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PubMed2004

Dendritic branching and homogenization of actin networks mediated by arp2/3 complex.

The cytoskeleton of motile cells exploits accessory proteins to locally modulate its organization and micromechanics. Here, we demonstrate that the Arp2/3 complex plays the role, unique among other cytoskeleton proteins, of an actin network "homogenizer," promoting the extremely rapid formation of homogeneous and stiff networks. Nanotracking of microspheres imbedded in F-actin networks reveals that the Arp2/3 complex promotes the formation of networks that are remarkably more homogeneous than control networks, a distinctive feature that coordinates a dramatic burst of elasticity. These results suggest that the Arp2/3 complex possesses a unique function of stabilizing membrane protrusions through the formation of homogeneous and stiff actin cytoskeleton at the leading edge of crawling cells.

Actin Cytoskeleton↗
PubMed2005

Mechanical shear can accelerate the gelation of actin filament networks.

Rearrangements of the filamentous actin cytoskeleton at the leading edge of motile cells occur under large mechanical stresses. Contrary to conventional wisdom, we show that mechanical deformations applied during gelation can accelerate the rate of gelation and produce F-actin networks that are stiffer and mechanically more resilient than those polymerized under low or high shear deformations. Above a threshold shear strain amplitude, F-actin networks collapse and become soft and liquidlike. This effect of shear-induced strengthening of polymerizing networks depends on the state of hydrolysis of the actin-bound adenosine triphosphate.

Actin Cytoskeleton↗
PubMed2005

The role of protein and surfactant interactions in membrane-protein crystallization.

The interactions leading to crystallization of the integral membrane protein bacteriorhodopsin solubilized in n-octyl-beta-D-glucoside were investigated. Osmotic second virial coefficients were measured by self-interaction chromatography in the presence of sodium malonate, sodium formate and ammonium sulfate. Attractive protein-detergent complex (PDC) interactions were observed as the surfactant cloud-point temperature was approached for each salt, suggesting that surfactant interactions may play an important role in promoting PDC crystallization. Dynamic light scattering and tensiometry measurements show that the interaction trends are strongly influenced by micelle structure and surfactant phase behavior, both of which are sensitive to salt and surfactant concentration. Overall, detailed investigations using a combination of experimental techniques can provide insight into the complex nature of PDC interactions, which is essential to developing rational approaches to membrane-protein crystallization.

Bacteriorhodopsins↗
PubMed2005

Quantitative imaging by confocal scanning fluorescence microscopy of protein crystallization via liquid-liquid phase separation.

Metastable states such as liquid-liquid phase separation, aggregation and gelation can affect protein crystallization but their positive or negative effects are only partially understood. In this work, mixtures of PEG (MW 10 kDa) and a large model protein, glucose isomerase (MW 173 kDa), have been studied to characterize the effect of a metastable liquid-liquid phase separation on protein crystallization. Fluorescence labeling allowed confocal fluorescence microscopy observations and quantification of the partitioning of the protein and PEG between the liquid phases and showed two steps in the crystallization process. Two crystallization mechanisms within the liquid domain were revealed, yielding two different polymorphs. With one polymorph, few crystals nucleated and grew droplet-by-droplet in the dispersed concentrated liquid phase, while for the other homogeneous crystal nucleation and growth occurred independently and simultaneously in numerous droplets of the concentrated phase. The results demonstrate the substantial possible complexity of crystallization behavior, as well as its sensitivity to the location of the conditions on the phase diagram and to the physicochemical properties of the system.

Aldose-Ketose Isomerases↗
PubMed2005

In vivo haematopoietic potential of human neural stem cells.

The fetal sheep model was used to compare the in vivo haematopoietic potential of human neural stem cells (NSC) versus bone marrow (BM)-derived haematopoietic stem cells (HSC). To this end, sheep were transplanted with either 8 x 10(5) NSC (n = 11) or HSC, CD34(+)Lin(-) (n = 5), and subsequently analysed for haematopoietic chimaerism. While HSC-transplanted sheep displayed robust donor-derived haematopoiesis starting at less than 2 months post-transplant, NSC recipients exhibited haematopoietic engraftment at much later time points. Nevertheless, chimaerism persisted in both groups throughout the course of this study. Transplantation of secondary recipients with human CD45(+)/HLA-DR(+) cells from the BM of NSC primary recipients at 14 and 16 months post-transplant demonstrated that long-term engrafting HSC were present in these animals. At 6 months post-transplant, both NSC- and HSC-transplanted sheep were mobilised with granulocyte colony-stimulating factor. In contrast to HSC-transplanted animals, levels of human blood cells in peripheral blood of NSC-transplanted sheep remained low throughout mobilisation. Our results show that, although human NSC were able to give rise to multilineage haematopoiesis in our model, the levels, timing of blood cell production and the ability to respond to cytokine mobilisation were different, suggesting that human NSCs latent haematopoietic potential is inherently different from that of true HSC.

Animals↗
PubMed2004

Bone cell survival in microgravity: evidence that modeled microgravity increases osteoblast sensitivity to apoptogens.

Studies were performed to evaluate the effects of modeled microgravity on the induction of osteoblast apoptosis. MC3T3-E1 osteoblast-like cells were cultured in alginate carriers in the NASA-approved high aspect ratio vessel (HARV). This system subjects the cells to a time-averaged gravitational field (vector-averaged gravity) to simulate low gravity conditions. Cells were cultured in the HARV for five days, and then examined for apoptosis. In simulated microgravity, the cells remained vital, although analysis of expressed genes indicated that there was loss of the mature osteoblast phenotype. Additionally, we noted that there was a loss of the mitochondrial membrane potential, a low level of the antiapoptotic protein Bcl-2, as well as Akt protein, and the redox status of the cells was disturbed. All of these parameters indicated that vector-averaged gravity disrupts mitochondrial function, thereby sensitizing osteoblasts to apoptosis. We then used a challenge assay to evaluate the apoptotic sensitivity of the cells subjected to vector-averaged gravity. When challenged with staurosporine, cells subjected to vector-averaged gravity evidenced elevated levels of cell death relative to control cell populations. Another objective of the study was to improve upon conventional carriers by using alginate encapsulation to support cells in the HARV. We have demonstrated that the alginate carrier system affords a more robust system than surface-seeded carriers. This new system has the advantage of shielding cells from mechanical damage and fluid shear stresses on cells in the HARV, permitting carefully controlled studies of the effects of vector-averaged gravity.

3T3 Cells↗
PubMed2004

Modeling of phosphate ion transfer to the surface of osteoblasts under normal gravity and simulated microgravity conditions.

We have modeled the transport and accumulation of phosphate ions at the remodeling site of a trabecular bone consisting of osteoclasts and osteoblasts situated adjacent to each other in straining flows. Two such flows are considered; one corresponds to shear levels representative of trabecular bone conditions at normal gravity, the other corresponds to shear level that is representative of microgravity conditions. The latter is evaluated indirectly using a simulated microgravity environment prevailing in a rotating wall vessel bioreactor (RWV) designed by NASA. By solving the hydrodynamic equations governing the particle motion in a RWV using a direct numerical simulation (DNS) technique, the shear stress values on the surface of the microcarriers are found. In our present species transfer model, osteoclasts release phosphate ions (Pi) among other ions at bone resorption sites. Some of the ions so released are absorbed by the osteoblast, some accumulate at the osteoblast surface, and the remainder are advected away. The consumption of Pi by osteoblasts is assumed to follow Michaelis-Menten (MM) kinetics aided by a NaPi cotransporter system. MM kinetics views the NaPi cotransporter as a system for transporting extracellular Pi into the osteoblast. Our results show, for the conditions investigated here, the net accumulation of phosphate ions at the osteoblast surface under simulated microgravity conditions is higher by as much as a factor of three. Such increased accumulation may lead to enhanced apoptosis and may help explain the increased bone loss observed under microgravity conditions.

Animals↗
PubMed2004

Modeled microgravity inhibits osteogenic differentiation of human mesenchymal stem cells and increases adipogenesis.

Space flight-induced bone loss has been attributed to a decrease in osteoblast function, without a significant change in bone resorption. To determine the effect of microgravity (MG) on bone, we used the Rotary Cell Culture System [developed by the National Aeronautics and Space Administration (NASA)] to model MG. Cultured mouse calvariae demonstrated a 3-fold decrease in alkaline phosphatase (ALP) activity and failed to mineralize after 7 d of MG. ALP and osteocalcin gene expression were also decreased. To determine the effects of MG on osteoblastogenesis, we cultured human mesenchymal stem cells (hMSC) on plastic microcarriers, and osteogenic differentiation was induced immediately before the initiation of modeled MG. A marked suppression of hMSC differentiation into osteoblasts was observed because the cells failed to express ALP, collagen 1, and osteonectin. The expression of runt-related transcription factor 2 was also inhibited. Interestingly, we found that peroxisome proliferator-activated receptor gamma (PPARgamma2), which is known to be important for adipocyte differentiation, adipsin, leptin, and glucose transporter-4 are highly expressed in response to MG. These changes were not corrected after 35 d of readaptation to normal gravity. In addition, MG decreased ERK- and increased p38-phosphorylation. These pathways are known to regulate the activity of runt-related transcription factor 2 and PPARgamma2, respectively. Taken together, our findings indicate that modeled MG inhibits the osteoblastic differentiation of hMSC and induces the development of an adipocytic lineage phenotype. This work will increase understanding and aid in the prevention of bone loss, not only in MG but also potentially in age-and disuse-related osteoporosis.

Adipocytes↗
PubMed2004

Micro-organization and visco-elasticity of the interphase nucleus revealed by particle nanotracking.

The microstructure of the nucleus, one of the most studied but least understood cellular organelles, is the subject of much debate. Through the use of particle nanotracking, we detect and quantify the micro-organization as well as the viscoelastic properties of the intranuclear region in single, live, interphase somatic cells. We find that the intranuclear region is much stiffer than the cytoplasm; it is also more elastic than viscous, which reveals that the intranuclear region displays an unexpectedly strong solid-like behavior. The mean shear viscosity and elasticity of the intranuclear region of Swiss 3T3 fibroblasts are 520 Poise (P) and 180 dyn/cm(2), respectively. These measurements determine a lower bound of the propulsive forces (3-15 picoNewton) required for nuclear organelles such as promyelocytic-leukemia bodies to undergo processive transport within the nucleus by overcoming friction forces set by the intranuclear viscosity. Dynamic analysis of the spontaneous movements of nanospheres embedded in the nucleus reveals the presence of putative transient nuclear microdomains of mean size 290+/-50 nm, which are mostly absent in the cytoplasm. The strong elastic character and micro-organization of the intranuclear region revealed by particle nanotracking analysis may help the nucleus to preserve its structural coherence. These studies also highlight the difference between the low interstitial nucleoplasmic viscosity, which controls the transport of nuclear proteins and molecules, and the much higher mesoscale viscosity, which affects the diffusion and directed transport of nuclear organelles and re-organization of interphase chromosomes.

3T3 Cells↗
PubMed2005

Engineering a mimicry of bone marrow tissue ex vivo.

Hematopoietic stem cells reside in specific niches in the bone marrow and give rise to either more stem cells or maturing hematopoietic progeny depending on the signals provided in the bone marrow microenvironment. This microenvironment is comprised of cellular components as well as soluble constituents called cytokines. The use of cytokines alone for the ex vivo expansion of stem cells in flat, two-dimensional culture flasks, dishes or bags is inadequate and, given the three-dimensionality of the in vivo bone marrow microenvironment, inappropriate. Three-dimensional culture conditions can therefore provide an ex vivo mimicry of bone marrow, recapitulate the desired niche, and provide a suitable environment for stem cell expansion and differentiation. Choice of scaffold, manipulation and reproducibility of the scaffold properties and directed structuring of the niche, by choosing pore size and porosity may inform the resident stem cells of their fate in a directed fashion. The use of bioreactors for cultivation of hematopoietic cells will allow for culture control, optimization, standardization, scale-up, and a "hands-off" operation making the end-product dependable, predictable and free of contaminants, and therefore suitable for human use and therapeutic applications.

Biomimetics↗
PubMedDate not supplied

Restructuring dynamics of DU 145 and LNCaP prostate cancer spheroids.

Neoplastic cells acquire multidrug resistance as they assemble into multicellular spheroids. Image analysis and Monte Carlo simulation provided an insight into the adhesion and motility events during spheroid restructuring in liquid-overlay culture of DU 145 and LNCaP human prostate cancer cells. Irregularly shaped, two-dimensional aggregates restructured through incremental cell movements into three-dimensional spheroids. Of the two cultures examined, restructuring was more pronounced for DU 145 aggregates. Motile DU 145 cells formed spheroids with a minimum cell overlay of 30% for 25-mers as estimated by simulation versus 5% for adhesive LNCaP cells in aggregates of the same size. Over 72 h, the texture ratio increased from 0.55 +/- 0.05 for DU 145 aggregates with projected areas exceeding 2000 microm2 to a value approaching 0.75 +/- 0.02 (P < 0.05). For LNCaP aggregates of comparable size, the increase in texture ratio was more modest, less than 15% during the same time period (P < 0.05). Combined, these data suggest that motility events govern the overall rate of spheroid restructuring. This information has application to the chemosensitization of solid tumors and kinetic modeling of spheroid production.

Cell Aggregation↗
PubMed2005

Depth-varying density and organization of chondrocytes in immature and mature bovine articular cartilage assessed by 3d imaging and analysis.

Articular cartilage is a heterogeneous tissue, with cell density and organization varying with depth from the surface. The objectives of the present study were to establish a method for localizing individual cells in three-dimensional (3D) images of cartilage and quantifying depth-associated variation in cellularity and cell organization at different stages of growth. Accuracy of nucleus localization was high, with 99% sensitivity relative to manual localization. Cellularity (million cells per cm3) decreased from 290, 310, and 150 near the articular surface in fetal, calf, and adult samples, respectively, to 120, 110, and 50 at a depth of 1.0 mm. The distance/angle to the nearest neighboring cell was 7.9 microm/31 degrees , 7.1 microm/31 degrees , and 9.1 microm/31 degrees for cells at the articular surface of fetal, calf, and adult samples, respectively, and increased/decreased to 11.6 microm/31 degrees , 12.0 microm/30 degrees , and 19.2 microm/25 degrees at a depth of 0.7 mm. The methodologies described here may be useful for analyzing the 3D cellular organization of cartilage during growth, maturation, aging, degeneration, and regeneration.

Animals↗
PubMed2004

In vitro physical stimulation of tissue-engineered and native cartilage.

Because of the limited availability of donor cartilage for resurfacing defects in articular surfaces, there is tremendous interest in the in vitro bioengineering of cartilage replacements for clinical applications. However, attaining mechanical properties in engineered cartilaginous constructs that approach those of native cartilage has not been previously achieved when constructs are cultured under free-swelling conditions. One approach toward stimulating the development of constructs that are mechanically more robust is to expose them to physical environments that are similar, in certain ways, to those encountered by native cartilage. This is a strategy motivated by observations in numerous short-term experiments that certain mechanical signals are potent stimulators of cartilage metabolism. On the other hand, excess mechanical loading can have a deleterious effect on cartilage. Culture conditions that include a physical stimulation component are made possible by the use of specialized bioreactors. This chapter addresses some of the issues involved in using bioreactors as integral components of cartilage tissue engineering and in studying the physical regulation of cartilage. We first consider the generation of cartilaginous constructs in vitro. Next we describe the rationale and design of bioreactors that can impart either mechanical deformation or fluid-induced mechanical signals.

Animals↗
PubMed2004

Mechanical characterization of native and tissue-engineered cartilage.

Cartilage functions as a low-friction, wear-resistant, load-bearing tissue. During a normal gait cycle, one cartilage surface rolls and slides against another, all the while being loaded and unloaded. The durability of the tissue also makes for an impressive material to study. However, when cartilage is damaged or diseased, the tissue has little capacity to repair itself. The goal of cell-based repair strategies to replace damaged or diseased tissue requires that the functional biomechanical properties of normal (developing or mature), diseased, and repair cartilage be restored. This chapter addresses some of the major methods used to assess the biomechanical properties of native and tissue-engineered cartilage. First, the traditional methods of testing by compression, tension, shear, and indentation are reviewed. Next, additional methods to evaluate interfacial mechanics and lubrication are described. Thus, a variety of mechanical tests may be used to assess functional properties for normal, diseased, and tissue-engineered cartilage.

Animals↗
PubMed2004

Geometric and material determinants of patterning efficiency by dielectrophoresis.

Dielectrophoretic (DEP) forces have been used extensively to manipulate, separate, and localize biological cells and bioparticles via high-gradient electric fields. However, minimization of DEP exposure time is desirable, because of possible untoward effects on cell behavior. Toward this goal, this article investigates the geometric and material determinants of particle patterning kinetics and efficiency. In particular, the time required to achieve a steady-state pattern is theoretically modeled and experimentally validated for a planar, interdigitated bar electrode array energized in a standing-wave configuration. This measure of patterning efficiency is calculated from an improved Fourier series solution of DEP force, in which realistic boundary conditions and a finite chamber height are imposed to reflect typical microfluidic applications. The chamber height, electrode spacing, and fluid viscosity and conductivity are parameters that profoundly affect patterning efficiency, and optimization can reduce electric field exposure by orders of magnitude. Modeling strategies are generalizable to arbitrary electrode design as well as to conditions where DEP force may not act alone to cause particle motion. This improved understanding of DEP patterning kinetics provides a framework for new advances in the development of DEP-based biological devices and assays with minimal perturbation of cell behavior.

Biopolymers↗
PubMed2005

Relating surfactant properties to activity and solubilization of the human adenosine a3 receptor.

The effects of various surfactants on the activity and stability of the human adenosine A3 receptor (A3) were investigated. The receptor was expressed using stably transfected HEK293 cells at a concentration of 44 pmol functional receptor per milligram membrane protein and purified using over 50 different nonionic surfactants. A strong correlation was observed between a surfactant's ability to remove A3 from the membrane and the ability of the surfactant to remove A3 selectively relative to other membrane proteins. The activity of A3 once purified also correlates well with the selectivity of the surfactant used. The effects of varying the surfactant were much stronger than those achieved by including A3 ligands in the purification scheme. Notably, all surfactants that gave high efficiency, selectivity and activity fall within a narrow range of hydrophile-lipophile balance values. This effect may reflect the ability of the surfactant to pack effectively at the hydrophobic transmembrane interface. These findings emphasize the importance of identifying appropriate surfactants for a particular membrane protein, and offer promise for the development of rapid, efficient, and systematic methods to facilitate membrane protein purification.

Binding, Competitive↗
PubMed2004

Formation and structure of transplantable tissue constructs generated in simulated microgravity from Sertoli cells and neuron precursors.

Cell transplantation therapy for Parkinson's disease (PD) has received much attention as a potential treatment protocol for this neurodegenerative condition. Although there have been promising successes with this approach, it remains problematic, especially regarding the inability to provide immediate trophic support to the newly grafted cells and the inability to prevent acute and/or long-term graft rejection by the host. To address these issues of cell graftability, we have created a novel tissue construct from isolated rat Sertoli cells (SC) and the NTerra-2 immortalized human neuron precursor cell line (NT2) utilizing NASA-developed simulated microgravity technology. The two cell types were cocultured at a 1:4 (SC/NT2) ratio in the High Aspect Rotating Vessel (HARV) biochamber for 3 days, after which a disc-shaped aggregate (1-4 mm diameter) was formed. Sertoli neuron aggregated cells (SNAC) were collected by gravity sedimentation and processed either for light and electron microscopy or for fluorescent immunocytochemistry. Intra-SNAC clusters of SC and NT2 cells were identified by anti-human mitochondrial protein (huMT--specific for NT2 cells) and cholera toxin subunit B (CTb--specific for SC). There was little evidence of cell death throughout the aggregate and the absence of central necrosis, as might be expected in such a large aggregate in vitro. Ultrastructurally, SC did not express junctional modifications with NT2 cells nor with adjacent SC as is typical of SC in vivo and, in some protocols, in vitro. NT2 cells, however, showed distinct intercellular junction-like densities with adjacent NT2 cells, often defining canaliculi-like channels between the microvillus borders of the cells. The results show that the use of simulated microgravity coculture provides a culture environment suitable for the formation of a unique and viable Sertoli-NT2 (i.e., SNAC) tissue construct displaying intra-aggregate cellular organization. The structural integration of SC with NT2 cells provides a novel transplantable tissue source, which can be tested to determine if SC will suppress rejection of the grafted NT2 cells and provide for their short- and long-term trophic support in situ in the treatment of experimental PD.

Animals↗