collagen hydrogels populated with rat aortic smooth muscle cells (RASMCs) were created either as a compacting disk or constrained hemisphere. Both models were adhered to silicone membranes precoated with collagen I, fibronectin, or Cell-Tak and assayed for adhesion characteristics. The best performing model was then exposed to 48 h of 10% strain at 1Hz to simulate wall strain profiles found in vascular aneurysms, with static cultures serving as controls. The finite strain profile at the level of the membrane and the free surface of the construct was quantified using microbeads. The results indicate that the hemisphere model adhered with Cell-Tak had the most stable adhesion, followed by fibronectin and collagen I. Disk models did not adhere well under any coating condition. Uniform strain propagation was possible up to a maximum area strain of 20% with this system. RASMC responded to 10% equibiaxial strain by becoming less elongated, and immunohistochemistry suggested that stretched RASMC shifted to a more synthetic phenotype in comparison to static controls. These results suggest that equibiaxial strain may induce smooth muscle cell differentiation. We conclude that this system is effective in stimulating cells with cyclic equibiaxial strain in 3D cultures, and can be applied to a variety of biomaterial and tissue engineering applications.

WARP Is a Novel Multimeric Component of the Chondrocyte Pericellular Matrix That Interacts with Perlecan

WARP is a novel member of the von Willebrand factor A domain superfamily of extracellular matrix proteins that is expressed by chondrocytes. WARP is restricted to the presumptive articular cartilage zone prior to joint cavitation and to the articular cartilage and fibrocartilaginous elements in the joint, spine, and sternum during mouse embryonic development. In mature articular cartilage, WARP is highly specific for the chondrocyte pericellular microenvironment and co-localizes with perlecan, a prominent component of the chondrocyte pericellular region. WARP is present in the guanidine-soluble fraction of cartilage matrix extracts as a disulfide-bonded multimer, indicating that WARP is a strongly interacting component of the cartilage matrix. To investigate how WARP is integrated with the pericellular environment, we studied WARP binding to mouse perlecan using solid phase and surface plasmon resonance analysis. WARP interacts with domain III-2 of the perlecan core protein and the heparan sulfate chains of the perlecan domain I with K(D) values in the low nanomolar range. We conclude that WARP forms macromolecular structures that interact with perlecan to contribute to the assembly and/or maintenance of "permanent" cartilage structures during development and in mature cartilages.

Advanced microscopic imaging methods to investigate cortical development and the etiology of mental retardation

Studies on human patients and animal models of disease have shown that disruptions in prenatal and early postnatal brain development are a root cause of mental retardation. Since proper brain development is achieved by a strict spatiotemporal control of neurogenesis, cell migration, and patterning of synapses, abnormalities in one or more of these events during prenatal development can lead to cognitive dysfunction after birth. Many of underlying causes of mental retardation must therefore be studied in developing brains. To aid in this research, live imaging using laser scanning microscopy (LSM) has recently allowed neuroscientists to delve deeply into the complex three-dimensional environment of the living brain to record dynamic cellular events over time. This review will highlight recent examples of how LSM is being applied to elucidate both normal and abnormal cortical development.

Three-dimensional nonlinear regularized inversion of the induced polarization data based on the Cole–Cole model

The quantitative interpretation of induced polarization (IP) data in a complex three-dimensional (3D) environment is a very challenging problem. The analysis of IP phenomena is usually based on models with frequency dependent conductivity distributions. In this paper, we develop a technique for 3D nonlinear inversion of IP data based on the Cole–Cole relaxation model. Our method takes into account, though in an approximate way, the nonlinearity of electromagnetic induction and IP phenomena, and inverts the observed data for the Cole–Cole model parameters. The solution of the 3D IP inverse problem is based on both smooth and focusing regularized inversion, which helps to generate more reliable images of the subsurface structures. The method is tested on synthetic models with anomalous conductivity and intrinsic chargeability, and is also applied to a practical 3D IP survey. We demonstrate that both the electrical conductivity and the chargeability distributions, as well as the other parameters of the Cole–Cole model, can be recovered from the observed IP data simultaneously. The recovered parameters of the Cole–Cole model can be used for the discrimination of the different types of mineralization, which is an important goal in mineral exploration.

Can We Build Artificial Stem Cell Compartments?

Animals carry stem cells throughout their entire life, from embryogenesis to senescence. Their function during development and adulthood consists basically of forming and sustaining functional tissues while maintaining a small self-renewing population. They reside in a complex three-dimensional environment consisting of other nearby cells extracellular matrix components, endogenous or exogenous soluble factors, and physical, structural, or mechanical properties of the tissues they inhabit. Can we artificially recreate tissue development such that stem cells can both self-renew and be instructed to mature properly? The main factors required to regulate the maintenance and differentiation of some types of stem cells are known. In addition, new bioengineered synthetic materials that mimic extracellular matrix components can be used as initial scaffolding for building stem cell microenvironments.

Study on the Computer Network and Virtual Reality Technology for e-Learning ----Baed on WebDeGrator System

The purpose of this research is to apply the network and virtual reality (VR) technologies to computer-aided geometry design (CAGO) curriculum by integrating network, CAD and VR into an e-learning environment. Through wireless technologies, it is expected that the computer graphics (CG) course can be transit to anywhere. VR technologies provide a novel method to enhance user visualization of complex three-dimensional graphics and environments. We have developed an e-learning system to simulate and adjust computer graphics through various complex curves and surfaces algorithms, it be named WebDeGrator, which is an e-learning system for CAD design and application, such as fitting and adapting NURBS curve and surface, fundament mathematics of tools, robot model in VR, visualization for bioinformatics… etc. It’s related to educationa1 and VR technologies will be presented and discussed herein.

Seismic wave-field observations at a dense, small-aperture array located on a landslide in the Santa Cruz Mountains, California

Abstract A rectangular (4 by 5) array of short-period three-component seismometers with 15-m spacing was deployed to record several U.S. Geological Survey calibration explosions detonated around the Santa Cruz Mountains. The array was located at a site where an earlier station had recorded frequency-dependent polarized site resonances for aftershocks of the 1989 Loma Prieta earthquake. The site is on a hillside believed to be a landslide structure, with the near surface consisting of poorly sorted sediments and weathered rocks with dipping subsurface layers. The primary objective was to explore the site effects in this complex three-dimensional soft-rock environment, characteristic of much of the Loma Prieta source region. The direct P waves from four nearby (15 to 20 km) explosions at easterly azimuths from the array show counterclockwise arrival azimuth anomalies of 30° to 50°. These deflections are attributed to the presence of more than one dipping velocity contrast beneath the array, with dips of from 10° to 50° and dip directions generally toward the south. One such boundary may correspond to the landslide slip surface, and the presence of dipping velocity contrasts underlying the site is probably responsible for some of the observed directional site resonance. A slowness vector analysis demonstrates that arrivals early in the P coda have similar azimuthal anomalies, while later scattered arrivals come from many azimuths. Particle motions indicate that the more coherent arrivals in the coda are comprised of scattered P waves and Rayleigh waves, probably associated with scattering from the rough topography in the region. The coda displays greater spatial coherency along the hill strike than down the slope, consistent with a wedge-shaped landslide. The overall wave-field spatial coherence, CCC(f, Δx), decreases with increasing frequency, f, and spatial offset, Δx, and on average can be well represented by CCC(f, Δx) = e−cfΔx, with c = 0.6 km−1 Hz−1 for the vertical P wave in the first 1-sec window. This behavior is comparable to that found for previously studied hard-rock locations.

A terrain-covering algorithm for an AUV

An efficient, on-line terrain-covering algorithm is presented for a robot (AUV) moving in an unknown three-dimensional underwater environment. Such an algorithm is necessary for producing mosaicked images of the ocean floor. The basis of this three-dimensional motion planning algorithm is a new planar algorithm for nonsimply connected areas with boundaries of arbitrary shape. We show that this algorithm generalizes naturally to complex three-dimensional environments in which the terrain to be covered is projectively planar. This planar algorithm represents an improvement over previous algorithms because it results in a shorter path length for the robot and does not assume a polygonal environment. The path length of our algorithm is shown to be linear in the size of the area to be covered; the amount of memory required by the robot to implement the algorithm is linear in the size of the description of the boundary of the area. An example is provided that demonstrates the algorithm’s performance in a nonsimply connected, nonplanar environment.

Spacing between doses on a thrice-daily regimen.

Cancer invasion and metastasis occur in a complex three-dimensional (3D) environment, with reciprocal feedback from the surrounding host tissue and vasculature-governing behavior. In this study, we used a novel intravital method that revealed spatiotemporal regulation of Src activity in response to the anti-invasive Src inhibitor dasatinib. A fluorescence lifetime imaging microscopy–fluorescence resonance energy transfer (FLIM-FRET) Src biosensor was used to monitor drug-targeting efficacy in a transgenic p53-mutant mouse model of pancreatic cancer. In contrast to conventional techniques, FLIM-FRET analysis allowed for accurate, time-dependent, live monitoring of drug efficacy and clearance in live tumors. In 3D organotypic cultures, we showed that a spatially distinct gradient of Src activity exists within invading tumor cells, governed by the depth of penetration into complex matrices. In parallel, this gradient was also found to exist within live tumors, where Src activity is enhanced at the invasive border relative to the tumor cortex. Upon treatment with dasatinib, we observed a switch in activity at the invasive borders, correlating with impaired metastatic capacity in vivo. Src regulation was governed by the proximity of cells to the host vasculature, as cells distal to the vasculature were regulated differentially in response to drug treatment compared with cells proximal to the vasculature. Overall, our results in live tumors revealed that a threshold of drug penetrance exists in vivo and that this can be used to map areas of poor drug-targeting efficiency within specific tumor microenvironments. We propose that using FLIM-FRET in this capacity could provide a useful preclinical tool in animal models before clinical translation. Cancer Res; 73(15); 4674–86. ©2013 AACR.

A quantitative coronal plane evaluation of craniofacial growth and spatial relations during secondary palate development in the rat

Movement of the lateral palatine processes from the vertical to the horizontal plane occurs in a complex three-dimensional environment. Selected coronal sections of foetal Sprague-Dawley rat heads were magnified and measured. Mean group outlines were mapped and graphic composite illustrations made to demonstrate relative changes. During the two-day period about the time of palatal closure, although the height of the oro-nasal cavity doubled, there was little change in the width. As the height increased, the position of the tongue became lower relative to the palato-maxillary processes and a space developed above the tongue in the middle-palate region. During shelf elevation, the width of the oral cavity and the maxilla decreased, but the vertical dimension was unchanged.