Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

Directory of Open Access Journals (Sweden)

Luyan Ma

2009-03-01

Full Text Available Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell-cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications.

VANGL2 interacts with integrin αv to regulate matrix metalloproteinase activity and cell adhesion to the extracellular matrix.

Science.gov (United States)

Jessen, Tammy N; Jessen, Jason R

2017-12-15

Planar cell polarity (PCP) proteins are implicated in a variety of morphogenetic processes including embryonic cell migration and potentially cancer progression. During zebrafish gastrulation, the transmembrane protein Vang-like 2 (VANGL2) is required for PCP and directed cell migration. These cell behaviors occur in the context of a fibrillar extracellular matrix (ECM). While it is thought that interactions with the ECM regulate cell migration, it is unclear how PCP proteins such as VANGL2 influence these events. Using an in vitro cell culture model system, we previously showed that human VANGL2 negatively regulates membrane type-1 matrix metalloproteinase (MMP14) and activation of secreted matrix metalloproteinase 2 (MMP2). Here, we investigated the functional relationship between VANGL2, integrin αvβ3, and MMP2 activation. We provide evidence that VANGL2 regulates cell surface integrin αvβ3 expression and adhesion to fibronectin, laminin, and vitronectin. Inhibition of MMP14/MMP2 activity suppressed the cell adhesion defect in VANGL2 knockdown cells. Furthermore, our data show that MMP14 and integrin αv are required for increased proteolysis by VANGL2 knockdown cells. Lastly, we have identified integrin αvβ3 as a novel VANGL2 binding partner. Together, these findings begin to dissect the molecular underpinnings of how VANGL2 regulates MMP activity and cell adhesion to the ECM. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Theory of the particle matrix elements for Helium atom scattering in surfaces

International Nuclear Information System (INIS)

Khater, A.; Toennies, J.P.

2000-01-01

Full text.A brief review is presented for the recent development of the theory of the particle transition matrix elements, basic to the cross section for Helium and inert particle scattering at thermal energies in solid surfaces. the Jackson and Mott matrix elements are presented and discussed for surface scattering processes, habitually classified as elastic and inelastic. Modified transition matrix elements, introduced originally to account for the cut-off effects, are presented in a direct and simple manner. the Debye-Waller factor is introduced and discussed. A recent calculation for the particle transition matrix elements is presented for the specular and inelastic transition matrix elements and the corresponding inelastic scattering cross section is compared in detail to experimental data. the specular and inelastic transition matrix elements are found to be intrinsically similar owing to the intermediate role of a proposed virtual particle squeezed state near the surface

Characterization and fuel cell performance analysis of polyvinylalcohol-mordenite mixed-matrix membranes for direct methanol fuel cell use

Energy Technology Data Exchange (ETDEWEB)

Uctug, Fehmi Goerkem, E-mail: gorkem.uctug@bahcesehir.edu.t [University of Manchester, School of Chemical Engineering and Analytical Science, M60 1QD (United Kingdom); Holmes, Stuart M. [University of Manchester, School of Chemical Engineering and Analytical Science, M60 1QD (United Kingdom)

2011-10-01

Highlights: > We investigated the availability of PVA-mordenite membranes for DMFC use. > We measured the methanol permeability of PVA-mordenite membranes via pervaporation. > We did the fuel cell testing of these membranes, which had not been done before. > We showed that PVA-mordenite membranes have poorer DMFC performance than Nafion. > Membrane performance can be improved by increasing the proton conductivity of PVA. - Abstract: Polyvinylalcohol-mordenite (PVA-MOR) mixed matrix membranes were synthesized for direct methanol fuel cell (DMFC) use. For the structural and the morphological characterization, Scanning Electron Microscopy and Thermal Gravimetric Analysis methods were used. Zeolite distribution within the polymer matrix was found to be homogeneous. An impedance spectroscope was used to measure the proton conductivity. In order to obtain information about methanol permeation characteristics, swelling tests and a series of pervaporation experiments were carried out. 60-40 wt% PVA-MOR membranes were found to give the optimum transport properties. Proton conductivity of these membranes was found to be slightly lower than that of Nafion117{sup TM} whereas their methanol permeability was at least two orders of magnitude lower than Nafion117{sup TM}. DMFC performance of the PVA-MOR membranes was also measured. The inferior DMFC performance of PVA-MOR membranes was linked to drying in the fuel cell medium and the consequent proton conductivity loss. Their performance was improved by adding a dilute solution of sulfuric acid into the feed methanol solution. Future studies on the improvement of the proton conductivity of PVA-MOR membranes, especially via sulfonation of the polymer matrix, can overcome the low-performance problem associated with insufficient proton conductivity.

Characterization and fuel cell performance analysis of polyvinylalcohol-mordenite mixed-matrix membranes for direct methanol fuel cell use

International Nuclear Information System (INIS)

Uctug, Fehmi Goerkem; Holmes, Stuart M.

2011-01-01

Highlights: → We investigated the availability of PVA-mordenite membranes for DMFC use. → We measured the methanol permeability of PVA-mordenite membranes via pervaporation. → We did the fuel cell testing of these membranes, which had not been done before. → We showed that PVA-mordenite membranes have poorer DMFC performance than Nafion. → Membrane performance can be improved by increasing the proton conductivity of PVA. - Abstract: Polyvinylalcohol-mordenite (PVA-MOR) mixed matrix membranes were synthesized for direct methanol fuel cell (DMFC) use. For the structural and the morphological characterization, Scanning Electron Microscopy and Thermal Gravimetric Analysis methods were used. Zeolite distribution within the polymer matrix was found to be homogeneous. An impedance spectroscope was used to measure the proton conductivity. In order to obtain information about methanol permeation characteristics, swelling tests and a series of pervaporation experiments were carried out. 60-40 wt% PVA-MOR membranes were found to give the optimum transport properties. Proton conductivity of these membranes was found to be slightly lower than that of Nafion117 TM whereas their methanol permeability was at least two orders of magnitude lower than Nafion117 TM . DMFC performance of the PVA-MOR membranes was also measured. The inferior DMFC performance of PVA-MOR membranes was linked to drying in the fuel cell medium and the consequent proton conductivity loss. Their performance was improved by adding a dilute solution of sulfuric acid into the feed methanol solution. Future studies on the improvement of the proton conductivity of PVA-MOR membranes, especially via sulfonation of the polymer matrix, can overcome the low-performance problem associated with insufficient proton conductivity.

Synergistic cytotoxic effects of antibodies directed against different cell surface determinants

Energy Technology Data Exchange (ETDEWEB)

Elliott, E V; Pindar, A; Stevenson, F K; Stevenson, G T [Southampton General Hospital (UK). Tenovus Research Lab.

1978-03-01

Three antibody populations were raised in rabbits against surface antigens on guinea-pig L/sub 2/C leukaemic lymphocytes: against idiotypic determinants on the lambda chain of the surface immunoglobulin, against C region determinants on the lambda chain, and against the surface antigens recognised by conventional anti-lymphocyte sera. Complement and K-cell cytotoxicities effected by the antibodies on L/sub 2/C cells were studied in vitro. In both cytotoxic systems mixtures of the antibodies revealed synergy, in that the titres of the mixtures exceeded predicted additive titres of their components. The synergy was greater when the mixed antibodies were directed to determinants on the same molecule rather than to determinants on different molecules.

Adherence of extracellular matrix components to modified surfaces of titanium alloys

International Nuclear Information System (INIS)

Stelzer, C; Uhlmann, E; Meinke, M; Lademann, J; Hansen, U

2009-01-01

The adherence of biological materials on metal surfaces is of special importance in biology and medicine. The underlying interactions between surface and biological materials (e.g. extracellular matrix components or cells) are responsible for the application as a medical device. Numerous products are made of pure titanium and titanium alloys. This paper shows the influence of a laser production technology on machined surfaces of TiAl 6 V 4 and the resulting adherence of biological material on the basis of the surface characterisation. In this study, different machined TiAl 6 V 4 surfaces were used for coatings with extracellular matrix components. For this process, different coating with collagen I monomers and a complex mixture of extracellular matrix proteins derived from the dermal-epidermal basement membrane zone were analysed. The efficiency of the coating was analysed by different methods and the results are presented in this paper

Mechanosensing of matrix by stem cells: From matrix heterogeneity, contractility, and the nucleus in pore-migration to cardiogenesis and muscle stem cells in vivo.

Science.gov (United States)

Smith, Lucas; Cho, Sangkyun; Discher, Dennis E

2017-11-01

Stem cells are particularly 'plastic' cell types that are induced by various cues to become specialized, tissue-functional lineages by switching on the expression of specific gene programs. Matrix stiffness is among the cues that multiple stem cell types can sense and respond to. This seminar-style review focuses on mechanosensing of matrix elasticity in the differentiation or early maturation of a few illustrative stem cell types, with an intended audience of biologists and physical scientists. Contractile forces applied by a cell's acto-myosin cytoskeleton are often resisted by the extracellular matrix and transduced through adhesions and the cytoskeleton ultimately into the nucleus to modulate gene expression. Complexity is added by matrix heterogeneity, and careful scrutiny of the evident stiffness heterogeneity in some model systems resolves some controversies concerning matrix mechanosensing. Importantly, local stiffness tends to dominate, and 'durotaxis' of stem cells toward stiff matrix reveals a dependence of persistent migration on myosin-II force generation and also rigid microtubules that confer directionality. Stem and progenitor cell migration in 3D can be further affected by matrix porosity as well as stiffness, with nuclear size and rigidity influencing niche retention and fate choices. Cell squeezing through rigid pores can even cause DNA damage and genomic changes that contribute to de-differentiation toward stem cell-like states. Contraction of acto-myosin is the essential function of striated muscle, which also exhibit mechanosensitive differentiation and maturation as illustrated in vivo by beating heart cells and by the regenerative mobilization of skeletal muscle stem cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cadherin adhesion, tissue tension, and noncanonical Wnt signaling regulate fibronectin matrix organization.

Science.gov (United States)

Dzamba, Bette J; Jakab, Karoly R; Marsden, Mungo; Schwartz, Martin A; DeSimone, Douglas W

2009-03-01

In this study we demonstrate that planar cell polarity signaling regulates morphogenesis in Xenopus embryos in part through the assembly of the fibronectin (FN) matrix. We outline a regulatory pathway that includes cadherin adhesion and signaling through Rac and Pak, culminating in actin reorganization, myosin contractility, and tissue tension, which, in turn, directs the correct spatiotemporal localization of FN into a fibrillar matrix. Increased mechanical tension promotes FN fibril assembly in the blastocoel roof (BCR), while reduced BCR tension inhibits matrix assembly. These data support a model for matrix assembly in tissues where cell-cell adhesions play an analogous role to the focal adhesions of cultured cells by transferring to integrins the tension required to direct FN fibril formation at cell surfaces.

Senescent intervertebral disc cells exhibit perturbed matrix homeostasis phenotype.

Science.gov (United States)

Ngo, Kevin; Patil, Prashanti; McGowan, Sara J; Niedernhofer, Laura J; Robbins, Paul D; Kang, James; Sowa, Gwendolyn; Vo, Nam

2017-09-01

Aging greatly increases the risk for intervertebral disc degeneration (IDD) as a result of proteoglycan loss due to reduced synthesis and enhanced degradation of the disc matrix proteoglycan (PG). How disc matrix PG homeostasis becomes perturbed with age is not known. The goal of this study is to determine whether cellular senescence is a source of this perturbation. We demonstrated that disc cellular senescence is dramatically increased in the DNA repair-deficient Ercc1 -/Δ mouse model of human progeria. In these accelerated aging mice, increased disc cellular senescence is closely associated with the rapid loss of disc PG. We also directly examine PG homeostasis in oxidative damage-induced senescent human cells using an in vitro cell culture model system. Senescence of human disc cells treated with hydrogen peroxide was confirmed by growth arrest, senescence-associated β-galactosidase activity, γH2AX foci, and acquisition of senescence-associated secretory phenotype. Senescent human disc cells also exhibited perturbed matrix PG homeostasis as evidenced by their decreased capacity to synthesize new matrix PG and enhanced degradation of aggrecan, a major matrix PG. of the disc. Our in vivo and in vitro findings altogether suggest that disc cellular senescence is an important driver of PG matrix homeostatic perturbation and PG loss. Published by Elsevier B.V.

Mechanotransduction across the cell surface and through the cytoskeleton

Science.gov (United States)

Wang, N.; Butler, J. P.; Ingber, D. E.

1993-01-01

Mechanical stresses were applied directly to cell surface receptors with a magnetic twisting device. The extracellular matrix receptor, integrin beta 1, induced focal adhesion formation and supported a force-dependent stiffening response, whereas nonadhesion receptors did not. The cytoskeletal stiffness (ratio of stress to strain) increased in direct proportion to the applied stress and required intact microtubules and intermediate filaments as well as microfilaments. Tensegrity models that incorporate mechanically interdependent struts and strings that reorient globally in response to a localized stress mimicked this response. These results suggest that integrins act as mechanoreceptors and transmit mechanical signals to the cytoskeleton. Mechanotransduction, in turn, may be mediated simultaneously at multiple locations inside the cell through force-induced rearrangements within a tensionally integrated cytoskeleton.

Multiscale grooved titanium processed with femtosecond laser influences mesenchymal stem cell morphology, adhesion, and matrix organization.

Science.gov (United States)

Dumas, Virginie; Rattner, Aline; Vico, Laurence; Audouard, Eric; Dumas, Jean Claude; Naisson, Pierre; Bertrand, Philippe

2012-11-01

The femtosecond laser processing enabled the structuring of six types of surfaces on titanium-6aluminium-4vanadium (Ti-6Al-4V) plates. The obtained hierarchical features consisted of a combination of microgrooves and oriented nanostructures. By adjusting beam properties such as laser polarization, the width of the microgrooves (20 or 60 μm) and the orientation of the nanostructures (parallel or perpendicular to the microgrooves) can be precisely controlled. Mesenchymal stem cells (MSCs) grown on these structured surfaces produced cytoplasmic extensions with focal contacts, while on the smooth titanium, the cells were found to be well spread and without any focal contact 12 h postseeding. The 600-nm wide nanostructures on their own were sufficient to orient the MSCs. For the multiscale structured areas, when the orientation of the nanostructures was orthogonal in relation to the microgrooves, there was an important decrease in or even a loss of cell alignment signifying that cells were sensitive to the directional nanostructures in the microgrooves. At 7 days, cell proliferation was not affected but the direction of nanostructures controlled the matrix organization. The ultrafast laser, as a new method for producing micro-nanohybrid surfaces, is a promising approach to promote desired tissue organization for tissue engineering. Copyright © 2012 Wiley Periodicals, Inc.

Heading-vector navigation based on head-direction cells and path integration.

Science.gov (United States)

Kubie, John L; Fenton, André A

2009-05-01

Insect navigation is guided by heading vectors that are computed by path integration. Mammalian navigation models, on the other hand, are typically based on map-like place representations provided by hippocampal place cells. Such models compute optimal routes as a continuous series of locations that connect the current location to a goal. We propose a "heading-vector" model in which head-direction cells or their derivatives serve both as key elements in constructing the optimal route and as the straight-line guidance during route execution. The model is based on a memory structure termed the "shortcut matrix," which is constructed during the initial exploration of an environment when a set of shortcut vectors between sequential pairs of visited waypoint locations is stored. A mechanism is proposed for calculating and storing these vectors that relies on a hypothesized cell type termed an "accumulating head-direction cell." Following exploration, shortcut vectors connecting all pairs of waypoint locations are computed by vector arithmetic and stored in the shortcut matrix. On re-entry, when local view or place representations query the shortcut matrix with a current waypoint and goal, a shortcut trajectory is retrieved. Since the trajectory direction is in head-direction compass coordinates, navigation is accomplished by tracking the firing of head-direction cells that are tuned to the heading angle. Section 1 of the manuscript describes the properties of accumulating head-direction cells. It then shows how accumulating head-direction cells can store local vectors and perform vector arithmetic to perform path-integration-based homing. Section 2 describes the construction and use of the shortcut matrix for computing direct paths between any pair of locations that have been registered in the shortcut matrix. In the discussion, we analyze the advantages of heading-based navigation over map-based navigation. Finally, we survey behavioral evidence that nonhippocampal

Matrix mechanics and fluid shear stress control stem cells fate in three dimensional microenvironment.

Science.gov (United States)

Chen, Guobao; Lv, Yonggang; Guo, Pan; Lin, Chongwen; Zhang, Xiaomei; Yang, Li; Xu, Zhiling

2013-07-01

Stem cells have the ability to self-renew and to differentiate into multiple mature cell types during early life and growth. Stem cells adhesion, proliferation, migration and differentiation are affected by biochemical, mechanical and physical surface properties of the surrounding matrix in which stem cells reside and stem cells can sensitively feel and respond to the microenvironment of this matrix. More and more researches have proven that three dimensional (3D) culture can reduce the gap between cell culture and physiological environment where cells always live in vivo. This review summarized recent findings on the studies of matrix mechanics that control stem cells (primarily mesenchymal stem cells (MSCs)) fate in 3D environment, including matrix stiffness and extracellular matrix (ECM) stiffness. Considering the exchange of oxygen and nutrients in 3D culture, the effect of fluid shear stress (FSS) on fate decision of stem cells was also discussed in detail. Further, the difference of MSCs response to matrix stiffness between two dimensional (2D) and 3D conditions was compared. Finally, the mechanism of mechanotransduction of stem cells activated by matrix mechanics and FSS in 3D culture was briefly pointed out.

Decolorization of textile dye RB19 using volcanic rock matrix immobilized Bacillus thuringiensis cells with surface displayed laccase.

Science.gov (United States)

Wan, Juan; Sun, Xiaowen; Liu, Cheng; Tang, Mengjun; Li, Lin; Ni, Hong

2017-06-01

A triplicate volcanic rock matrix-Bacillus thuringiensis-laccase WlacD (VRMs-Bt-WlacD) dye decolorization system was developed. WlacD was displayed on the B. thuringiensis MB174 cell surface to prepare a whole-cell laccase biocatalyst by using two repeat N-terminal domains of autolysin Mbg (Mbgn) 2 as the anchoring motif. Immunofluorescence microscopic assays confirmed that the fusion protein (Mbgn) 2 -WlacD was anchored on the surface of the recombinant B. thuringiensis MB174. After optimization by a single factor test, L 9 (3 4 )-orthogonal test, Plackett-Burman test, steepest ascent method, and Box-Behnken response surface methodology, the whole-cell specific laccase activity of B. thuringiensis MB174 was improved to 555.2 U L -1 , which was 2.25 times than that of the primary culture condition. Optimized B. thuringiensis MB174 cells were further adsorbed by VRMs to prepare VRMs-Bt-WlacD, an immobilized whole-cell laccase biocatalyst. Decolorization capacity of as-prepared VRMs-Bt-WlacD toward an initial concentration of 500 mg L -1 of an textile dye reactive blue 19 (RB19) aqueous solution reached 72.36% at a solid-to-liquid ratio of 10 g-100 mL. Repeated decolorization-activation operations showed the high decolorization capacity of VRMs-Bt-WlacD and have the potential for large-scale or continuous operations.

Methods of conditioning direct methanol fuel cells

Science.gov (United States)

Rice, Cynthia; Ren, Xiaoming; Gottesfeld, Shimshon

2005-11-08

Methods for conditioning the membrane electrode assembly of a direct methanol fuel cell ("DMFC") are disclosed. In a first method, an electrical current of polarity opposite to that used in a functioning direct methanol fuel cell is passed through the anode surface of the membrane electrode assembly. In a second method, methanol is supplied to an anode surface of the membrane electrode assembly, allowed to cross over the polymer electrolyte membrane of the membrane electrode assembly to a cathode surface of the membrane electrode assembly, and an electrical current of polarity opposite to that in a functioning direct methanol fuel cell is drawn through the membrane electrode assembly, wherein methanol is oxidized at the cathode surface of the membrane electrode assembly while the catalyst on the anode surface is reduced. Surface oxides on the direct methanol fuel cell anode catalyst of the membrane electrode assembly are thereby reduced.

Gelatin-based laser direct-write technique for the precise spatial patterning of cells.

Science.gov (United States)

Schiele, Nathan R; Chrisey, Douglas B; Corr, David T

2011-03-01

Laser direct-writing provides a method to pattern living cells in vitro, to study various cell-cell interactions, and to build cellular constructs. However, the materials typically used may limit its long-term application. By utilizing gelatin coatings on the print ribbon and growth surface, we developed a new approach for laser cell printing that overcomes the limitations of Matrigel™. Gelatin is free of growth factors and extraneous matrix components that may interfere with cellular processes under investigation. Gelatin-based laser direct-write was able to successfully pattern human dermal fibroblasts with high post-transfer viability (91% ± 3%) and no observed double-strand DNA damage. As seen with atomic force microscopy, gelatin offers a unique benefit in that it is present temporarily to allow cell transfer, but melts and is removed with incubation to reveal the desired application-specific growth surface. This provides unobstructed cellular growth after printing. Monitoring cell location after transfer, we show that melting and removal of gelatin does not affect cellular placement; cells maintained registry within 5.6 ± 2.5 μm to the initial pattern. This study demonstrates the effectiveness of gelatin in laser direct-writing to create spatially precise cell patterns with the potential for applications in tissue engineering, stem cell, and cancer research.

N-sulfation of heparan sulfate is critical for syndecan-4-mediated podocyte cell-matrix interactions

NARCIS (Netherlands)

Sugar, T.; Wassenhove-McCarthy, D.J.; Orr, A.W.; Green, J.; Kuppevelt, T.H. van; McCarthy, K.J.

2016-01-01

Previous research has shown that podocytes unable to assemble heparan sulfate on cell surface proteoglycan core proteins have compromised cell-matrix interactions. This report further explores the role of N-sulfation of intact heparan chains in podocyte-matrix interactions. For the purposes of this

Carbonate fuel cell matrix

Science.gov (United States)

Farooque, Mohammad; Yuh, Chao-Yi

1996-01-01

A carbonate fuel cell matrix comprising support particles and crack attenuator particles which are made platelet in shape to increase the resistance of the matrix to through cracking. Also disclosed is a matrix having porous crack attenuator particles and a matrix whose crack attenuator particles have a thermal coefficient of expansion which is significantly different from that of the support particles, and a method of making platelet-shaped crack attenuator particles.

Laser-Sintered Constructs with Bio-inspired Porosity and Surface Micro/Nano-Roughness Enhance Mesenchymal Stem Cell Differentiation and Matrix Mineralization In Vitro.

Science.gov (United States)

Cheng, Alice; Cohen, David J; Boyan, Barbara D; Schwartz, Zvi

2016-12-01

Direct metal laser sintering can produce porous Ti-6Al-4V orthopedic and dental implants. The process requires reduced resources and time and can provide greater structural control than machine manufacturing. Implants in bone are colonized by mesenchymal stem cells (MSCs), which can differentiate into osteoblasts and contribute to osseointegration. This study examined osteoblast differentiation and matrix mineralization of human MSCs cultured on laser-sintered Ti-6Al-4V constructs with varying porosity and at different time scales. 2D solid disks and low, medium and high porosity (LP, MP, and HP) 3D constructs based on a human trabecular bone template were laser sintered from Ti-6Al-4V powder and further processed to have micro- and nanoscale roughness. hMSCs exhibited greater osteoblastic differentiation and local factor production on all 3D porous constructs compared to 2D surfaces, which was sustained for 9 days without use of exogenous factors. hMSCs cultured for 8 weeks on MP constructs in osteogenic medium (OM), OM supplemented with BMP2 or collagen-coated MP constructs in OM exhibited bone-like extracellular matrix mineralization. Use of bio-inspired porosity for the 3D architecture of additively manufactured Ti-6Al-4V enhanced osteogenic differentiation of hMSCs beyond surface roughness alone. This study suggests that a 3D architecture may enhance the osseointegration of orthopedic and dental implants in vivo.

Design of experiments approach to engineer cell-secreted matrices for directing osteogenic differentiation.

Science.gov (United States)

Decaris, Martin L; Leach, J Kent

2011-04-01

The presentation of extracellular matrix (ECM) proteins provides an opportunity to instruct the phenotype and behavior of responsive cells. Decellularized cell-secreted matrix coatings (DM) represent a biomimetic culture surface that retains the complexity of the natural ECM. Microenvironmental culture conditions alter the composition of these matrices and ultimately the ability of DMs to direct cell fate. We employed a design of experiments (DOE) multivariable analysis approach to determine the effects and interactions of four variables (culture duration, cell seeding density, oxygen tension, and media supplementation) on the capacity of DMs to direct the osteogenic differentiation of human mesenchymal stem cells (hMSCs). DOE analysis revealed that matrices created with extended culture duration, ascorbate-2-phosphate supplementation, and in ambient oxygen tension exhibited significant correlations with enhanced hMSC differentiation. We validated the DOE model results using DMs predicted to have superior (DM1) or lesser (DM2) osteogenic potential for naïve hMSCs. Compared to cells on DM2, hMSCs cultured on DM1 expressed 2-fold higher osterix levels and deposited 3-fold more calcium over 3 weeks. Cells on DM1 coatings also exhibited greater proliferation and viability compared to DM2-coated substrates. This study demonstrates that DOE-based analysis is a powerful tool for optimizing engineered systems by identifying significant variables that have the greatest contribution to the target output.

Modeling and predictions of biphasic mechanosensitive cell migration altered by cell-intrinsic properties and matrix confinement.

Science.gov (United States)

Pathak, Amit

2018-04-12

Motile cells sense the stiffness of their extracellular matrix (ECM) through adhesions and respond by modulating the generated forces, which in turn lead to varying mechanosensitive migration phenotypes. Through modeling and experiments, cell migration speed is known to vary with matrix stiffness in a biphasic manner, with optimal motility at an intermediate stiffness. Here, we present a two-dimensional cell model defined by nodes and elements, integrated with subcellular modeling components corresponding to mechanotransductive adhesion formation, force generation, protrusions and node displacement. On 2D matrices, our calculations reproduce the classic biphasic dependence of migration speed on matrix stiffness and predict that cell types with higher force-generating ability do not slow down on very stiff matrices, thus disabling the biphasic response. We also predict that cell types defined by lower number of total receptors require stiffer matrices for optimal motility, which also limits the biphasic response. For a cell type with robust biphasic migration on 2D surface, simulations in channel-like confined environments of varying width and height predict faster migration in more confined matrices. Simulations performed in shallower channels predict that the biphasic mechanosensitive cell migration response is more robust on 2D micro-patterns as compared to the channel-like 3D confinement. Thus, variations in the dimensionality of matrix confinement alters the way migratory cells sense and respond to the matrix stiffness. Our calculations reveal new phenotypes of stiffness- and topography-sensitive cell migration that critically depend on both cell-intrinsic and matrix properties. These predictions may inform our understanding of various mechanosensitive modes of cell motility that could enable tumor invasion through topographically heterogeneous microenvironments. © 2018 IOP Publishing Ltd.

Characterization of Polyethylene-Graft-Sulfonated Polyarylsulfone Proton Exchange Membranes for Direct Methanol Fuel Cell Applications.

Science.gov (United States)

Kim, Hyung Kyu; Zhang, Gang; Nam, Changwoo; Chung, T C Mike

2015-12-04

This paper examines polymer film morphology and several important properties of polyethylene-graft-sulfonated polyarylene ether sulfone (PE-g-s-PAES) proton exchange membranes (PEMs) for direct methanol fuel cell applications. Due to the extreme surface energy differences between a semi-crystalline and hydrophobic PE backbone and several amorphous and hydrophilic s-PAES side chains, the PE-g-s-PAES membrane self-assembles into a unique morphology, with many proton conductive s-PAES channels embedded in the stable and tough PE matrix and a thin hydrophobic PE layer spontaneously formed on the membrane surfaces. In the bulk, these membranes show good mechanical properties (tensile strength >30 MPa, Young's modulus >1400 MPa) and low water swelling (λ 3 mmol/g in the s-PAES domains. On the surface, the thin hydrophobic and semi-crystalline PE layer shows some unusual barrier (protective) properties. In addition to exhibiting higher through-plane conductivity (up to 160 mS/cm) than in-plane conductivity, the PE surface layer minimizes methanol cross-over from anode to cathode with reduced fuel loss, and stops the HO• and HO₂• radicals, originally formed at the anode, entering into PEM matrix. Evidently, the thin PE surface layer provides a highly desirable protecting layer for PEMs to reduce fuel loss and increase chemical stability. Overall, the newly developed PE-g-s-PAES membranes offer a desirable set of PEM properties, including conductivity, selectivity, mechanical strength, stability, and cost-effectiveness for direct methanol fuel cell applications.

Integrins and extracellular matrix in mechanotransduction

Directory of Open Access Journals (Sweden)

Ramage L

2011-12-01

Full Text Available Lindsay RamageQueen’s Medical Research Institute, University of Edinburgh, Edinburgh, UKAbstract: Integrins are a family of cell surface receptors which mediate cell–matrix and cell–cell adhesions. Among other functions they provide an important mechanical link between the cells external and intracellular environments while the adhesions that they form also have critical roles in cellular signal-transduction. Cell–matrix contacts occur at zones in the cell surface where adhesion receptors cluster and when activated the receptors bind to ligands in the extracellular matrix. The extracellular matrix surrounds the cells of tissues and forms the structural support of tissue which is particularly important in connective tissues. Cells attach to the extracellular matrix through specific cell-surface receptors and molecules including integrins and transmembrane proteoglycans. Integrins work alongside other proteins such as cadherins, immunoglobulin superfamily cell adhesion molecules, selectins, and syndecans to mediate cell–cell and cell–matrix interactions and communication. Activation of adhesion receptors triggers the formation of matrix contacts in which bound matrix components, adhesion receptors, and associated intracellular cytoskeletal and signaling molecules form large functional, localized multiprotein complexes. Cell–matrix contacts are important in a variety of different cell and tissue properties including embryonic development, inflammatory responses, wound healing, and adult tissue homeostasis. This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.Keywords: ligand binding, α subunit, ß subunit, focal adhesion, cell differentiation, mechanical loading, cell–matrix interaction

Metal Matrix Composite Material by Direct Metal Deposition

Science.gov (United States)

Novichenko, D.; Marants, A.; Thivillon, L.; Bertrand, P. H.; Smurov, I.

Direct Metal Deposition (DMD) is a laser cladding process for producing a protective coating on the surface of a metallic part or manufacturing layer-by-layer parts in a single-step process. The objective of this work is to demonstrate the possibility to create carbide-reinforced metal matrix composite objects. Powders of steel 16NCD13 with different volume contents of titanium carbide are tested. On the base of statistical analysis, a laser cladding processing map is constructed. Relationships between the different content of titanium carbide in a powder mixture and the material microstructure are found. Mechanism of formation of various precipitated titanium carbides is investigated.

MMP-sensitive PEG diacrylate hydrogels with spatial variations in matrix properties stimulate directional vascular sprout formation.

Directory of Open Access Journals (Sweden)

Michael V Turturro

Full Text Available The spatial presentation of immobilized extracellular matrix (ECM cues and matrix mechanical properties play an important role in directed and guided cell behavior and neovascularization. The goal of this work was to explore whether gradients of elastic modulus, immobilized matrix metalloproteinase (MMP-sensitivity, and YRGDS cell adhesion ligands are capable of directing 3D vascular sprout formation in tissue engineered scaffolds. PEGDA hydrogels were engineered with mechanical and biofunctional gradients using perfusion-based frontal photopolymerization (PBFP. Bulk photopolymerized hydrogels with uniform mechanical properties, degradation, and immobilized biofunctionality served as controls. Gradient hydrogels exhibited an 80.4% decrease in elastic modulus and a 56.2% decrease in immobilized YRGDS. PBFP hydrogels also demonstrated gradients in hydrogel degradation with degradation times ranging from 10-12 hours in the more crosslinked regions to 4-6 hours in less crosslinked regions. An in vitro model of neovascularization, composed of co-culture aggregates of endothelial and smooth muscle cells, was used to evaluate the effect of these gradients on vascular sprout formation. Aggregate invasion in gradient hydrogels occurred bi-directionally with sprout alignment observed in the direction parallel to the gradient while control hydrogels with homogeneous properties resulted in uniform invasion. In PBFP gradient hydrogels, aggregate sprout length was found to be twice as long in the direction parallel to the gradient as compared to the perpendicular direction after three weeks in culture. This directionality was found to be more prominent in gradient regions of increased stiffness, crosslinked MMP-sensitive peptide presentation, and immobilized YRGDS concentration.

Complete horizontal skin cell resurfacing and delayed vertical cell infiltration into porcine reconstructive tissue matrix compared to bovine collagen matrix and human dermis.

Science.gov (United States)

Mirastschijski, Ursula; Kerzel, Corinna; Schnabel, Reinhild; Strauss, Sarah; Breuing, Karl-Heinz

2013-10-01

Xenogenous dermal matrices are used for hernia repair and breast reconstruction. Full-thickness skin replacement is needed after burn or degloving injuries with exposure of tendons or bones. The authors used a human skin organ culture model to study whether porcine reconstructive tissue matrix (Strattice) is effective as a dermal tissue replacement. Skin cells or split-thickness skin grafts were seeded onto human deepidermized dermis, Strattice, and Matriderm. Cellular resurfacing and matrix infiltration were monitored by live fluorescence imaging, histology, and electron microscopy. Proliferation, apoptosis, cell differentiation, and adhesion were analyzed by immunohistochemistry. Epithelial resurfacing and vertical proliferation were reduced and delayed with both bioartificial matrices compared with deepidermized dermis; however, no differences in apoptosis, cell differentiation, or basement membrane formation were found. Vertical penetration was greatest on Matriderm, whereas no matrix infiltration was found on Strattice in the first 12 days. Uncompromised horizontal resurfacing was greatest with Strattice but was absent with Matriderm. Strattice showed no stimulatory effect on cellular inflammation. Matrix texture and surface properties governed cellular performance on tissues. Although dense dermal compaction delayed vertical cellular ingrowth for Strattice, it allowed uncompromised horizontal resurfacing. Dense dermal compaction may slow matrix decomposition and result in prolonged biomechanical stability of the graft. Reconstructive surgeons should choose the adequate matrix substitute depending on biomechanical requirements at the recipient site. Strattice may be suitable as a dermal replacement at recipient sites with high mechanical load requirements.

Direct Surface Analysis of Fungal Species by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Energy Technology Data Exchange (ETDEWEB)

Valentine, Nancy B.(BATTELLE (PACIFIC NW LAB)); Wahl, Jon H.(BATTELLE (PACIFIC NW LAB)); Kingsley, Mark T.(BATTELLE (PACIFIC NW LAB)); Wahl, Karen L.(BATTELLE (PACIFIC NW LAB))

2001-12-01

Intact spores and/or hyphae of Aspergillus niger, Rhizopus oryzae, Trichoderma reesei and Phanerochaete chrysosporium are analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). This study investigates various methods of sample preparation and matrices to determine optimum collection and analysis criteria for fungal analysis by MALDI-MS. Fungi are applied to the MALDI sample target as untreated, sonicated, acid/heat treated, or blotted directly from the fungal culture with double-stick tape. Ferulic acid or sinapinic acid matrix solution is layered over the dried samples and analyzed by MALDI-MS. Statistical analysis of the data show that simply using double stick tape to collect and transfer to a MALDI sample plate typically worked as well as the other preparation methods, but requires the least sample handling.

Soluble extracellular matrix metalloproteinase inducer (EMMPRIN, EMN) regulates cancer-related cellular functions by homotypic interactions with surface CD147.

Science.gov (United States)

Knutti, Nadine; Kuepper, Michael; Friedrich, Karlheinz

2015-11-01

EMMPRIN (extracellular matrix metalloproteinase inducer) is a widely expressed glycoprotein and a member of the immunoglobulin superfamily which exists in both a membrane-spanning and a soluble form. Homotypic interactions of EMMPRIN underlie its multiple roles in normal development and pathological situations such as viral infections, Alzheimer's disease and cancer. This study employed a recombinant soluble, fully glycosylated EMMPRIN domain (rhsEMN) as a tool to characterize the structural basis of EMMPRIN-EMMPRIN receptor (EMNR) contacts and their functional effects on MCF-7 breast carcinoma cells. rhsEMN did not form dimers in solution but bound to surface EMMPRIN (EMN) on MCF-7 cells with high affinity and was readily internalized. The interaction interface for the homotypic contact was localized to the N-terminal Ig domain. rhsEMN exerted a stimulatory effect on proliferation of MCF-7 cells whereas it reduced cell migration in a dose-dependent manner. These effects were accompanied by an upregulation of endogenous EMMPRIN as well as of matrix metalloproteinase-14 (MMP-14), a membrane-bound protease involved in the extracellular release of soluble EMMPRIN, indicating a regulatory feedback mechanism. The proliferation-promoting activity of rhsEMN was mimicked by a novel functional antibody directed to EMMPRIN, underscoring that crosslinking of cell surface EMMPRIN (EMNR) is crucial for eliciting intracellular signalling. Addressing malignancy-related signal transduction in HEK-293 cells, we could show that rhsEMN triggers the oncogenic Wnt pathway. © 2015 FEBS.

Grooved surface topography alters matrix-metalloproteinase production by human fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Brydone, Alistair S; Dominic Meek, R M [Department of Orthopaedics, Southern General Hospital, 1345 Govan Road, Glasgow G51 4TF (United Kingdom); Dalby, Matthew J; Berry, Catherine C; McNamara, Laura E, E-mail: alibrydone@gmail.com [Centre for Cell Engineering, Joseph Black Building, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ (United Kingdom)

2011-06-15

Extracellular matrix (ECM) remodelling is an essential physiological process in which matrix-metalloproteinases (MMPs) have a key role. Manipulating the manner in which cells produce MMPs and ECMs may enable the creation of a desired tissue type, i.e. effect repair, or the prevention of tissue invasion (e.g. metastasis). The aim of this project was to determine if culturing fibroblasts on grooved topography altered collagen deposition or MMP production. Human fibroblasts were seeded on planar or grooved polycaprolactone substrates (grooves were 12.5 {mu}m wide with varying depths of 240 nm, 540 nm or 2300 nm). Cell behaviour and collagen production were studied using fluorescence microscopy and the spent culture medium was assessed using gel zymography to detect MMPs. Total collagen deposition was high on the 240 nm deep grooves, but decreased as the groove depth increased, i.e. as cell contact guidance decreased. There was an increase in gelatinase on the 2300 nm deep grooved topography and there was a difference in the temporal expression of MMP-3 observed on the planar surface compared to the 540 nm and 2300 nm topographies. These results show that topography can alter collagen and MMP production. A fuller understanding of these processes may permit the design of surfaces tailored to tissue regeneration e.g. tendon repair.

Grooved surface topography alters matrix-metalloproteinase production by human fibroblasts

International Nuclear Information System (INIS)

Brydone, Alistair S; Dominic Meek, R M; Dalby, Matthew J; Berry, Catherine C; McNamara, Laura E

2011-01-01

Extracellular matrix (ECM) remodelling is an essential physiological process in which matrix-metalloproteinases (MMPs) have a key role. Manipulating the manner in which cells produce MMPs and ECMs may enable the creation of a desired tissue type, i.e. effect repair, or the prevention of tissue invasion (e.g. metastasis). The aim of this project was to determine if culturing fibroblasts on grooved topography altered collagen deposition or MMP production. Human fibroblasts were seeded on planar or grooved polycaprolactone substrates (grooves were 12.5 μm wide with varying depths of 240 nm, 540 nm or 2300 nm). Cell behaviour and collagen production were studied using fluorescence microscopy and the spent culture medium was assessed using gel zymography to detect MMPs. Total collagen deposition was high on the 240 nm deep grooves, but decreased as the groove depth increased, i.e. as cell contact guidance decreased. There was an increase in gelatinase on the 2300 nm deep grooved topography and there was a difference in the temporal expression of MMP-3 observed on the planar surface compared to the 540 nm and 2300 nm topographies. These results show that topography can alter collagen and MMP production. A fuller understanding of these processes may permit the design of surfaces tailored to tissue regeneration e.g. tendon repair.

Silk Film Topography Directs Collective Epithelial Cell Migration

Science.gov (United States)

Rosenblatt, Mark I.

2012-01-01

The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

Inhibition of matrix metalloproteinase-14 in osteosarcoma cells by clodronate

NARCIS (Netherlands)

Heikkilä, P.; Teronen, O.; Hirn, M.Y.; Sorsa, T.; Tervahartiala, T.; Salo, T.; Konttinen, Y.T.; Halttunen, T.; Moilanen, M.; Hanemaaijer, R.; Laitinen, M.

2003-01-01

Background. Bisphosphonates reduce the bone metastasis formation and angiogenesis but the exact molecular mechanisms involved are unclear. Progelatinase A (proMMP-2; 78 KDa) is activated up during the tumor spread and metastasis by a cell surface-associated matrix metalloproteinase (membrane-type

How does the extracellular matrix direct gene expression

Energy Technology Data Exchange (ETDEWEB)

Bissell, M J; Hall, H G; Parry, G

1982-01-01

Based on the existing literature, a model is presented that postulates a ''dynamic reciprocity'' between the extracellular matrix (ECM) on the one hand and the cytoskeleton and the nuclear matrix on the other hand. The ECM is postulated to exert physical and chemical influences on the geometry and the biochemistry of the cell via transmembrane receptors so as to alter the pattern of gene expression by changing the association of the cytoskeleton with mRNA and the interaction of the chromatin with the nuclear matrix. This, in turn, would affect the ECM, which would affect the cell.

Characterization of Polyethylene-Graft-Sulfonated Polyarylsulfone Proton Exchange Membranes for Direct Methanol Fuel Cell Applications

Directory of Open Access Journals (Sweden)

Hyung Kyu Kim

2015-12-01

Full Text Available This paper examines polymer film morphology and several important properties of polyethylene-graft-sulfonated polyarylene ether sulfone (PE-g-s-PAES proton exchange membranes (PEMs for direct methanol fuel cell applications. Due to the extreme surface energy differences between a semi-crystalline and hydrophobic PE backbone and several amorphous and hydrophilic s-PAES side chains, the PE-g-s-PAES membrane self-assembles into a unique morphology, with many proton conductive s-PAES channels embedded in the stable and tough PE matrix and a thin hydrophobic PE layer spontaneously formed on the membrane surfaces. In the bulk, these membranes show good mechanical properties (tensile strength >30 MPa, Young’s modulus >1400 MPa and low water swelling (λ < 15 even with high IEC >3 mmol/g in the s-PAES domains. On the surface, the thin hydrophobic and semi-crystalline PE layer shows some unusual barrier (protective properties. In addition to exhibiting higher through-plane conductivity (up to 160 mS/cm than in-plane conductivity, the PE surface layer minimizes methanol cross-over from anode to cathode with reduced fuel loss, and stops the HO• and HO2• radicals, originally formed at the anode, entering into PEM matrix. Evidently, the thin PE surface layer provides a highly desirable protecting layer for PEMs to reduce fuel loss and increase chemical stability. Overall, the newly developed PE-g-s-PAES membranes offer a desirable set of PEM properties, including conductivity, selectivity, mechanical strength, stability, and cost-effectiveness for direct methanol fuel cell applications.

Integrin-mediated adhesion of human mesenchymal stem cells to extracellular matrix proteins adsorbed to polymer surfaces

International Nuclear Information System (INIS)

Dånmark, S; Mustafa, K; Finne-Wistrand, A; Albertsson, A-C; Patarroyo, M

2012-01-01

In vitro, degradable aliphatic polyesters are widely used as cell carriers for bone tissue engineering, despite their lack of biological cues. Their biological active surface is rather determined by an adsorbed layer of proteins from the surrounding media. Initial cell fate, including adhesion and proliferation, which are key properties for efficient cell carriers, is determined by the adsorbed layer of proteins. Herein we have investigated the ability of human bone marrow derived stem cells (hBMSC) to adhere to extracellular matrix (ECM) proteins, including fibronectin and vitronectin which are present in plasma and serum. hBMSC expressed integrins for collagens, laminins, fibronectin and vitronectin. Accordingly, hBMSC strongly adhered to these purified ECM proteins by using the corresponding integrins. Although purified fibronectin and vitronectin adsorbed to aliphatic polyesters to a lower extent than to cell culture polystyrene, these low levels were sufficient to mediate adhesion of hBMSC. It was found that plasma- and serum-coated polystyrene adsorbed significant levels of both fibronectin and vitronectin, and fibronectin was identified as the major adhesive component of plasma for hBMSC; however, aliphatic polyesters adsorbed minimal levels of fibronectin under similar conditions resulting in impaired cell adhesion. Altogether, the results suggest that the efficiency of aliphatic polyesters cell carriers could be improved by increasing their ability to adsorb fibronectin. (paper)

Synergetic effect of yeast cell-surface expression of cellulase and expansin-like protein on direct ethanol production from cellulose

Science.gov (United States)

2013-01-01

Background Numerous studies have examined the direct fermentation of cellulosic materials by cellulase-expressing yeast; however, ethanol productivity in these systems has not yet reached an industrial level. Certain microorganisms, such as the cellulolytic fungus Trichoderma reesei, produce expansin-like proteins, which have a cellulose-loosening effect that may increase the breakdown of cellulose. Here, to improve the direct conversion of cellulose to ethanol, yeast Saccharomyces cerevisiae co-displaying cellulase and expansin-like protein on the cell surface were constructed and examined for direct ethanol fermentation performance. Results The cellulase and expansin-like protein co-expressing strain showed 246 mU/g-wet cell of phosphoric acid swollen cellulose (PASC) degradation activity, which corresponded to 2.9-fold higher activity than that of a cellulase-expressing strain. This result clearly demonstrated that yeast cell-surface expressed cellulase and expansin-like protein act synergistically to breakdown cellulose. In fermentation experiments examining direct ethanol production from PASC, the cellulase and expansin-like protein co-expressing strain produced 3.4 g/L ethanol after 96 h of fermentation, a concentration that was 1.4-fold higher than that achieved by the cellulase-expressing strain (2.5 g/L). Conclusions The PASC degradation and fermentation ability of an engineered yeast strain was markedly improved by co-expressing cellulase and expansin-like protein on the cell surface. To our knowledge, this is the first report to demonstrate the synergetic effect of co-expressing cellulase and expansin-like protein on a yeast cell surface, which may be a promising strategy for constructing direct ethanol fermenting yeast from cellulose. PMID:23835302

Oriented collagen fibers direct tumor cell intravasation

KAUST Repository

Han, Weijing

2016-09-24

In this work, we constructed a Collagen I-Matrigel composite extracellular matrix (ECM). The composite ECM was used to determine the influence of the local collagen fiber orientation on the collective intravasation ability of tumor cells. We found that the local fiber alignment enhanced cell-ECM interactions. Specifically, metastatic MDA-MB-231 breast cancer cells followed the local fiber alignment direction during the intravasation into rigid Matrigel (∼10 mg/mL protein concentration).

Hypoxic stellate cells of pancreatic cancer stroma regulate extracellular matrix fiber organization and cancer cell motility.

Science.gov (United States)

Sada, Masafumi; Ohuchida, Kenoki; Horioka, Kohei; Okumura, Takashi; Moriyama, Taiki; Miyasaka, Yoshihiro; Ohtsuka, Takao; Mizumoto, Kazuhiro; Oda, Yoshinao; Nakamura, Masafumi

2016-03-28

Desmoplasia and hypoxia in pancreatic cancer mutually affect each other and create a tumor-supportive microenvironment. Here, we show that microenvironment remodeling by hypoxic pancreatic stellate cells (PSCs) promotes cancer cell motility through alteration of extracellular matrix (ECM) fiber architecture. Three-dimensional (3-D) matrices derived from PSCs under hypoxia exhibited highly organized parallel-patterned matrix fibers compared with 3-D matrices derived from PSCs under normoxia, and promoted cancer cell motility by inducing directional migration of cancer cells due to the parallel fiber architecture. Microarray analysis revealed that procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) in PSCs was the gene that potentially regulates ECM fiber architecture under hypoxia. Stromal PLOD2 expression in surgical specimens of pancreatic cancer was confirmed by immunohistochemistry. RNA interference-mediated knockdown of PLOD2 in PSCs blocked parallel fiber architecture of 3-D matrices, leading to decreased directional migration of cancer cells within the matrices. In conclusion, these findings indicate that hypoxia-induced PLOD2 expression in PSCs creates a permissive microenvironment for migration of cancer cells through architectural regulation of stromal ECM in pancreatic cancer. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Quantitative image analysis for investigating cell-matrix interactions

Science.gov (United States)

Burkel, Brian; Notbohm, Jacob

2017-07-01

The extracellular matrix provides both chemical and physical cues that control cellular processes such as migration, division, differentiation, and cancer progression. Cells can mechanically alter the matrix by applying forces that result in matrix displacements, which in turn may localize to form dense bands along which cells may migrate. To quantify the displacements, we use confocal microscopy and fluorescent labeling to acquire high-contrast images of the fibrous material. Using a technique for quantitative image analysis called digital volume correlation, we then compute the matrix displacements. Our experimental technology offers a means to quantify matrix mechanics and cell-matrix interactions. We are now using these experimental tools to modulate mechanical properties of the matrix to study cell contraction and migration.

Cells behaviors and genotoxicity on topological surface

International Nuclear Information System (INIS)

Yang, N.; Yang, M.K.; Bi, S.X.; Chen, L.; Zhu, Z.Y.; Gao, Y.T.; Du, Z.

2013-01-01

To investigate different cells behaviors and genotoxicity, which were driven by specific microenvironments, three patterned surfaces (pillars, wide grooves and narrow grooves) and one smooth surface were prepared by template-based technique. Vinculin is a membrane-cytoskeletal protein in focal adhesion plaques and associates with cell–cell and cell–matrix junctions, which can promote cell adhesion and spreading. The immunofluorescence staining of vinculin revealed that the narrow grooves patterned substrate was favorable for L929 cell adhesion. For cell multiplication, the narrow grooves surface was fitted for the proliferation of L929, L02 and MSC cells, the pillars surface was only in favor of L929 cells to proliferate during 7 days of cell cultivation. Cell genetic toxicity was evaluated by cellular micronuclei test (MNT). The results indicated that topological surfaces were more suitable for L929 cells to proliferate and maintain the stability of genome. On the contrary, the narrow grooves surface induced higher micronuclei ratio of L02 and MSC cells than other surfaces. With the comprehensive results of cell multiplication and MNT, it was concluded that the wide grooves surface was best fitted for L02 cells to proliferate and have less DNA damages, and the smooth surface was optimum for the research of MSC cells in vitro. - Highlights: • Different cells behaviors on microstructure surfaces were discussed in this paper. • The expression of cell protein of Vinculin was studied in this research. • Cellular micronuclei test was applied to evaluate cells' genotoxicity. • Cell genotoxicity was first studied in the research field of topological surfaces

Hydrophilic polyurethane matrix promotes chondrogenesis of mesenchymal stem cells.

Science.gov (United States)

Nalluri, Sandeep M; Krishnan, G Rajesh; Cheah, Calvin; Arzumand, Ayesha; Yuan, Yuan; Richardson, Caley A; Yang, Shuying; Sarkar, Debanjan

2015-09-01

Segmental polyurethanes exhibit biphasic morphology and can control cell fate by providing distinct matrix guided signals to increase the chondrogenic potential of mesenchymal stem cells (MSCs). Polyethylene glycol (PEG) based hydrophilic polyurethanes can deliver differential signals to MSCs through their matrix phases where hard segments are cell-interactive domains and PEG based soft segments are minimally interactive with cells. These coordinated communications can modulate cell-matrix interactions to control cell shape and size for chondrogenesis. Biphasic character and hydrophilicity of polyurethanes with gel like architecture provide a synthetic matrix conducive for chondrogenesis of MSCs, as evidenced by deposition of cartilage-associated extracellular matrix. Compared to monophasic hydrogels, presence of cell interactive domains in hydrophilic polyurethanes gels can balance cell-cell and cell-matrix interactions. These results demonstrate the correlation between lineage commitment and the changes in cell shape, cell-matrix interaction, and cell-cell adhesion during chondrogenic differentiation which is regulated by polyurethane phase morphology, and thus, represent hydrophilic polyurethanes as promising synthetic matrices for cartilage regeneration. Copyright © 2015 Elsevier B.V. All rights reserved.

Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments

Science.gov (United States)

Baker, Brendon M.; Trappmann, Britta; Wang, William Y.; Sakar, Mahmut S.; Kim, Iris L.; Shenoy, Vivek B.; Burdick, Jason A.; Chen, Christopher S.

2015-12-01

To investigate how cells sense stiffness in settings structurally similar to native extracellular matrices, we designed a synthetic fibrous material with tunable mechanics and user-defined architecture. In contrast to flat hydrogel surfaces, these fibrous materials recapitulated cell-matrix interactions observed with collagen matrices including stellate cell morphologies, cell-mediated realignment of fibres, and bulk contraction of the material. Increasing the stiffness of flat hydrogel surfaces induced mesenchymal stem cell spreading and proliferation; however, increasing fibre stiffness instead suppressed spreading and proliferation for certain network architectures. Lower fibre stiffness permitted active cellular forces to recruit nearby fibres, dynamically increasing ligand density at the cell surface and promoting the formation of focal adhesions and related signalling. These studies demonstrate a departure from the well-described relationship between material stiffness and spreading established with hydrogel surfaces, and introduce fibre recruitment as a previously undescribed mechanism by which cells probe and respond to mechanics in fibrillar matrices.

The emergence of extracellular matrix mechanics and cell traction forces as important regulators of cellular self-organization.

Science.gov (United States)

Checa, Sara; Rausch, Manuel K; Petersen, Ansgar; Kuhl, Ellen; Duda, Georg N

2015-01-01

Physical cues play a fundamental role in a wide range of biological processes, such as embryogenesis, wound healing, tumour invasion and connective tissue morphogenesis. Although it is well known that during these processes, cells continuously interact with the local extracellular matrix (ECM) through cell traction forces, the role of these mechanical interactions on large scale cellular and matrix organization remains largely unknown. In this study, we use a simple theoretical model to investigate cellular and matrix organization as a result of mechanical feedback signals between cells and the surrounding ECM. The model includes bi-directional coupling through cellular traction forces to deform the ECM and through matrix deformation to trigger cellular migration. In addition, we incorporate the mechanical contribution of matrix fibres and their reorganization by the cells. We show that a group of contractile cells will self-polarize at a large scale, even in homogeneous environments. In addition, our simulations mimic the experimentally observed alignment of cells in the direction of maximum stiffness and the building up of tension as a consequence of cell and fibre reorganization. Moreover, we demonstrate that cellular organization is tightly linked to the mechanical feedback loop between cells and matrix. Cells with a preference for stiff environments have a tendency to form chains, while cells with a tendency for soft environments tend to form clusters. The model presented here illustrates the potential of simple physical cues and their impact on cellular self-organization. It can be used in applications where cell-matrix interactions play a key role, such as in the design of tissue engineering scaffolds and to gain a basic understanding of pattern formation in organogenesis or tissue regeneration.

INERT-MATRIX FUEL: ACTINIDE ''BURNING'' AND DIRECT DISPOSAL

International Nuclear Information System (INIS)

Rodney C. Ewing; Lumin Wang

2002-01-01

Excess actinides result from the dismantlement of nuclear weapons (Pu) and the reprocessing of commercial spent nuclear fuel (mainly 241 Am, 244 Cm and 237 Np). In Europe, Canada and Japan studies have determined much improved efficiencies for burnup of actinides using inert-matrix fuels. This innovative approach also considers the properties of the inert-matrix fuel as a nuclear waste form for direct disposal after one-cycle of burn-up. Direct disposal can considerably reduce cost, processing requirements, and radiation exposure to workers

Distinct roles for dystroglycan, beta1 integrin and perlecan in cell surface laminin organization

DEFF Research Database (Denmark)

Henry, M D; Satz, J S; Brakebusch, C

2001-01-01

Dystroglycan (DG) is a cell surface receptor for several extracellular matrix (ECM) molecules including laminins, agrin and perlecan. Recent data indicate that DG function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface...... integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that beta1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells......, the formation of complex laminin-1 structures is defective, implicating perlecan in the laminin matrix assembly process. Moreover, laminin and perlecan reciprocally modulate the organization of the other on the cell surface. Taken together, the data support a model whereby DG serves as a receptor essential...

Cell surface of sea urchin micromeres and primary mesenchyme

International Nuclear Information System (INIS)

DeSimone, D.W.

1985-01-01

The cell surface and extracellular matrix (ECM) of the sea urchin embryo were studied during the early morphogenetic events involved in the differentiation of the micromere cell lineage. Sixteen-cell and early cleavage stage blastomeres were isolated and the protein composition of their cell surfaces examined by 125 I-labelling followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Micromere-specific cell surface proteins are reported for Arbacia punctulata, Strongylocentrotus droebachiensis, and Strongylocentrotus purpuratus. Cell surface glycoproteins were characterized on the basis of lectin binding specificity with a novel lectin affinity transfer technique. Using this procedure, cell-type specific surface proteins, which are also lectin-binding specific, can be detected. In addition, fluorescein conjugated lectins were microinjected into the blastocoels of living S. drobachiensis and Lytechinus pictus embryos and the patterns of lectin bindings observed by fluorescence microscopy. The evidence presented in this thesis suggests that the differentiation of the primary mesenchyme cells is correlated with changes in the molecular composition of the cell-surface and the ECM

Modeling the formation of cell-matrix adhesions on a single 3D matrix fiber.

Science.gov (United States)

Escribano, J; Sánchez, M T; García-Aznar, J M

2015-11-07

Cell-matrix adhesions are crucial in different biological processes like tissue morphogenesis, cell motility, and extracellular matrix remodeling. These interactions that link cell cytoskeleton and matrix fibers are built through protein clutches, generally known as adhesion complexes. The adhesion formation process has been deeply studied in two-dimensional (2D) cases; however, the knowledge is limited for three-dimensional (3D) cases. In this work, we simulate different local extracellular matrix properties in order to unravel the fundamental mechanisms that regulate the formation of cell-matrix adhesions in 3D. We aim to study the mechanical interaction of these biological structures through a three dimensional discrete approach, reproducing the transmission pattern force between the cytoskeleton and a single extracellular matrix fiber. This numerical model provides a discrete analysis of the proteins involved including spatial distribution, interaction between them, and study of the different phenomena, such as protein clutches unbinding or protein unfolding. Copyright © 2015 Elsevier Ltd. All rights reserved.

Response of cells on surface-induced nanopatterns: fibroblasts and mesenchymal progenitor cells.

Science.gov (United States)

Khor, Hwei Ling; Kuan, Yujun; Kukula, Hildegard; Tamada, Kaoru; Knoll, Wolfgang; Moeller, Martin; Hutmacher, Dietmar W

2007-05-01

Ultrathin films of a poly(styrene)-block-poly(2-vinylpyrindine) diblock copolymer (PS-b-P2VP) and poly(styrene)-block-poly(4-vinylpyrindine) diblock copolymer (PS-b-P4VP) were used to form surface-induced nanopattern (SINPAT) on mica. Surface interaction controlled microphase separation led to the formation of chemically heterogeneous surface nanopatterns on dry ultrathin films. Two distinct nanopatterned surfaces, namely, wormlike and dotlike patterns, were used to investigate the influence of topography in the nanometer range on cell adhesion, proliferation, and migration. Atomic force microscopy was used to confirm that SINPAT was stable under cell culture conditions. Fibroblasts and mesenchymal progenitor cells were cultured on the nanopatterned surfaces. Phase contrast and confocal laser microscopy showed that fibroblasts and mesenchymal progenitor cells preferred the densely spaced wormlike patterns. Atomic force microscopy showed that the cells remodelled the extracellular matrix differently as they migrate over the two distinctly different nanopatterns.

The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix.

Science.gov (United States)

Williams, B Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

2012-07-01

Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells.

Direct comparison of phosphate uptake by adnate and loosely attached microalgae within an intact biofilm matrix

International Nuclear Information System (INIS)

Burkholder, J.M.; Wetzel, R.G.; Klomparens, K.L.

1990-01-01

We report a direct comparison of phosphate uptake by adnate and loosely attached microalgae in an intact biofilm matrix, with resolution at the level of individual cells. Track scanning electron microscope autoradiography enabled assay of [ 33 P]phosphate uptake from the overlying water by adnate algae left undisturbed on mature leaves of the macrophyte Potamogeton illinoensis or on artificial plant mimics. The epiphyte communities developed in either phosphate-poor or moderately phosphate-enriched water, and they were assayed on both natural and artificial plants. All adnate taxa examined from both natural and artificial plants in both habitats took up significantly less radiolabel when assayed beneath the overlying matrix than when they were exposed to the water upon removal of the overstory material. Track scanning electron microscope autoradiography and track light microscope autoradiography were intercalibrated to enable comparison of [ 33 P]phosphate uptake by adnate and loosely attached components of the epiphyte matrix. Loosley attached cells on substrata from both habitats took up significantly more radiolabel than did underlying adnate cells, indicating that access to phosphate supplies from the water depended on the position of microbial cells in the matrix. In this short-term assay, the adnate microalgae were relatively isolated from the water column nutrient source

The influence of matrix properties on growth and morphogenesis of human pancreatic ductal epithelial cells in 3D

Science.gov (United States)

Raza, Asad; Ki, Chang Seok; Lin, Chien-Chi

2013-01-01

A highly tunable synthetic biomimetic hydrogel platform was developed to study the growth and morphogenesis of pancreatic ductal epithelial cells (PDEC) under the influence of a myriad of instructive cues. A PDEC line, PANC-1, was used as a model system to illustrate the importance of matrix compositions on cell fate determination. PANC-1 is an immortalized ductal epithelial cell line widely used in the study of pancreatic tumor cell behaviors. PANC-1 cells are also increasingly explored as a potential cell source for endocrine differentiation. Thus far, most studies related to PANC-1, among other PDEC lines, are performed on 2D culture surfaces. Here, we evaluated the effect of matrix compositions on PANC-1 cell growth and morphogenesis in 3D. Specifically, PANC-1 cells were encapsulated in PEG-based hydrogels prepared by step-growth thiol-ene photopolymerization. It was found that thiol-ene hydrogels provided a cytocompatible environment for encapsulation and 3D culture of PANC-1 cells. In contrast to a monolayer morphology on 2D culture surfaces, PANC-1 cells formed clusters in 3D thiol-ene hydrogels within 4 days of culture. After culturing for 10 days, however, the growth and structures of these clusters were significantly impacted by gel matrix properties, including sensitivity of the matrix to proteases, stiffness of the matrix, and ECM-mimetic motifs. The use of matrix metalloproteinase (MMP) sensitive linker or the immobilization of fibronectin-derived RGDS ligand in the matrix promoted PANC-1 cell growth and encouraged them to adopt ductal cyst-like structures. On the other hand, the encapsulated cells formed smaller and more compact aggregates in non-MMP responsive gels. The incorporation of laminin-derived YIGSR peptide did not enhance cell growth and caused the cells to form compact aggregates. Immobilized YIGSR also enhanced the expression of epithelial cell markers including β-catenin and E-cadherin. These studies have established PEG

Compressive Elasticity of Three-Dimensional Nanofiber Matrix Directs Mesenchymal Stem Cell Differentiation to Vascular Cells with Endothelial or Smooth Muscle Cell Markers

OpenAIRE

Wingate, Kathryn; Bonani, Walter; Tan, Yan; Bryant, Stephanie J.; Tan, Wei

2012-01-01

The importance of mesenchymal stem cell (MSC) in vascular regeneration is becoming increasingly recognized. However, few in vitro studies have been performed to identify the effects of environmental elasticity on the differentiation of MSC into vascular cell types. We utilized electrospinning and photopolymerization techniques to fabricate a 3D PEGdma nanofiber hydrogel matrix with a tunable elasticity for use as a cellular substrate. Compression testing demonstrated that the elastic modulus ...

A molecular smart surface for spatio-temporal studies of cell mobility.

Science.gov (United States)

Lee, Eun-ju; Luo, Wei; Chan, Eugene W L; Yousaf, Muhammad N

2015-01-01

Active migration in both healthy and malignant cells requires the integration of information derived from soluble signaling molecules with positional information gained from interactions with the extracellular matrix and with other cells. How a cell responds and moves involves complex signaling cascades that guide the directional functions of the cytoskeleton as well as the synthesis and release of proteases that facilitate movement through tissues. The biochemical events of the signaling cascades occur in a spatially and temporally coordinated manner then dynamically shape the cytoskeleton in specific subcellular regions. Therefore, cell migration and invasion involve a precise but constantly changing subcellular nano-architecture. A multidisciplinary effort that combines new surface chemistry and cell biological tools is required to understand the reorganization of cytoskeleton triggered by complex signaling during migration. Here we generate a class of model substrates that modulate the dynamic environment for a variety of cell adhesion and migration experiments. In particular, we use these dynamic substrates to probe in real-time how the interplay between the population of cells, the initial pattern geometry, ligand density, ligand affinity and integrin composition affects cell migration and growth. Whole genome microarray analysis indicates that several classes of genes ranging from signal transduction to cytoskeletal reorganization are differentially regulated depending on the nature of the surface conditions.

In vitro behavior of human mesenchymal stem cells on poly(N-isopropylacrylamide) based biointerfaces obtained by matrix assisted pulsed laser evaporation

Science.gov (United States)

Icriverzi, Madalina; Rusen, Laurentiu; Sima, Livia Elena; Moldovan, Antoniu; Brajnicov, Simona; Bonciu, Anca; Mihailescu, Natalia; Dinescu, Maria; Cimpean, Anisoara; Roseanu, Anca; Dinca, Valentina

2018-05-01

The use of smart coatings with tunable characteristics in bioengineering fields is directly correlated with the surface chemical and topographical properties, the method of preparation, and also with the type of cells implied for the specific application. In this work, a versatile surface modification technique based on the use of lasers (Matrix-Assisted Pulsed Laser Evaporation (MAPLE)) was used to yield poly(N-isopropylacrylamide) (pNIPAM) and its derivatives (amine, azide and amide terminated pNIPAM) functional and termoresponsive thin films. Surface properties of pNIPAM and its derivative films such as morphology, roughness and hydrophobic/hydrophilic character, as well as the thermoresponsive capacity were investigated by atomic force microscopy and contact angle measurements. The chemical characteristics of the pNIPAM based thin films were analysed by Fourier Transform Infrared Spectroscopy (FTIR). The chemical functionality was kept for all the samples obtained by MAPLE and the thermoresponse was demonstrated by the change in the contact angle and thickness values when the temperature was shifted from 37 °C to 24 °C for all the materials tested, with a smaller change for maleimide terminated pNIPAM. Biological assays performed in vitro (fluorescence microscopy and Scanning Electron Microscopy (SEM)) confirmed the conditioning of the early mesenchymal stem cell (MSC) growth by specific chemistry of the coatings. The cell imaging analysis revealed no cytotoxic effect of pNIPAM surfaces irrespective of type of functionalization. An increased proliferation rate of the cells grown on pNIPAM-azide surfaces and a lower cell density on pNIPAM-maleimide surfaces compared to the pNIPAM surfaces was observed, which can direct their use to potential surfaces in regenerative medicine approaches.

Google matrix analysis of directed networks

Science.gov (United States)

Ermann, Leonardo; Frahm, Klaus M.; Shepelyansky, Dima L.

2015-10-01

In the past decade modern societies have developed enormous communication and social networks. Their classification and information retrieval processing has become a formidable task for the society. Because of the rapid growth of the World Wide Web, and social and communication networks, new mathematical methods have been invented to characterize the properties of these networks in a more detailed and precise way. Various search engines extensively use such methods. It is highly important to develop new tools to classify and rank a massive amount of network information in a way that is adapted to internal network structures and characteristics. This review describes the Google matrix analysis of directed complex networks demonstrating its efficiency using various examples including the World Wide Web, Wikipedia, software architectures, world trade, social and citation networks, brain neural networks, DNA sequences, and Ulam networks. The analytical and numerical matrix methods used in this analysis originate from the fields of Markov chains, quantum chaos, and random matrix theory.

Teaching the extracellular matrix and introducing online databases within a multidisciplinary course with i-cell-MATRIX: A student-centered approach.

Science.gov (United States)

Sousa, João Carlos; Costa, Manuel João; Palha, Joana Almeida

2010-03-01

The biochemistry and molecular biology of the extracellular matrix (ECM) is difficult to convey to students in a classroom setting in ways that capture their interest. The understanding of the matrix's roles in physiological and pathological conditions study will presumably be hampered by insufficient knowledge of its molecular structure. Internet-available resources can bridge the division between the molecular details and ECM's biological properties and associated processes. This article presents an approach to teach the ECM developed for first year medical undergraduates who, working in teams: (i) Explore a specific molecular component of the matrix, (ii) identify a disease in which the component is implicated, (iii) investigate how the component's structure/function contributes to ECM' supramolecular organization in physiological and in pathological conditions, and (iv) share their findings with colleagues. The approach-designated i-cell-MATRIX-is focused on the contribution of individual components to the overall organization and biological functions of the ECM. i-cell-MATRIX is student centered and uses 5 hours of class time. Summary of results and take home message: A "1-minute paper" has been used to gather student feedback on the impact of i-cell-MATRIX. Qualitative analysis of student feedback gathered in three consecutive years revealed that students appreciate the approach's reliance on self-directed learning, the interactivity embedded and the demand for deeper insights on the ECM. Learning how to use internet biomedical resources is another positive outcome. Ninety percent of students recommend the activity for subsequent years. i-cell-MATRIX is adaptable by other medical schools which may be looking for an approach that achieves higher student engagement with the ECM. Copyright © 2010 International Union of Biochemistry and Molecular Biology, Inc.

Cell differentiation and matrix organization in engineered teeth.

Science.gov (United States)

Nait Lechguer, A; Couble, M L; Labert, N; Kuchler-Bopp, S; Keller, L; Magloire, H; Bleicher, F; Lesot, H

2011-05-01

Embryonic dental cells were used to check a series of criteria to be achieved for tooth engineering. Implantation of cultured cell-cell re-associations led to crown morphogenesis, epithelial histogenesis, organ vascularization, and root and periodontium development. The present work aimed to investigate the organization of predentin/dentin, enamel, and cementum which formed and mineralized after implantation. These implants were processed for histology, transmission electron microscopy, x-ray microanalysis, and electron diffraction. After two weeks of implantation, the re-associations showed gradients of differentiating odontoblasts. There were ciliated, polarized, and extended cell processes in predentin/dentin. Ameloblasts became functional. Enamel crystals showed a typical oriented arrangement in the inner and outer enamel. In the developing root, odontoblasts differentiated, cementogenesis occurred, and periodontal ligament fibroblasts interacted with the root surface and newly formed bone. The implantation of cultured dental cell re-associations allows for reproduction of complete functional differentiation at the cell, matrix, and mineral levels.

Basigin-2 Is a Cell Surface Receptor for Soluble Basigin Ligand*S⃞

Science.gov (United States)

Belton, Robert J.; Chen, Li; Mesquita, Fernando S.; Nowak, Romana A.

2008-01-01

The metastatic spread of a tumor is dependent upon the ability of the tumor to stimulate surrounding stromal cells to express enzymes required for tissue remodeling. The immunoglobulin superfamily protein basigin (EMMPRIN/CD147) is a cell surface glycoprotein expressed by tumor cells that stimulates matrix metalloproteinase and vascular endothelial growth factor expression in stromal cells. The ability of basigin to stimulate expression of molecules involved in tissue remodeling and angiogenesis makes basigin a potential target for the development of strategies to block metastasis. However, the identity of the cell surface receptor for basigin remains controversial. The goal of this study was to determine the identity of the receptor for basigin. Using a novel recombinant basigin protein (rBSG) corresponding to the extracellular domain of basigin, it was demonstrated that the native, nonglycosylated rBSG protein forms dimers in solution. Furthermore, rBSG binds to the surface of uterine fibroblasts, activates the ERK1/2 signaling pathway, and induces expression of matrix metalloproteinases 1, 2, and 3. Proteins that interact with rBSG were isolated using a biotin label transfer technique and sequenced by matrix-assisted laser desorption ionization tandem mass spectrophotometry. The results demonstrate that rBSG interacts with basigin expressed on the surface of fibroblasts and is subsequently internalized. During internalization, rBSG associates with a novel form of human basigin (basigin-3). It was concluded that cell surface basigin functions as a membrane receptor for soluble basigin and this homophilic interaction is not dependent upon glycosylation of the basigin ligand. PMID:18434307

Decellularized matrix from tumorigenic human mesenchymal stem cells promotes neovascularization with galectin-1 dependent endothelial interaction.

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Jorge S Burns

Full Text Available BACKGROUND: Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC strain (hMSC-TERT20 immortalized by retroviral vector mediated human telomerase (hTERT gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+ and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. CONCLUSIONS: Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1

Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction

Science.gov (United States)

Burns, Jorge S.; Kristiansen, Malthe; Kristensen, Lars P.; Larsen, Kenneth H.; Nielsen, Maria O.; Christiansen, Helle; Nehlin, Jan; Andersen, Jens S.; Kassem, Moustapha

2011-01-01

Background Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. Methodology/Principal Findings Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. Conclusions Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was

Membrane-type-3 matrix metalloproteinase (MT3-MMP functions as a matrix composition-dependent effector of melanoma cell invasion.

Directory of Open Access Journals (Sweden)

Olga Tatti

Full Text Available In primary human melanoma, the membrane-type matrix metalloproteinase, MT3-MMP, is overexpressed in the most aggressive nodular-type tumors. Unlike MT1-MMP and MT2-MMP, which promote cell invasion through basement membranes and collagen type I-rich tissues, the function of MT3-MMP in tumor progression remains unclear. Here, we demonstrate that MT3-MMP inhibits MT1-MMP-driven melanoma cell invasion in three-dimensional collagen, while yielding an altered, yet MT1-MMP-dependent, form of expansive growth behavior that phenocopies the formation of nodular cell colonies. In melanoma cell lines originating from advanced primary or metastatic lesions, endogenous MT3-MMP expression was associated with limited collagen-invasive potential. In the cell lines with highest MT3-MMP expression relative to MT1-MMP, collagen-invasive activity was increased following stable MT3-MMP gene silencing. Consistently, MT3-MMP overexpression in cells derived from less advanced superficially spreading melanoma lesions, or in the MT3-MMP knockdown cells, reduced MT1-MMP-dependent collagen invasion. Rather than altering MT1-MMP transcription, MT3-MMP interacted with MT1-MMP in membrane complexes and reduced its cell surface expression. By contrast, as a potent fibrinolytic enzyme, MT3-MMP induced efficient invasion of the cells in fibrin, a provisional matrix component frequently found at tumor-host tissue interfaces and perivascular spaces of melanoma. Since MT3-MMP was significantly upregulated in biopsies of human melanoma metastases, these results identify MT3-MMP as a matrix-dependent modifier of the invasive tumor cell functions during melanoma progression.

Cell adhesion over two distinct surfaces varied with chemical and mechanical properties

International Nuclear Information System (INIS)

Huang, Chih-Ling; Liao, Jiunn-Der; Yang, Chia-Fen; Chang, Chia-Wei; Ju, Ming-Shaung; Lin, Chou-Ching K.

2009-01-01

Chitosan is widely recognized as a natural and proper scaffold material; however, as a base substrate, it shows little promotion effect for the growth of cultured fibroblast cells. In this study, chitosan in a film form was prepared and used as a cell-culturing matrix, followed by patterning the evaporated Au upon it. Micro-scale Au clusters of ∼ 150 μm in diameter and ∼ 20 nm in thickness were then patterned and adhered upon the chitosan matrix. Physical and chemical properties of Au/chitosan were characterized. In particular, nano-indentation with dynamic contact module was applied to measure the nano-hardness of the tailored surfaces on Au/chitosan. Fibroblast cells were thereafter cultured on Au/chitosan. Experimental results demonstrated that as compared with the chitosan matrix, Au clusters and their boundary area exhibited favorable to promote cell adhesion, spreading, and growth. As well, nano-hardness on the boundary area of Au/chitosan significantly enhanced, while the cultured fibroblast cells aggregated upon Au clusters and the boundary area. In combination with the possible chemical and mechanical changes resulted by the evaporation of Au clusters upon the chitosan matrix, a selectively-enhanced Au/chitosan to promote fibroblast cells proliferation was created. Such design is anticipated for enabling a surface for scaffold materials with the cell-guidable function.

Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties

DEFF Research Database (Denmark)

Couchman, J R; Austria, R; Woods, A

1988-01-01

In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin......-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased...... sulfation, reduced affinity for fibronectin and decreased half-life on the cell surface when compared to the normal counterpart. Our conclusions based on this data are that these altered properties may, in part, account for the adhesion defect in the ricin-resistant mutant. Whether this results from...

A Nafion-Ceria Composite Membrane Electrolyte for Reduced Methanol Crossover in Direct Methanol Fuel Cells

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Parthiban Velayutham

2017-02-01

Full Text Available An alternative Nafion composite membrane was prepared by incorporating various loadings of CeO2 nanoparticles into the Nafion matrix and evaluated its potential application in direct methanol fuel cells (DMFCs. The effects of CeO2 in the Nafion matrix were systematically studied in terms of surface morphology, thermal and mechanical stability, proton conductivity and methanol permeability. The composite membrane with optimum filler content (1 wt. % CeO2 exhibits a proton conductivity of 176 mS·cm−1 at 70 °C, which is about 30% higher than that of the unmodified membrane. Moreover, all the composite membranes possess a much lower methanol crossover compared to pristine Nafion membrane. In a single cell DMFC test, MEA fabricated with the optimized composite membrane delivered a peak power density of 120 mW·cm−2 at 70 °C, which is about two times higher in comparison with the pristine Nafion membrane under identical operating conditions.

Direct determination of scattering time delays using the R-matrix propagation method

International Nuclear Information System (INIS)

Walker, R.B.; Hayes, E.F.

1989-01-01

A direct method for determining time delays for scattering processes is developed using the R-matrix propagation method. The procedure involves the simultaneous generation of the global R matrix and its energy derivative. The necessary expressions to obtain the energy derivative of the S matrix are relatively simple and involve many of the same matrix elements required for the R-matrix propagation method. This method is applied to a simple model for a chemical reaction that displays sharp resonance features. The test results of the direct method are shown to be in excellent agreement with the traditional numerical differentiation method for scattering energies near the resonance energy. However, for sharp resonances the numerical differentiation method requires calculation of the S-matrix elements at many closely spaced energies. Since the direct method presented here involves calculations at only a single energy, one is able to generate accurate energy derivatives and time delays much more efficiently and reliably

Regulation of cell cycle progression by cell-cell and cell-matrix forces

NARCIS (Netherlands)

Uroz, Marina; Wistorf, Sabrina; Serra-Picamal, Xavier; Conte, Vito; Sales-Pardo, Marta; Roca-Cusachs, Pere; Guimerà, Roger; Trepat, Xavier

2018-01-01

It has long been proposed that the cell cycle is regulated by physical forces at the cell-cell and cell-extracellular matrix (ECM) interfaces 1-12 . However, the evolution of these forces during the cycle has never been measured in a tissue, and whether this evolution affects cell cycle progression

3D cancer cell migration in a confined matrix

Science.gov (United States)

Alobaidi, Amani; Sun, Bo

Cancer cell migration is widely studied in 2D motion, which does not mimic the invasion processes in vivo. More recently, 3D cell migration studies have been performed. The ability of cancer cells to migrate within the extracellular matrix depends on the physical and biochemical features of the extracellular matrix. We present a model of cell motility in confined matrix geometry. The aim of the study is to study cancer migration in collagen matrix, as a soft tissue, to investigate their motility within the confined and surrounding collagen environment. Different collagen concentrations have been used to show the ability of these cancer cells to move through such a complex structure by measuring Cancer cell migration velocity as well as the displacement. Graduate student physics department.

Three-dimensional matrix fiber alignment modulates cell migration and MT1-MMP utility by spatially and temporally directing protrusions

Science.gov (United States)

Fraley, Stephanie I.; Wu, Pei-Hsun; He, Lijuan; Feng, Yunfeng; Krisnamurthy, Ranjini; Longmore, Gregory D.; Wirtz, Denis

2015-10-01

Multiple attributes of the three-dimensional (3D) extracellular matrix (ECM) have been independently implicated as regulators of cell motility, including pore size, crosslink density, structural organization, and stiffness. However, these parameters cannot be independently varied within a complex 3D ECM protein network. We present an integrated, quantitative study of these parameters across a broad range of complex matrix configurations using self-assembling 3D collagen and show how each parameter relates to the others and to cell motility. Increasing collagen density resulted in a decrease and then an increase in both pore size and fiber alignment, which both correlated significantly with cell motility but not bulk matrix stiffness within the range tested. However, using the crosslinking enzyme Transglutaminase II to alter microstructure independently of density revealed that motility is most significantly predicted by fiber alignment. Cellular protrusion rate, protrusion orientation, speed of migration, and invasion distance showed coupled biphasic responses to increasing collagen density not predicted by 2D models or by stiffness, but instead by fiber alignment. The requirement of matrix metalloproteinase (MMP) activity was also observed to depend on microstructure, and a threshold of MMP utility was identified. Our results suggest that fiber topography guides protrusions and thereby MMP activity and motility.

Surface-modified Y zeolite-filled chitosan membrane for direct methanol fuel cell

Energy Technology Data Exchange (ETDEWEB)

Wu, Hong; Zheng, Bin; Zheng, Xiaohong; Wang, Jingtao; Yuan, Weikang; Jiang, Zhongyi [Key Laboratory for Green Chemical Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China)

2007-11-15

Hybrid membranes composed of chitosan (CS) as organic matrix and surface-modified Y zeolite as inorganic filler are prepared and their applicability for DMFC is demonstrated by methanol permeability, proton conductivity and swelling property. Y zeolite is modified using silane coupling agents, 3-aminopropyl-triethoxysilane (APTES) and 3-mercaptopropyl-trimethoxysilane (MPTMS), to improve the organic-inorganic interfacial morphology. The mercapto group on MPTMS-modified Y zeolite is further oxidized into sulfonic group. Then, the resultant surface-modified Y zeolites with either aminopropyl groups or sulfonicpropyl groups are mixed with chitosan in acetic acid solution and cast into membranes. The transitional phase generated between chitosan matrix and zeolite filler reduces or even eliminates the nonselective voids commonly exist at the interface. The hybrid membranes exhibit a significant reduction in methanol permeability compared with pure chitosan and Nafion117 membranes, and this reduction extent becomes more pronounced with the increase of methanol concentration. By introducing -SO{sub 3}H groups onto zeolite surface, the conductivity of hybrid membranes is increased up to 2.58 x 10{sup -2} S cm{sup -1}. In terms of the overall selectivity index ({beta} = {sigma}/P), the hybrid membrane is comparable with Nafion117 at low methanol concentration (2 mol L{sup -1}) and much better (three times) at high methanol concentration (12 mol L{sup -1}). (author)

Single Cell Force Spectroscopy for Quantification of Cellular Adhesion on Surfaces

Science.gov (United States)

Christenson, Wayne B.

Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is alpha Mbeta2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering

Atypical protein kinase C activity is required for extracellular matrix degradation and invasion by Src-transformed cells.

Science.gov (United States)

Rodriguez, Elena M; Dunham, Elizabeth E; Martin, G Steven

2009-10-01

Atypical protein kinase C (aPKC) isoforms have been shown to mediate Src-dependent signaling in response to growth factor stimulation. To determine if aPKC activity contributes to the transformed phenotype of cells expressing oncogenic Src, we have examined the activity and function of aPKCs in 3T3 cells expressing viral Src (v-Src). aPKC activity and tyrosine phosphorylation were found to be elevated in some but not all clones of mouse fibroblasts expressing v-Src. aPKC activity was inhibited either by addition of a membrane-permeable pseudosubstrate, by expression of a dominant-negative aPKC, or by RNAi-mediated knockdown of specific aPKC isoforms. aPKC activity contributes to morphological transformation and stress fiber disruption, and is required for migration of Src-transformed cells and for their ability to polarize at the edge of a monolayer. The lambda isoform of aPKC is specifically required for invasion through extracellular matrix in Boyden chamber assays and for degradation of the extracellular matrix in in situ zymography assays. Tyrosine phosphorylation of aPKClambda is required for its ability to promote cell invasion. The defect in invasion upon aPKC inhibition appears to result from a defect in the assembly and/or function of podosomes, invasive adhesions on the ventral surface of the cell that are sites of protease secretion. aPKC was also found to localize to podosomes of v-Src transformed cells, suggesting a direct role for aPKC in podosome assembly and/or function. We conclude that basal or elevated aPKC activity is required for the ability of Src-transformed cells to degrade and invade the extracellular matrix. Copyright 2009 Wiley-Liss, Inc.

Cell-surface proteoglycan in sea urchin primary mesenchyme cell migration

International Nuclear Information System (INIS)

Lane, M.C.

1989-01-01

Early in the development of the sea urchin embryo, the primary mesenchyme cells (PMC) migrate along the basal lamina of the blastocoel. Migration is inhibited in L. pictus embryos cultured in sulfate-free seawater and in S. purpuratus embryos exposed to exogenous β-D-xylosides. An in vitro assay was developed to test the migratory capacity of normal PMC on normal and treated blastocoelic matrix. Sulfate deprivation and exposure to exogenous xyloside render PMC nonmotile on either matrix. Materials removed from the surface of normal PMC by treatment with 1 M urea restored migratory ability to defective cells, whereas a similar preparation isolated from the surface of epithelial cells at the same stage did not. Migration also resumed when cells were removed from the xyloside or returned to normal seawater. The urea extract was partially purified and characterized by radiolabeling, gel electrophoresis, fluorography, ion exchange chromatography, and western blotting. The PMC synthesize a large chondroitin sulfate/dermatan sulfate proteoglycan that is present in an active fraction isolated by chromatography. Chondroitinase ABC digestion of live cells blocked migration reversibly, further supporting the identification of the chondroitin sulfate/dermatan sulfate proteoglycan as the active component in the urea extract. Much of the incorporated sulfate was distributed along the filopodia in 35 SO 4 -labelled PMC by autoradiography. The morphology of normal and treated S. purpuratus PMC was examined by scanning electron microscopy, and differences in spreading, particularly of the extensive filopodia present on the cells, was observed. A model for the role of the chondroitin sulfate/dermatan sulfate proteoglycan in cell detachment during migration is proposed

A Novel Silicon-based Wideband RF Nano Switch Matrix Cell and the Fabrication of RF Nano Switch Structures

Directory of Open Access Journals (Sweden)

Yi Xiu YANG

2011-12-01

Full Text Available This paper presents the concept of RF nano switch matrix cell and the fabrication of RF nano switch. The nano switch matrix cell can be implemented into complex switch matrix for signal routing. RF nano switch is the decision unit for the matrix cell; in this research, it is fabricated on a tri-layer high-resistivity-silicon substrate using surface micromachining approach. Electron beam lithography is introduced to define the pattern and IC compatible deposition process is used to construct the metal layers. Silicon-based nano switch fabricated by IC compatible process can lead to a high potential of system integration to perform a cost effective system-on-a-chip solution. In this paper, simulation results of the designed matrix cell are presented; followed by the details of the nano structure fabrication and fabrication challenges optimizations; finally, measurements of the fabricated nano structure along with analytical discussions are also discussed.

Molecular polymorphism of a cell surface proteoglycan: distinct structures on simple and stratified epithelia.

Science.gov (United States)

Sanderson, R D; Bernfield, M

1988-12-01

Epithelial cells are organized into either a single layer (simple epithelia) or multiple layers (stratified epithelia). Maintenance of these cellular organizations requires distinct adhesive mechanisms involving many cell surface molecules. One such molecule is a cell surface proteoglycan, named syndecan, that contains both heparan sulfate and chondroitin sulfate chains. This proteoglycan binds cells to fibrillar collagens and fibronectin and thus acts as a receptor for interstitial matrix. The proteoglycan is restricted to the basolateral surface of simple epithelial cells, but is located over the entire surface of stratified epithelial cells, even those surfaces not contacting matrix. We now show that the distinct localization in simple and stratified epithelia correlates with a distinct proteoglycan structure. The proteoglycan from simple epithelia (modal molecular size, 160 kDa) is larger than that from stratified epithelia (modal molecular size, 92 kDa), but their core proteins are identical in size and immunoreactivity. The proteoglycan from simple epithelia has more and larger heparan sulfate and chondroitin sulfate chains than the proteoglycan from stratified epithelia. Thus, the cell surface proteoglycan shows a tissue-specific structural polymorphism due to distinct posttranslational modifications. This polymorphism likely reflects distinct proteoglycan functions in simple and stratified epithelia, potentially meeting the different adhesive requirements of the cells in these different organizations.

Surface morphology of thin lysozyme films produced by matrix-assisted pulsed laser evaporation (MAPLE)

DEFF Research Database (Denmark)

Purice, Andreea; Schou, Jørgen; Pryds, Nini

2007-01-01

Thin films of the protein, lysozyme, have been deposited by the matrix-assisted pulsed laser evaporation (MAPLE) technique. Frozen targets of 0.3-1.0 wt.% lysozyme dissolved in ultrapure water were irradiated by laser light at 355 mn with a fluence of 2 J/cm(2). The surface quality of the thin....... The concentration of lysozyme in the ice matrix apparently does not play any significant role for the morphology of the film. The morphology obtained with MAPLE has been compared with results for direct laser irradiation of a pressed lysozyme sample (i.e. pulsed laser deposition (PLD)). (C) 2007 Elsevier B.V. All...

Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation

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Srsen Vlastimil

2009-04-01

Full Text Available Abstract Background Muscle fibres are formed by elongation and fusion of myoblasts into myotubes. During this differentiation process, the cytoskeleton is reorganized, and proteins of the centrosome re-localize to the surface of the nucleus. The exact timing of this event, and the underlying molecular mechanisms are still poorly understood. Results We performed studies on mouse myoblast cell lines that were induced to differentiate in culture, to characterize the early events of centrosome protein re-localization. We demonstrate that this re-localization occurs already at the single cell stage, prior to fusion into myotubes. Centrosome proteins that accumulate at the nuclear surface form an insoluble matrix that can be reversibly disassembled if isolated nuclei are exposed to mitotic cytoplasm from Xenopus egg extract. Our microscopy data suggest that this perinuclear matrix of centrosome proteins consists of a system of interconnected fibrils. Conclusion Our data provide new insights into the reorganization of centrosome proteins during muscular differentiation, at the structural and biochemical level. Because we observe that centrosome protein re-localization occurs early during differentiation, we believe that it is of functional importance for the reorganization of the cytoskeleton in the differentiation process.

Molten carbonate fuel cell integral matrix tape and bubble barrier

International Nuclear Information System (INIS)

Reiser, C.A.; Maricle, D.L.

1983-01-01

A molten carbonate fuel cell matrix material is described made up of a matrix tape portion and a bubble barrier portion. The matrix tape portion comprises particles inert to molten carbonate electrolyte, ceramic particles and a polymeric binder, the matrix tape being flexible, pliable and having rubber-like compliance at room temperature. The bubble barrier is a solid material having fine porosity preferably being bonded to the matrix tape. In operation in a fuel cell, the polymer binder burns off leaving the matrix and bubble barrier providing superior sealing, stability and performance properties to the fuel cell stack

A role for topographic cues in the organization of collagenous matrix by corneal fibroblasts and stem cells.

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Dimitrios Karamichos

Full Text Available Human corneal fibroblasts (HCF and corneal stromal stem cells (CSSC each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7. Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200-300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes.

Porous matrix structures for alkaline electrolyte fuel cells

Science.gov (United States)

Vine, R. W.; Narsavage, S. T.

1975-01-01

A number of advancements have been realized by a continuing research program to develop higher chemically stable porous matrix structures with high bubble pressure (crossover resistance) for use as separators in potassium hydroxide electrolyte fuel cells. More uniform, higher-bubble-pressure asbestos matrices were produced by reconstituting Johns-Manville asbestos paper; Fybex potassium titanate which was found compatible with 42% KOH at 250 F for up to 3000 hr; good agreement was found between bubble pressures predicted by an analytical study and those measured with filtered structures; Teflon-bonded Fybex matrices with bubble pressures greater than 30 psi were obtained by filtering a water slurry of the mixture directly onto fuel cell electrodes; and PBI fibers have satisfactory compatibility with 42% KOH at 250 F.

Alterations in the extracellular matrix organization associated with the reexpression of tumorigenicity in human cell hybrids.

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Der, C J; Stanbridge, E J

1980-10-15

The expression of fibronectin on the cell surface was evaluated on a series of intraspecific human cell hybrids formed between HeLa and normal fibroblast strains. Although these hybrids continued to express many of the in vitro transformation properties of their corresponding tumorigenic HeLa parent, they were now unable to form tumors when inoculated into athymic nude mice. From these suppressed hybrid populations, rare tumorigenic segregant subpopulations arose which had regained their tumorigenic capacity. A comparison of the expression of fibronectin on the cell surface was made between these tumorigenic segregant cell lines and their corresponding non-tumorigenic HeLa/fibroblast hybrid. Following specific immunofluorescent staining for fibronectin, a striking alteration in the cell surface organization was observed to correspond with the reexpression of tumorigenicity in these hybrids. Tumorigenic HeLa/fibroblast hybrids were also significantly altered in both their cellular and colonial morphology. Double immunofluorescent staining to simultaneously visualize both surface fibronectin and collagen revealed that these two extracellular matrix proteins displayed an extensive degree of codistribution and expressed a coordinate shift in organization which correlated with the appearance of tumorigenic segregant hybrid populations. These observations are in agreement with the apparently close structural association between fibronectin and collagen and suggest that the organization of these two components in the extracellular matrix may be an important determinant for in vivo growth potential.

Efficiency of a concentric matrix track detector surface scanning

International Nuclear Information System (INIS)

Bek-Uzarov, Dj.; Nikezic, D.; Kostic, D.; Krstic, D.; Cuknic, O.

1995-01-01

Heavy particle ionizing radiation track counting on the surface of a solid state round surface detector is made using the microscope and scanning step by step by a round field of vision. The whole solid state detector surface could not be fully or completely covered by round fields of visions. Therefore detector surface could be divided on the two parts, the larger surface, being under fields of vision, really scanned and no scanned missed or omitted surface. The ratio between omitted and scanned surfaces is so called track scanning efficiency. The knowledge of really counted, or scanned surface is a important value for evaluating the real surface track density an exposed solid state track detector. In the paper a matrix of a concentric field of vision made around the first microscope field of vision placed in center of the round disc of the scanned track detector is proposed. In a such scanning matrix the real scanned surface could be easy calculated and by the microscope scanning made as well. By this way scanned surface is very precisely obtained as well. Precise knowledge of scanned and omitted surface allows to obtain more precise scanning efficiency factor as well as real surface track density, the main parameter in solid state track detection measurements. (author)

Surface deformation during an action potential in pearled cells

Science.gov (United States)

Mussel, Matan; Fillafer, Christian; Ben-Porath, Gal; Schneider, Matthias F.

2017-11-01

Electric pulses in biological cells (action potentials) have been reported to be accompanied by a propagating cell-surface deformation with a nanoscale amplitude. Typically, this cell surface is covered by external layers of polymer material (extracellular matrix, cell wall material, etc.). It was recently demonstrated in excitable plant cells (Chara braunii) that the rigid external layer (cell wall) hinders the underlying deformation. When the cell membrane was separated from the cell wall by osmosis, a mechanical deformation, in the micrometer range, was observed upon excitation of the cell. The underlying mechanism of this mechanical pulse has, to date, remained elusive. Herein we report that Chara cells can undergo a pearling instability, and when the pearled fragments were excited even larger and more regular cell shape changes were observed (˜10 -100 μ m in amplitude). These transient cellular deformations were captured by a curvature model that is based on three parameters: surface tension, bending rigidity, and pressure difference across the surface. In this paper these parameters are extracted by curve-fitting to the experimental cellular shapes at rest and during excitation. This is a necessary step to identify the mechanical parameters that change during an action potential.

Diffusion of MMPs on the surface of collagen fibrils: the mobile cell surface-collagen substratum interface.

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Ivan E Collier

Full Text Available Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP(2/TIMP-2/MMP-2 can initiate (MT1-MMP and complete (MMP-2 degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP(2/TIMP-2/MMP-2 represents a Mobile Cell Surface-Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions.

Macrophage involvement affects matrix stiffness-related influences on cell osteogenesis under three-dimensional culture conditions.

Science.gov (United States)

He, Xiao-Tao; Wu, Rui-Xin; Xu, Xin-Yue; Wang, Jia; Yin, Yuan; Chen, Fa-Ming

2018-04-15

of a scaffold plays critical roles in modulating both reparative cells, such as mesenchymal stem cells (MSCs), and immune cells, such as macrophages (Mφs). Although the influences of material stiffness on either Mφs or MSCs, have been extensively described, how the two cell types respond to matrix cues to dynamically affect each other in a three-dimensional (3D) biosystem remains largely unknown. Here, we report our findings that, in a platform wherein Mφs and bone marrow-derived MSCs coexist, matrix stiffness can influence stem cell fate through both direct matrix-associated regulation and indirect Mφ-based modulation. Our data support future studies of the MSC-Mφ-matrix interplay in the 3D context to optimize matrix parameters for the development of the next biomaterial. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Tantalum coating on TiO2 nanotubes induces superior rate of matrix mineralization and osteofunctionality in human osteoblasts

International Nuclear Information System (INIS)

Frandsen, Christine J.; Brammer, Karla S.; Noh, Kunbae; Johnston, Gary; Jin, Sungho

2014-01-01

Nanostructured surface geometries have been the focus of a multitude of recent biomaterial research, and exciting findings have been published. However, only a few publications have directly compared nanostructures of various surface chemistries. The work herein directly compares the response of human osteoblast cells to surfaces of identical nanotube geometries with two well-known orthopedic biomaterials: titanium oxide (TiO 2 ) and tantalum (Ta). The results reveal that the Ta surface chemistry on the nanotube architecture enhances alkaline phosphatase activity, and promotes a ∼ 30% faster rate of matrix mineralization and bone-nodule formation when compared to results on bare TiO 2 nanotubes. This study implies that unique combinations of surface chemistry and nanostructure may influence cell behavior due to distinctive physico-chemical properties. These findings are of paramount importance to the orthopedics field for understanding cell behavior in response to subtle alterations in nanostructure and surface chemistry, and will enable further insight into the complex manipulation of biomaterial surfaces. With increased focus in the field of orthopedic materials research on nanostructured surfaces, this study emphasizes the need for careful and systematic review of variations in surface chemistry in concurrence with nanotopographical changes. - Highlights: • A TiO 2 nanotube surface structure was coated with tantalum. • Osteoblast cell response was compared between the tantalum coated and as-formed TiO 2 nanotube surface. • We observed superior rates of bone matrix mineralization and osteoblast maturation on the tantalum coated nanotube surface

Surface acoustic wave actuated cell sorting (SAWACS).

Science.gov (United States)

Franke, T; Braunmüller, S; Schmid, L; Wixforth, A; Weitz, D A

2010-03-21

We describe a novel microfluidic cell sorter which operates in continuous flow at high sorting rates. The device is based on a surface acoustic wave cell-sorting scheme and combines many advantages of fluorescence activated cell sorting (FACS) and fluorescence activated droplet sorting (FADS) in microfluidic channels. It is fully integrated on a PDMS device, and allows fast electronic control of cell diversion. We direct cells by acoustic streaming excited by a surface acoustic wave which deflects the fluid independently of the contrast in material properties of deflected objects and the continuous phase; thus the device underlying principle works without additional enhancement of the sorting by prior labelling of the cells with responsive markers such as magnetic or polarizable beads. Single cells are sorted directly from bulk media at rates as fast as several kHz without prior encapsulation into liquid droplet compartments as in traditional FACS. We have successfully directed HaCaT cells (human keratinocytes), fibroblasts from mice and MV3 melanoma cells. The low shear forces of this sorting method ensure that cells survive after sorting.

Direct Torque Control of Matrix Converter Fed Induction Motor Drive

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JAGADEESAN Karpagam

2011-10-01

Full Text Available This paper presents the Direct TorqueControl (DTC of induction motor drive using matrixconverters. DTC is a high performance motor controlscheme with fast torque and flux responses. However,the main disadvantage of conventional DTC iselectromagnetic torque ripple. In this paper, directtorque control for Induction Motors using MatrixConverters is analysed and points out the problem ofthe electromagnetic torque ripple which is one of themost important drawbacks of the Direct TorqueControl. Besides, the matrix converter is a single-stageac-ac power conversion device without dc-link energystorage elements. Matrix converter (MC may becomea good alternative to voltage-source inverter (VSI.This work combines the advantages of the matrixconverter with those of the DTC technique, generatingthe required voltage vectors under unity input powerfactor operation. Simulation results demonstrates theeffectiveness of the torque control.

Compressive elasticity of three-dimensional nanofiber matrix directs mesenchymal stem cell differentiation to vascular cells with endothelial or smooth muscle cell markers.

Science.gov (United States)

Wingate, K; Bonani, W; Tan, Y; Bryant, S J; Tan, W

2012-04-01

The importance of mesenchymal stem cells (MSC) in vascular regeneration is becoming increasingly recognized. However, few in vitro studies have been performed to identify the effects of environmental elasticity on the differentiation of MSC into vascular cell types. Electrospinning and photopolymerization techniques were used to fabricate a three-dimensional (3-D) polyethylene glycol dimethacrylate nanofiber hydrogel matrix with tunable elasticity for use as a cellular substrate. Compression testing demonstrated that the elastic modulus of the hydrated 3-D matrices ranged from 2 to 15 kPa, similar to the in vivo elasticity of the intima basement membrane and media layer. MSC seeded on rigid matrices (8-15 kPa) showed an increase in cell area compared with those seeded on soft matrices (2-5 kPa). Furthermore, the matrix elasticity guided the cells to express different vascular-specific phenotypes with high differentiation efficiency. Around 95% of MSC seeded on the 3-D matrices with an elasticity of 3 kPa showed Flk-1 endothelial markers within 24h, while only 20% of MSC seeded on the matrices with elasticity >8 kPa demonstrated Flk-1 marker. In contrast, ∼80% of MSC seeded on 3-D matrices with elasticity >8 kPa demonstrated smooth muscle α-actin marker within 24h, while fewer than 10% of MSC seeded on 3-D matrices with elasticity elasticity of the substrate could be a powerful tool for vascular tissue regeneration. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Atomic layer deposition of ruthenium surface-coating on porous platinum catalysts for high-performance direct ethanol solid oxide fuel cells

Science.gov (United States)

Jeong, Heon Jae; Kim, Jun Woo; Jang, Dong Young; Shim, Joon Hyung

2015-09-01

Pt-Ru bi-metallic catalysts are synthesized by atomic layer deposition (ALD) of Ru surface-coating on sputtered Pt mesh. The catalysts are evaluated in direct ethanol solid oxide fuel cells (DESOFCs) in the temperature range of 300-500 °C. Island-growth of the ALD Ru coating is confirmed by transmission electron microscopy and X-ray photoelectron spectroscopy (XPS) analyses. The performance of the DESOFCs is evaluated based on the current-voltage output and electrochemical impedance spectroscopy. Genuine reduction of the polarization impedance, and enhanced power output with improved surface kinetics are achieved with the optimized ALD Ru surface-coating compared to bare Pt. The chemical composition of the Pt/ALD Ru electrode surface after fuel cell operation is analyzed via XPS. Enhanced cell performance is clearly achieved, attributed to the effective Pt/ALD Ru bi-metallic catalysis, including oxidation of Cdbnd O by Ru, and de-protonation of ethanol and cleavage of C-C bonds by Pt, as supported by surface morphology analysis which confirms formation of a large amount of carbon on bare Pt after the ethanol-fuel-cell test.

Physical, Spatial, and Molecular Aspects of Extracellular Matrix of In Vivo Niches and Artificial Scaffolds Relevant to Stem Cells Research

Directory of Open Access Journals (Sweden)

Maria Akhmanova

2015-01-01

Full Text Available Extracellular matrix can influence stem cell choices, such as self-renewal, quiescence, migration, proliferation, phenotype maintenance, differentiation, or apoptosis. Three aspects of extracellular matrix were extensively studied during the last decade: physical properties, spatial presentation of adhesive epitopes, and molecular complexity. Over 15 different parameters have been shown to influence stem cell choices. Physical aspects include stiffness (or elasticity, viscoelasticity, pore size, porosity, amplitude and frequency of static and dynamic deformations applied to the matrix. Spatial aspects include scaffold dimensionality (2D or 3D and thickness; cell polarity; area, shape, and microscale topography of cell adhesion surface; epitope concentration, epitope clustering characteristics (number of epitopes per cluster, spacing between epitopes within cluster, spacing between separate clusters, cluster patterns, and level of disorder in epitope arrangement, and nanotopography. Biochemical characteristics of natural extracellular matrix molecules regard diversity and structural complexity of matrix molecules, affinity and specificity of epitope interaction with cell receptors, role of non-affinity domains, complexity of supramolecular organization, and co-signaling by growth factors or matrix epitopes. Synergy between several matrix aspects enables stem cells to retain their function in vivo and may be a key to generation of long-term, robust, and effective in vitro stem cell culture systems.

Tantalum coating on TiO{sub 2} nanotubes induces superior rate of matrix mineralization and osteofunctionality in human osteoblasts

Energy Technology Data Exchange (ETDEWEB)

Frandsen, Christine J.; Brammer, Karla S. [Materials Science and Engineering, University of California at San Diego, La Jolla, CA 92093 (United States); Noh, Kunbae [Corporate Research Institute, Cheil Industries, Inc., Gocheon-Dong, Uiwang-Si, Gyeonggi-Do, 437-711 (Korea, Republic of); Johnston, Gary [Materials Science and Engineering, University of California at San Diego, La Jolla, CA 92093 (United States); Jin, Sungho, E-mail: jin@ucsd.edu [Materials Science and Engineering, University of California at San Diego, La Jolla, CA 92093 (United States); Mechanical and Aerospace Engineering, University of California at San Diego, La Jolla, CA 92093 (United States)

2014-04-01

Nanostructured surface geometries have been the focus of a multitude of recent biomaterial research, and exciting findings have been published. However, only a few publications have directly compared nanostructures of various surface chemistries. The work herein directly compares the response of human osteoblast cells to surfaces of identical nanotube geometries with two well-known orthopedic biomaterials: titanium oxide (TiO{sub 2}) and tantalum (Ta). The results reveal that the Ta surface chemistry on the nanotube architecture enhances alkaline phosphatase activity, and promotes a ∼ 30% faster rate of matrix mineralization and bone-nodule formation when compared to results on bare TiO{sub 2} nanotubes. This study implies that unique combinations of surface chemistry and nanostructure may influence cell behavior due to distinctive physico-chemical properties. These findings are of paramount importance to the orthopedics field for understanding cell behavior in response to subtle alterations in nanostructure and surface chemistry, and will enable further insight into the complex manipulation of biomaterial surfaces. With increased focus in the field of orthopedic materials research on nanostructured surfaces, this study emphasizes the need for careful and systematic review of variations in surface chemistry in concurrence with nanotopographical changes. - Highlights: • A TiO{sub 2} nanotube surface structure was coated with tantalum. • Osteoblast cell response was compared between the tantalum coated and as-formed TiO{sub 2} nanotube surface. • We observed superior rates of bone matrix mineralization and osteoblast maturation on the tantalum coated nanotube surface.

Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture.

Science.gov (United States)

Wallace, Charles S; Strike, Sophie A; Truskey, George A

2007-09-01

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the alpha(5)beta(1)-integrin complex, whereas ECs used either alpha(5)beta(1)- or alpha(v)beta(3)-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC alpha(5)beta(1)-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.

High fidelity visualization of cell-to-cell variation and temporal dynamics in nascent extracellular matrix formation.

Science.gov (United States)

McLeod, Claire M; Mauck, Robert L

2016-12-12

Extracellular matrix dynamics are key to tissue morphogenesis, homeostasis, injury, and repair. The spatiotemporal organization of this matrix has profound biological implications, but is challenging to monitor using standard techniques. Here, we address these challenges by using noncanonical amino acid tagging to fluorescently label extracellular matrix synthesized in the presence of bio-orthogonal methionine analogs. This strategy labels matrix proteins with high resolution, without compromising their distribution or mechanical function. We demonstrate that the organization and temporal dynamics of the proteinaceous matrix depend on the biophysical features of the microenvironment, including the biomaterial scaffold and the niche constructed by cells themselves. Pulse labeling experiments reveal that, in immature constructs, nascent matrix is highly fibrous and interdigitates with pre-existing matrix, while in more developed constructs, nascent matrix lacks fibrous organization and is retained in the immediate pericellular space. Inhibition of collagen crosslinking increases matrix synthesis, but compromises matrix organization. Finally, these data demonstrate marked cell-to-cell heterogeneity amongst both chondrocytes and mesenchymal stem cells undergoing chondrogenesis. Collectively, these results introduce fluorescent noncanonical amino acid tagging as a strategy to investigate spatiotemporal matrix organization, and demonstrate its ability to identify differences in phenotype, microenvironment, and matrix assembly at the single cell level.

Cell surface hydrophobicity of dental plaque microorganisms in situ.

OpenAIRE

Rosenberg, M; Judes, H; Weiss, E

1983-01-01

The cell surface hydrophobicity of bacteria obtained directly from human tooth surfaces was assayed by measuring their adherence to liquid hydrocarbons. Fresh samples of supragingival dental plaque were washed and dispersed in buffer. Adherence of the plaque microorganisms to hexadecane, octane, and xylene was tested turbidimetrically and by direct microscopic observation. The results clearly show that the vast majority of bacteria comprising dental plaque exhibit pronounced cell surface hydr...

The Complex Interaction of Matrix Metalloproteinases in the Migration of Cancer Cells through Breast Tissue Stroma

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Kerry J. Davies

2014-01-01

Full Text Available Breast cancer mortality is directly linked to metastatic spread. The metastatic cell must exhibit a complex phenotype that includes the capacity to escape from the primary tumour mass, invade the surrounding normal tissue, and penetrate into the circulation before proliferating in the parenchyma of distant organs to produce a metastasis. In the normal breast, cellular structures change cyclically in response to ovarian hormones leading to regulated cell proliferation and apoptosis. Matrix metalloproteinases (MMPs are a family of zinc dependent endopeptidases. Their primary function is degradation of proteins in the extracellular matrix to allow ductal progression through the basement membrane. A complex balance between matrix metalloproteinases and their inhibitors regulate these changes. These proteinases interact with cytokines, growth factors, and tumour necrosis factors to stimulate branching morphologies in normal breast tissues. In breast cancer this process is disrupted facilitating tumour progression and metastasis and inhibiting apoptosis increasing the life of the metastatic cells. This paper highlights the role of matrix metalloproteinases in cell progression through the breast stroma and reviews the complex relationships between the different proteinases and their inhibitors in relation to breast cancer cells as they metastasise.

Characterization of Cell Surface and EPS Remodeling of Azospirillum brasilense Chemotaxis-like 1 Signal Transduction Pathway mutants by Atomic Force Microscopy

Energy Technology Data Exchange (ETDEWEB)

Billings, Amanda N [ORNL; Siuti, Piro [ORNL; Bible, Amber [University of Tennessee, Knoxville (UTK); Alexandre, Gladys [University of Tennessee, Knoxville (UTK); Retterer, Scott T [ORNL; Doktycz, Mitchel John [ORNL; Morrell-Falvey, Jennifer L [ORNL

2011-01-01

To compete in complex microbial communities, bacteria must quickly sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the modulation of multiple cellular responses, including motility, EPS production, and cell-to-cell interactions. Recently, the Che1 chemotaxis-like pathway from Azospirillum brasilense was shown to modulate flocculation. In A. brasilense, cell surface properties, including EPS production, are thought to play a direct role in promoting flocculation. Using atomic force microscopy (AFM), we have detected distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains that are absent in the wild type strain. Whereas the wild type strain produces a smooth mucosal extracellular matrix, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition and lectin-binding assays suggest that the composition of EPS components in the extracellular matrix differs between the cheA1 and cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that mutations in the Che1 pathway that result in increased flocculation are correlated with distinctive changes in the extracellular matrix structure produced by the mutants, including likely changes in the EPS structure and/or composition.

Temperature responsive functional polymeric thin films obtained by matrix assisted pulsed laser evaporation for cells attachment–detachment study

Energy Technology Data Exchange (ETDEWEB)

Rusen, L. [NILPRP, National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, RO-077125 Magurele, Bucharest (Romania); Dinca, V., E-mail: dinali@nipne.ro [NILPRP, National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, RO-077125 Magurele, Bucharest (Romania); Mitu, B. [NILPRP, National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, RO-077125 Magurele, Bucharest (Romania); Mustaciosu, C. [Horia Hulubei National Institute of Physics and Nuclear Engineering, IFIN HH, Magurele, Bucharest (Romania); Dinescu, M. [NILPRP, National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, RO-077125 Magurele, Bucharest (Romania)

2014-05-01

Multifunctional thin films used as thermoresponsive substrate for engineering cell sheets represent an important area in tissue engineering. As the morphology and the chemical characteristics of the thin films directly control their interaction with cells, it is important to correlate these characteristics with the biological answer. In this study, thermally sensitive poly(N-isopropylacrylamide), (pNIPAAm) thin films were prepared by matrix assisted pulsed laser evaporation and utilized in L929 cell adhesion and detachment studies. Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM) were used to determine the pNIPAAm thin films chemical and morphological characteristics. The FTIR data demonstrated that the functional groups in the MAPLE-deposited films remained intact for fluences in the range of 200–600 mJ cm{sup −2}. Within this fluence range, the AFM topographical studies showed that the roughness of the coatings was dependent on laser fluence and the obtained surfaces were characterized by a granular aspect. L929 cell viability studies onto the pNIPAAm coatings showed little or no toxic effect for fluences below 600 mJ cm{sup −2}, while for higher fluences, viability was decreased with more than 50%. The adhesion and detachment of the cell was found to be mainly dependent on the film surface morphology.

Diffusion of MMPs on the Surface of Collagen Fibrils: The Mobile Cell Surface – Collagen Substratum Interface

Science.gov (United States)

Collier, Ivan E.; Legant, Wesley; Marmer, Barry; Lubman, Olga; Saffarian, Saveez; Wakatsuki, Tetsuro; Elson, Elliot; Goldberg, Gregory I.

2011-01-01

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions. PMID:21912660

Integrative systems and synthetic biology of cell-matrix adhesion sites.

Science.gov (United States)

Zamir, Eli

2016-09-02

The complexity of cell-matrix adhesion convolves its roles in the development and functioning of multicellular organisms and their evolutionary tinkering. Cell-matrix adhesion is mediated by sites along the plasma membrane that anchor the actin cytoskeleton to the matrix via a large number of proteins, collectively called the integrin adhesome. Fundamental challenges for understanding how cell-matrix adhesion sites assemble and function arise from their multi-functionality, rapid dynamics, large number of components and molecular diversity. Systems biology faces these challenges in its strive to understand how the integrin adhesome gives rise to functional adhesion sites. Synthetic biology enables engineering intracellular modules and circuits with properties of interest. In this review I discuss some of the fundamental questions in systems biology of cell-matrix adhesion and how synthetic biology can help addressing them.

A density gradient of VAPG peptides on a cell-resisting surface achieves selective adhesion and directional migration of smooth muscle cells over fibroblasts.

Science.gov (United States)

Yu, Shan; Zuo, Xingang; Shen, Tao; Duan, Yiyuan; Mao, Zhengwei; Gao, Changyou

2018-05-01

Selective adhesion and migration of smooth muscle cells (SMCs) over fibroblasts (FIBs) is required to prevent adventitia fibrosis in vascular regeneration. In this study, a uniform cell-resisting layer of poly(ethylene glycol) (PEG) with a density gradient of azide groups was generated on a substrate by immobilizing two kinds of PEG molecules in a gradient manner. A density gradient of alkynyl-functionalized Val-Ala-Pro-Gly (VAPG) peptides was then prepared on the PEG layer via click chemistry. The VAPG density gradient was characterized by fluorescence imaging, revealing the gradual enhancement of the fluorescent intensity along the substrate direction. The adhesion and mobility of SMCs were selectively enhanced on the VAPG density gradient, leading to directional migration toward the higher peptide density (up to 84%). In contrast, the adhesion and mobility of FIBs were significantly weakened. The net displacement of SMCs also significantly increased compared with that on tissue culture polystyrene (TCPS) and that of FIBs on the gradient. The mitogen-activated protein kinase (MAPK) signaling pathways related to cell migration were studied, showing higher expressions of functional proteins from SMCs on the VAPG-modified surface in a density-dependent manner. For the first time the selective adhesion and directional migration of SMCs over FIBs was achieved by an elaborative design of a gradient surface, leading to a new insight in design of novel vascular regenerative materials. Selective cell adhesion and migration guided by regenerative biomaterials are extremely important for the regeneration of targeted tissues, which can avoid the drawbacks of incorrect and uncontrolled responses of tissue cells to implants. For example, selectivity of smooth muscle cells (SMCs) over fibroblasts (FIBs) is required to prevent adventitia fibrosis in vascular regeneration. Herein we prepare a uniform cell-repelling layer, on which SMCs-selective Val-Ala-Pro-Gly (VAPG) peptides

Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels

Science.gov (United States)

Khetan, Sudhir; Guvendiren, Murat; Legant, Wesley R.; Cohen, Daniel M.; Chen, Christopher S.; Burdick, Jason A.

2013-05-01

Although cell-matrix adhesive interactions are known to regulate stem cell differentiation, the underlying mechanisms, in particular for direct three-dimensional encapsulation within hydrogels, are poorly understood. Here, we demonstrate that in covalently crosslinked hyaluronic acid (HA) hydrogels, the differentiation of human mesenchymal stem cells (hMSCs) is directed by the generation of degradation-mediated cellular traction, independently of cell morphology or matrix mechanics. hMSCs within HA hydrogels of equivalent elastic moduli that permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favoured osteogenesis (adipogenesis). Moreover, switching the permissive hydrogel to a restrictive state through delayed secondary crosslinking reduced further hydrogel degradation, suppressed traction, and caused a switch from osteogenesis to adipogenesis in the absence of changes to the extended cellular morphology. Furthermore, inhibiting tension-mediated signalling in the permissive environment mirrored the effects of delayed secondary crosslinking, whereas upregulating tension induced osteogenesis even in the restrictive environment.

Effects of matrix elasticity and cell density on human mesenchymal stem cells differentiation.

Science.gov (United States)

Xue, Ruyue; Li, Julie Yi-Shuan; Yeh, Yiting; Yang, Li; Chien, Shu

2013-09-01

Human mesenchymal stem cells (hMSCs) can differentiate into various cell types, including osteogenic and chondrogenic cells. The matrix elasticity and cell seeding density are important factors in hMSCs differentiation. We cultured hMSCs at different seeding densities on polyacrylamide hydrogels with different stiffness corresponding to Young's moduli of 1.6 ± 0.3 and 40 ± 3.6 kPa. The promotion of osteogenic marker expression by hard gel is overridden by a high seeding density. Cell seeding density, however, did not influence the chondrogenic marker expressions induced by soft gel. These findings suggest that interplays between cell-matrix and cell-cell interactions contribute to hMSCs differentiation. The promotion of osteogenic differentiation on hard matrix was shown to be mediated through the Ras pathway. Inhibition of Ras (RasN17) significantly decreased ERK, Smad1/5/8 and AKT activation, and osteogenic markers expression. However, constitutively active Ras (RasV12) had little effect on osteogenic marker expression, suggesting that the Ras pathways are necessary but not sufficient for osteogenesis. Taken together, our results indicate that matrix elasticity and cell density are important microenvironmental cues driving hMSCs proliferation and differentiation. Copyright © 2013 Orthopaedic Research Society.

Matrix stiffness reverses the effect of actomyosin tension on cell proliferation.

Science.gov (United States)

Mih, Justin D; Marinkovic, Aleksandar; Liu, Fei; Sharif, Asma S; Tschumperlin, Daniel J

2012-12-15

The stiffness of the extracellular matrix exerts powerful effects on cell proliferation and differentiation, but the mechanisms transducing matrix stiffness into cellular fate decisions remain poorly understood. Two widely reported responses to matrix stiffening are increases in actomyosin contractility and cell proliferation. To delineate their relationship, we modulated cytoskeletal tension in cells grown across a physiological range of matrix stiffnesses. On both synthetic and naturally derived soft matrices, and across a panel of cell types, we observed a striking reversal of the effect of inhibiting actomyosin contractility, switching from the attenuation of proliferation on rigid substrates to the robust promotion of proliferation on soft matrices. Inhibiting contractility on soft matrices decoupled proliferation from cytoskeletal tension and focal adhesion organization, but not from cell spread area. Our results demonstrate that matrix stiffness and actomyosin contractility converge on cell spreading in an unexpected fashion to control a key aspect of cell fate.

Tuning cell adhesion by direct nanostructuring silicon into cell repulsive/adhesive patterns

International Nuclear Information System (INIS)

Premnath, Priyatha; Tavangar, Amirhossein; Tan, Bo; Venkatakrishnan, Krishnan

2015-01-01

Developing platforms that allow tuning cell functionality through incorporating physical, chemical, or mechanical cues onto the material surfaces is one of the key challenges in research in the field of biomaterials. In this respect, various approaches have been proposed and numerous structures have been developed on a variety of materials. Most of these approaches, however, demand a multistep process or post-chemical treatment. Therefore, a simple approach would be desirable to develop bio-functionalized platforms for effectively modulating cell adhesion and consequently programming cell functionality without requiring any chemical or biological surface treatment. This study introduces a versatile yet simple laser approach to structure silicon (Si) chips into cytophobic/cytophilic patterns in order to modulate cell adhesion and proliferation. These patterns are fabricated on platforms through direct laser processing of Si substrates, which renders a desired computer-generated configuration into patterns. We investigate the morphology, chemistry, and wettability of the platform surfaces. Subsequently, we study the functionality of the fabricated platforms on modulating cervical cancer cells (HeLa) behaviour. The results from in vitro studies suggest that the nanostructures efficiently repel HeLa cells and drive them to migrate onto untreated sites. The study of the morphology of the cells reveals that cells evade the cytophobic area by bending and changing direction. Additionally, cell patterning, cell directionality, cell channelling, and cell trapping are achieved by developing different platforms with specific patterns. The flexibility and controllability of this approach to effectively structure Si substrates to cell-repulsive and cell-adhesive patterns offer perceptible outlook for developing bio-functionalized platforms for a variety of biomedical devices. Moreover, this approach could pave the way for developing anti-cancer platforms that selectively repel

Tuning cell adhesion by direct nanostructuring silicon into cell repulsive/adhesive patterns

Energy Technology Data Exchange (ETDEWEB)

Premnath, Priyatha, E-mail: priyatha.premnath@ryerson.ca [Micro/Nanofabrication Laboratory, Department of Mechanical and Industrial Engineering, Ryerson University, 350 Victoria Street, Toronto, ON, Canada M5B 2K3 (Canada); Tavangar, Amirhossein, E-mail: atavanga@ryerson.ca [Micro/Nanofabrication Laboratory, Department of Mechanical and Industrial Engineering, Ryerson University, 350 Victoria Street, Toronto, ON, Canada M5B 2K3 (Canada); Tan, Bo, E-mail: tanbo@ryerson.ca [Nanocharacterization Laboratory, Department of Aerospace Engineering, Ryerson University, 350 Victoria Street, Toronto, ON, Canada M5B 2K3 (Canada); Venkatakrishnan, Krishnan, E-mail: venkat@ryerson.ca [Micro/Nanofabrication Laboratory, Department of Mechanical and Industrial Engineering, Ryerson University, 350 Victoria Street, Toronto, ON, Canada M5B 2K3 (Canada)

2015-09-10

Developing platforms that allow tuning cell functionality through incorporating physical, chemical, or mechanical cues onto the material surfaces is one of the key challenges in research in the field of biomaterials. In this respect, various approaches have been proposed and numerous structures have been developed on a variety of materials. Most of these approaches, however, demand a multistep process or post-chemical treatment. Therefore, a simple approach would be desirable to develop bio-functionalized platforms for effectively modulating cell adhesion and consequently programming cell functionality without requiring any chemical or biological surface treatment. This study introduces a versatile yet simple laser approach to structure silicon (Si) chips into cytophobic/cytophilic patterns in order to modulate cell adhesion and proliferation. These patterns are fabricated on platforms through direct laser processing of Si substrates, which renders a desired computer-generated configuration into patterns. We investigate the morphology, chemistry, and wettability of the platform surfaces. Subsequently, we study the functionality of the fabricated platforms on modulating cervical cancer cells (HeLa) behaviour. The results from in vitro studies suggest that the nanostructures efficiently repel HeLa cells and drive them to migrate onto untreated sites. The study of the morphology of the cells reveals that cells evade the cytophobic area by bending and changing direction. Additionally, cell patterning, cell directionality, cell channelling, and cell trapping are achieved by developing different platforms with specific patterns. The flexibility and controllability of this approach to effectively structure Si substrates to cell-repulsive and cell-adhesive patterns offer perceptible outlook for developing bio-functionalized platforms for a variety of biomedical devices. Moreover, this approach could pave the way for developing anti-cancer platforms that selectively repel

Tumor cell surface proteins

International Nuclear Information System (INIS)

Kennel, S.J.; Braslawsky, G.R.; Flynn, K.; Foote, L.J.; Friedman, E.; Hotchkiss, J.A.; Huang, A.H.L.; Lankford, P.K.

1982-01-01

Cell surface proteins mediate interaction between cells and their environment. Unique tumor cell surface proteins are being identified and quantified in several tumor systems to address the following questions: (i) how do tumor-specific proteins arise during cell transformation; (ii) can these proteins be used as markers of tumor cell distribution in vivo; (iii) can cytotoxic drugs be targeted specifically to tumor cells using antibody; and (iv) can solid state radioimmunoassay of these proteins provide a means to quantify transformation frequencies. A tumor surface protein of 180,000 M/sub r/ (TSP-180) has been identified on cells of several lung carcinomas of BALB/c mice. TSP-180 was not detected on normal lung tissue, embryonic tissue, or other epithelial or sarcoma tumors, but it was found on lung carcinomas of other strains of mice. Considerable amino acid sequence homology exists among TSP-180's from several cell sources, indicating that TSP-180 synthesis is directed by normal cellular genes although it is not expressed in normal cells. The regulation of synthesis of TSP-180 and its relationship to normal cell surface proteins are being studied. Monoclonal antibodies (MoAb) to TSP-180 have been developed. The antibodies have been used in immunoaffinity chromatography to isolate TSP-180 from tumor cell sources. This purified tumor antigen was used to immunize rats. Antibody produced by these animals reacted at different sites (epitopes) on the TSP-180 molecule than did the original MoAb. These sera and MoAb from these animals are being used to identify normal cell components related to the TSP-180 molecule

Microbial surface displayed enzymes based biofuel cell utilizing degradation products of lignocellulosic biomass for direct electrical energy.

Science.gov (United States)

Fan, Shuqin; Hou, Chuantao; Liang, Bo; Feng, Ruirui; Liu, Aihua

2015-09-01

In this work, a bacterial surface displaying enzyme based two-compartment biofuel cell for the direct electrical energy conversion from degradation products of lignocellulosic biomass is reported. Considering that the main degradation products of the lignocellulose are glucose and xylose, xylose dehydrogenase (XDH) displayed bacteria (XDH-bacteria) and glucose dehydrogenase (GDH) displayed bacteria (GDH-bacteria) were used as anode catalysts in anode chamber with methylene blue as electron transfer mediator. While the cathode chamber was constructed with laccase/multi-walled-carbon nanotube/glassy-carbon-electrode. XDH-bacteria exhibited 1.75 times higher catalytic efficiency than GDH-bacteria. This assembled enzymatic fuel cell exhibited a high open-circuit potential of 0.80 V, acceptable stability and energy conversion efficiency. Moreover, the maximum power density of the cell could reach 53 μW cm(-2) when fueled with degradation products of corn stalk. Thus, this finding holds great potential to directly convert degradation products of biomass into electrical energy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Layers and Multilayers of Self-Assembled Polymers: Tunable Engineered Extracellular Matrix Coatings for Neural Cell Growth.

Science.gov (United States)

Landry, Michael J; Rollet, Frédéric-Guillaume; Kennedy, Timothy E; Barrett, Christopher J

2018-03-12

Growing primary cells and tissue in long-term cultures, such as primary neural cell culture, presents many challenges. A critical component of any environment that supports neural cell growth in vivo is an appropriate 2-D surface or 3-D scaffold, typically in the form of a thin polymer layer that coats an underlying plastic or glass substrate and aims to mimic critical aspects of the extracellular matrix. A fundamental challenge to mimicking a hydrophilic, soft natural cell environment is that materials with these properties are typically fragile and are difficult to adhere to and stabilize on an underlying plastic or glass cell culture substrate. In this review, we highlight the current state of the art and overview recent developments of new artificial extracellular matrix (ECM) surfaces for in vitro neural cell culture. Notably, these materials aim to strike a balance between being hydrophilic and soft while also being thick, stable, robust, and bound well to the underlying surface to provide an effective surface to support long-term cell growth. We focus on improved surface and scaffold coating systems that can mimic the natural physicochemical properties that enhance neuronal survival and growth, applied as soft hydrophilic polymer coatings for both in vitro cell culture and for implantable neural probes and 3-D matrixes that aim to enhance stability and longevity to promote neural biocompatibility in vivo. With respect to future developments, we outline four emerging principles that serve to guide the development of polymer assemblies that function well as artificial ECMs: (a) design inspired by biological systems and (b) the employment of principles of aqueous soft bonding and self-assembly to achieve (c) a high-water-content gel-like coating that is stable over time in a biological environment and possesses (d) a low modulus to more closely mimic soft, compliant real biological tissue. We then highlight two emerging classes of thick material coatings that

Iduronic acid in chondroitin/dermatan sulfate affects directional migration of aortic smooth muscle cells.

Directory of Open Access Journals (Sweden)

Barbara Bartolini

Full Text Available Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA, catalyzed by two DS epimerases. Functional ablation of DS-epi1, the main epimerase in these cells, resulted in a major reduction of IdoA both on cell surface and in secreted CS/DS proteoglycans. Downregulation of IdoA led to delayed ability to re-populate wounded areas due to loss of directional persistence of migration. DS-epi1-/- aortic smooth muscle cells, however, had not lost the general property of migration showing even increased speed of movement compared to wild type cells. Where the cell membrane adheres to the substratum, stress fibers were denser whereas focal adhesion sites were fewer. Total cellular expression of focal adhesion kinase (FAK and phospho-FAK (pFAK was decreased in mutant cells compared to control cells. As many pathological conditions are dependent on migration, modulation of IdoA content may point to therapeutic strategies for diseases such as cancer and atherosclerosis.

Preparation and properties of hybrid direct methanol fuel cell membranes by embedding organophosphorylated titania submicrospheres into a chitosan polymer matrix

Energy Technology Data Exchange (ETDEWEB)

Wu, Hong [Key Laboratory for Green Chemical Technology, School of Chemical Engineering and Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072 (China); Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072 (China); Hou, Weiqiang; Wang, Jingtao; Xiao, Lulu; Jiang, Zhongyi [Key Laboratory for Green Chemical Technology, School of Chemical Engineering and Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072 (China)

2010-07-01

Organophosphorylated titania submicrospheres (OPTi) are prepared and incorporated into a chitosan (CS) matrix to fabricate hybrid membranes with enhanced methanol resistance and proton conductivity for application in direct methanol fuel cells (DMFC). The pristine monodispersed titania submicrospheres (TiO{sub 2}) of controllable particle size are synthesized through a modified sol-gel method and then phosphorylated by amino trimethylene phosphonic acid (ATMP) via chemical adsorption, which is confirmed by XPS, FTIR and TGA. The morphology and thermal property of the hybrid membranes are explored by SEM and TGA. The ionic cross-linking between the -PO{sub 3}H{sub 2} groups on OPTi and the -NH{sub 2} groups on CS lead to better compatibility between the inorganic fillers and the polymer matrix, as well as a decreased fractional free volume (FFV), which is verified by positron annihilation lifetime spectroscopy (PALS). The effects of particle size and content on the methanol permeability, proton conductivity, swelling and FFV of the membranes are investigated. Compared to pure CS membrane, the hybrid membranes exhibit an increased proton conductivity to an acceptable level of 0.01 S cm{sup -1} for DMFC application and a reduced methanol permeability of 5 x 10{sup -7} cm{sup 2} s{sup -1} at a 2 M methanol feed. (author)

Preparation and properties of hybrid direct methanol fuel cell membranes by embedding organophosphorylated titania submicrospheres into a chitosan polymer matrix

Science.gov (United States)

Wu, Hong; Hou, Weiqiang; Wang, Jingtao; Xiao, Lulu; Jiang, Zhongyi

Organophosphorylated titania submicrospheres (OPTi) are prepared and incorporated into a chitosan (CS) matrix to fabricate hybrid membranes with enhanced methanol resistance and proton conductivity for application in direct methanol fuel cells (DMFC). The pristine monodispersed titania submicrospheres (TiO 2) of controllable particle size are synthesized through a modified sol-gel method and then phosphorylated by amino trimethylene phosphonic acid (ATMP) via chemical adsorption, which is confirmed by XPS, FTIR and TGA. The morphology and thermal property of the hybrid membranes are explored by SEM and TGA. The ionic cross-linking between the -PO 3H 2 groups on OPTi and the -NH 2 groups on CS lead to better compatibility between the inorganic fillers and the polymer matrix, as well as a decreased fractional free volume (FFV), which is verified by positron annihilation lifetime spectroscopy (PALS). The effects of particle size and content on the methanol permeability, proton conductivity, swelling and FFV of the membranes are investigated. Compared to pure CS membrane, the hybrid membranes exhibit an increased proton conductivity to an acceptable level of 0.01 S cm -1 for DMFC application and a reduced methanol permeability of 5 × 10 -7 cm 2 s -1 at a 2 M methanol feed.

Matrix regulators in neural stem cell functions.

Science.gov (United States)

Wade, Anna; McKinney, Andrew; Phillips, Joanna J

2014-08-01

Neural stem/progenitor cells (NSPCs) reside within a complex and dynamic extracellular microenvironment, or niche. This niche regulates fundamental aspects of their behavior during normal neural development and repair. Precise yet dynamic regulation of NSPC self-renewal, migration, and differentiation is critical and must persist over the life of an organism. In this review, we summarize some of the major components of the NSPC niche and provide examples of how cues from the extracellular matrix regulate NSPC behaviors. We use proteoglycans to illustrate the many diverse roles of the niche in providing temporal and spatial regulation of cellular behavior. The NSPC niche is comprised of multiple components that include; soluble ligands, such as growth factors, morphogens, chemokines, and neurotransmitters, the extracellular matrix, and cellular components. As illustrated by proteoglycans, a major component of the extracellular matrix, the NSPC, niche provides temporal and spatial regulation of NSPC behaviors. The factors that control NSPC behavior are vital to understand as we attempt to modulate normal neural development and repair. Furthermore, an improved understanding of how these factors regulate cell proliferation, migration, and differentiation, crucial for malignancy, may reveal novel anti-tumor strategies. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. Copyright © 2014 Elsevier B.V. All rights reserved.

Intestinal Stem Cell Niche: The Extracellular Matrix and Cellular Components

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Laween Meran

2017-01-01

Full Text Available The intestinal epithelium comprises a monolayer of polarised columnar cells organised along the crypt-villus axis. Intestinal stem cells reside at the base of crypts and are constantly nourished by their surrounding niche for maintenance, self-renewal, and differentiation. The cellular microenvironment including the adjacent Paneth cells, stromal cells, smooth muscle cells, and neural cells as well as the extracellular matrix together constitute the intestinal stem cell niche. A dynamic regulatory network exists among the epithelium, stromal cells, and the matrix via complex signal transduction to maintain tissue homeostasis. Dysregulation of these biological or mechanical signals could potentially lead to intestinal injury and disease. In this review, we discuss the role of different intestinal stem cell niche components and dissect the interaction between dynamic matrix factors and regulatory signalling during intestinal stem cell homeostasis.

The ECM-Cell Interaction of Cartilage Extracellular Matrix on Chondrocytes

Directory of Open Access Journals (Sweden)

Yue Gao

2014-01-01

Full Text Available Cartilage extracellular matrix (ECM is composed primarily of the network type II collagen (COLII and an interlocking mesh of fibrous proteins and proteoglycans (PGs, hyaluronic acid (HA, and chondroitin sulfate (CS. Articular cartilage ECM plays a crucial role in regulating chondrocyte metabolism and functions, such as organized cytoskeleton through integrin-mediated signaling via cell-matrix interaction. Cell signaling through integrins regulates several chondrocyte functions, including differentiation, metabolism, matrix remodeling, responses to mechanical stimulation, and cell survival. The major signaling pathways that regulate chondrogenesis have been identified as wnt signal, nitric oxide (NO signal, protein kinase C (PKC, and retinoic acid (RA signal. Integrins are a large family of molecules that are central regulators in multicellular biology. They orchestrate cell-cell and cell-matrix adhesive interactions from embryonic development to mature tissue function. In this review, we emphasize the signaling molecule effect and the biomechanics effect of cartilage ECM on chondrogenesis.

Macro-mechanical material model for fiber reinforced metal matrix composites

CERN Document Server

Banks-Sills, L

1999-01-01

The stress-strain behavior of a metal matrix composite reinforced with unidirectional, continuous and periodic fibers is investigated. Three-dimensional micro-mechanical analyses of a unit cell by means of the finite element method $9 and homogenization-localization are carried out. These calculations allow the determination of material behavior of the in-plane, as well as the fiber directions. The fibers are assumed to be elastic and the matrix elasto-plastic. $9 The matrix material is governed by a von Mises yield surface, isotropic hardening and an associated flow rule. With the aid of these analyses, the foundation to a macro-mechanical material model is presented which is employed to $9 consider an elementary problem. The model includes an anisotropic yield surface with isotropic hardening and an associated flow rule. A beam in bending containing square fibers under plane strain conditions is analyzed by means of $9 the model. Two cases are considered: one in which the fibers are symmetric with respect t...

In vitro mesenchymal trilineage differentiation and extracellular matrix production by adipose and bone marrow derived adult equine multipotent stromal cells on a collagen scaffold.

Science.gov (United States)

Xie, Lin; Zhang, Nan; Marsano, Anna; Vunjak-Novakovic, Gordana; Zhang, Yanru; Lopez, Mandi J

2013-12-01

Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 days of culture in stromal or induction medium. Cell number was determined with DNA content, cell viability and spatial uniformity with confocal laser microscopy and cell phenotype and matrix production with light and scanning electron microscopy and mRNA levels. The MSC were positive for CD29 (>90 %), CD44 (>99 %), and CD105 (>60 %). Loading efficiencies were >70 %. The ASC and BMSC cell numbers on scaffolds were affected by culture in induction medium differently. Viable cells remained uniformly distributed in scaffolds for up to 21 days and could be directed to differentiate or to maintain an MSC phenotype. Micro- and ultrastructure showed lineage-specific cell and ECM changes. Lineage-specific mRNA levels differed between ASC and BMSC with induction and changed with time. Based on these results, equine ASC and BMSC differentiate into chondrogenic, osteogenic and adipogenic lineages and form ECM similarly on COLI scaffolds. The collected data supports the potential for equine MSC-COLI constructs to support diverse equine tissue formation for controlled biological studies.

Ascorbate-dependent impact on cell-derived matrix in modulation of stiffness and rejuvenation of infrapatellar fat derived stem cells toward chondrogenesis.

Science.gov (United States)

Pizzute, Tyler; Zhang, Ying; He, Fan; Pei, Ming

2016-08-10

Developing an in vitro microenvironment using cell-derived decellularized extracellular matrix (dECM) is a promising approach to efficiently expand adult stem cells for cartilage engineering and regeneration. Ascorbic acid serves as a critical stimulus for cells to synthesize collagens, which constitute the major component of dECM. In this study, we hypothesized that optimization of ascorbate treatment would maximize the rejuvenation effect of dECM on expanded stem cells from human infrapatellar fat pad in both proliferation and chondrogenic differentiation. In the duration regimen study, we found that dECM without L-ascorbic acid phosphate (AA) treatment, exhibiting lower stiffness measured by atomic force microscopy, yielded expanded cells with higher proliferation capacity but lower chondrogenic potential when compared to those with varied durations of AA treatment. dECM with 250 µM of AA treatment for 10 d had better rejuvenation in chondrogenic capacity if the deposited cells were from passage 2 rather than passage 5, despite no significant difference in matrix stiffness. In the dose regimen study, we found that dECMs deposited by varied concentrations of AA yielded expanded cells with higher proliferation capacity despite lower expression levels of stem cell related surface markers. Compared to cells expanded on tissue culture polystyrene, those on dECM exhibited greater chondrogenic potential, particularly for the dECMs with 50 µM and 250 µM of AA treatment. With the supplementation of ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor targeting procollagen synthesis, the dECM with 50 µM of AA treatment exhibited a dramatic decrease in the rejuvenation effect of expanded cell chondrogenic potential at both mRNA and protein levels despite no significant difference in matrix stiffness. Defined AA treatments during matrix preparation will benefit dECM-mediated stem cell engineering and future treatments for cartilage defects.

Extracellular matrix structure.

Science.gov (United States)

Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

2016-02-01

Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented. Copyright © 2015 Elsevier B.V. All rights reserved.

Electric field stimulation through a substrate influences Schwann cell and extracellular matrix structure

Science.gov (United States)

Nguyen, Hieu T.; Wei, Claudia; Chow, Jacqueline K.; Nguy, Lindsey; Nguyen, Hieu K.; Schmidt, Christine E.

2013-08-01

Objective. Electric field (EF) stimulation has been used to cue cell growth for tissue engineering applications. In this study, we explore the electrical parameters and extracellular mechanisms that elicit changes in cell behavior when stimulated through the substrate. Approach. Rat Schwann cell morphology was compared when exposed to EF through the media or a conductive indium tin oxide substrate. Ionic and structural effects were then analyzed on Matrigel and collagen I, respectively. Main results. When stimulating through media, cells had greater alignment perpendicular to the EF with higher current densities (106 mA cm-2 at 245 mV mm-1), and reached maximum alignment within 8 h. Stimulation through the substrate with EF (up to 110 mV mm-1) did not affect Schwann cell orientation, however the EF caused extracellular matrix (ECM) coatings on substrates to peel away, suggesting EF can physically change the ECM. Applying alternating current (ac) 2-1000 Hz signals through the media or substrate both caused cells to flatten and protrude many processes, without preferential alignment. Matrigel exposed to a substrate EF of 10 mV mm-1 for 2 h had a greater calcium concentration near the cathode, but quickly dissipated when the EF was removed. Schwann cells seeded 7 d after gels were exposed to substrate EF still aligned perpendicular to the EF direction. Microscopy of collagen I exposed to substrate EF shows alignment and bundling of fibrils. Significance. These findings demonstrate EF exposure can control Schwann cell alignment and morphology, change ECM bulk/surface architecture, and align ECM structures.

A versatile class of cell surface directional motors gives rise to gliding motility and sporulation in Myxococcus xanthus.

Directory of Open Access Journals (Sweden)

Morgane Wartel

2013-12-01

Full Text Available Eukaryotic cells utilize an arsenal of processive transport systems to deliver macromolecules to specific subcellular sites. In prokaryotes, such transport mechanisms have only been shown to mediate gliding motility, a form of microbial surface translocation. Here, we show that the motility function of the Myxococcus xanthus Agl-Glt machinery results from the recent specialization of a versatile class of bacterial transporters. Specifically, we demonstrate that the Agl motility motor is modular and dissociates from the rest of the gliding machinery (the Glt complex to bind the newly expressed Nfs complex, a close Glt paralogue, during sporulation. Following this association, the Agl system transports Nfs proteins directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by Agl-Nfs ensures its distribution around the spore. Thus, the Agl-Glt/Nfs machineries may constitute a novel class of directional bacterial surface transporters that can be diversified to specific tasks depending on the cognate cargo and machinery-specific accessories.

CADM1 controls actin cytoskeleton assembly and regulates extracellular matrix adhesion in human mast cells.

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Elena P Moiseeva

Full Text Available CADM1 is a major receptor for the adhesion of mast cells (MCs to fibroblasts, human airway smooth muscle cells (HASMCs and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM. Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion.

Cell stiffness, contractile stress and the role of extracellular matrix

International Nuclear Information System (INIS)

An, Steven S.; Kim, Jina; Ahn, Kwangmi; Trepat, Xavier; Drake, Kenneth J.; Kumar, Sarvesh; Ling, Guoyu; Purington, Carolyn; Rangasamy, Tirumalai; Kensler, Thomas W.; Mitzner, Wayne; Fredberg, Jeffrey J.; Biswal, Shyam

2009-01-01

Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genes in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses.

Cell stiffness, contractile stress and the role of extracellular matrix

Energy Technology Data Exchange (ETDEWEB)

An, Steven S., E-mail: san@jhsph.edu [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Kim, Jina [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Ahn, Kwangmi [Division of Biostatistics, Penn State College of Medicine, Hershey, PA 17033 (United States); Trepat, Xavier [CIBER, Enfermedades Respiratorias, 07110 Bunyola (Spain); Drake, Kenneth J. [Division of Molecular and Integrative Physiological Sciences, Harvard School of Public Health, Boston, MA 02115 (United States); Kumar, Sarvesh; Ling, Guoyu; Purington, Carolyn; Rangasamy, Tirumalai; Kensler, Thomas W.; Mitzner, Wayne [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Fredberg, Jeffrey J. [Division of Molecular and Integrative Physiological Sciences, Harvard School of Public Health, Boston, MA 02115 (United States); Biswal, Shyam [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Division of Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205 (United States)

2009-05-15

Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genes in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses.

Engineering the human pluripotent stem cell microenvironment to direct cell fate.

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Hazeltine, Laurie B; Selekman, Joshua A; Palecek, Sean P

2013-11-15

Human pluripotent stem cells (hPSCs), including both embryonic stem cells and induced pluripotent stem cells, offer a potential cell source for research, drug screening, and regenerative medicine applications due to their unique ability to self-renew or differentiate to any somatic cell type. Before the full potential of hPSCs can be realized, robust protocols must be developed to direct their fate. Cell fate decisions are based on components of the surrounding microenvironment, including soluble factors, substrate or extracellular matrix, cell-cell interactions, mechanical forces, and 2D or 3D architecture. Depending on their spatio-temporal context, these components can signal hPSCs to either self-renew or differentiate to cell types of the ectoderm, mesoderm, or endoderm. Researchers working at the interface of engineering and biology have identified various factors which can affect hPSC fate, often based on lessons from embryonic development, and they have utilized this information to design in vitro niches which can reproducibly direct hPSC fate. This review highlights culture systems that have been engineered to promote self-renewal or differentiation of hPSCs, with a focus on studies that have elucidated the contributions of specific microenvironmental cues in the context of those culture systems. We propose the use of microsystem technologies for high-throughput screening of spatial-temporal presentation of cues, as this has been demonstrated to be a powerful approach for differentiating hPSCs to desired cell types. Copyright © 2013 Elsevier Inc. All rights reserved.

Engineered matrix coatings to modulate the adhesion of CD133+ human hematopoietic progenitor cells.

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Franke, Katja; Pompe, Tilo; Bornhäuser, Martin; Werner, Carsten

2007-02-01

Interactions of hematopoietic progenitor cells (HPC) with their local microenvironments in the bone marrow are thought to control homing, differentiation, and self-renewal of the cells. To dissect the role of extracellular matrix (ECM) components of the niche microenvironment, a set of well-defined ECM coatings including fibronectin, heparin, heparan sulphate, hyaluronic acid, tropocollagen I, and co-fibrils of collagen I with heparin or hyaluronic acid was prepared and analysed with respect to the attachment of human CD133+ HPC in vitro. The extension of the adhesion areas of individual cells as well as the fraction of adherent cells were assessed by reflection interference contrast microscopy (RICM). Intense cell-matrix interactions were found on surfaces coated with fibronectin, heparin, heparan sulphate, and on the collagen I based co-fibrils. Insignificant adhesion was found for tropocollagen I and hyaluronic acid. The strongest adhesion of HPC was observed on fibronectin with contact areas of about 7 microm(2). Interaction of HPC with coatings consisting of heparin, heparan sulphate, and co-fibrils result in small circular shaped contact zones of 3 microm(2) pointing to another, less efficient, adhesion mechanism. Analysing the specificity of cell-matrix interaction by antibody blocking experiments suggests an integrin(alpha(5)beta(1))-specific adhesion on fibronectin, while adhesion on heparin was shown to be mediated by selectins (CD62L). Taken together, our data provide a basis for the design of advanced culture carriers supporting site-specific proliferation or differentiation of HPC.

Cartilage oligomeric matrix protein enhances matrix assembly during chondrogenesis of human mesenchymal stem cells.

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Haleem-Smith, Hana; Calderon, Raul; Song, Yingjie; Tuan, Rocky S; Chen, Faye H

2012-04-01

Cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5) is an abundant cartilage extracellular matrix (ECM) protein that interacts with major cartilage ECM components, including aggrecan and collagens. To test our hypothesis that COMP/TSP5 functions in the assembly of the ECM during cartilage morphogenesis, we have employed mesenchymal stem cell (MSC) chondrogenesis in vitro as a model to examine the effects of COMP over-expression on neo-cartilage formation. Human bone marrow-derived MSCs were transfected with either full-length COMP cDNA or control plasmid, followed by chondrogenic induction in three-dimensional pellet or alginate hydrogel culture. MSC chondrogenesis and ECM production was estimated based on quantitation of sulfated glycosaminoglycan (sGAG) accumulation, immunohistochemistry of the presence and distribution of cartilage ECM proteins, and real-time RT-PCR analyis of mRNA expression of cartilage markers. Our results showed that COMP over-expression resulted in increased total sGAG content during the early phase of MSC chondrogenesis, and increased immuno-detectable levels of aggrecan and collagen type II in the ECM of COMP-transfected pellet and alginate cultures, indicating more abundant cartilaginous matrix. COMP transfection did not significantly increase the transcript levels of the early chondrogenic marker, Sox9, or aggrecan, suggesting that enhancement of MSC cartilage ECM was effected at post-transcriptional levels. These findings strongly suggest that COMP functions in mesenchymal chondrogenesis by enhancing cartilage ECM organization and assembly. The action of COMP is most likely mediated not via direct changes in cartilage matrix gene expression but via interactions of COMP with other cartilage ECM proteins, such as aggrecan and collagens, that result in enhanced assembly and retention.

CARTILAGE OLIGOMERIC MATRIX PROTEIN ENHANCES MATRIX ASSEMBLY DURING CHONDROGENESIS OF HUMAN MESENCHYMAL STEM CELLS

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Haleem-Smith, Hana; Calderon, Raul; Song, Yingjie; Tuan, Rocky S.; Chen, Faye H.

2011-01-01

Cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5) is an abundant cartilage extracellular matrix (ECM) protein that interacts with major cartilage ECM components, including aggrecan and collagens. To test our hypothesis that COMP/TSP5 functions in the assembly of the ECM during cartilage morphogenesis, we have employed mesenchymal stem cell (MSC) chondrogenesis in vitro as a model to examine the effects of COMP over-expression on neo-cartilage formation. Human bone marrow-derived MSCs were transfected with either full-length COMP cDNA or control plasmid, followed by chondrogenic induction in three-dimensional pellet or alginate-hydrogel culture. MSC chondrogenesis and ECM production was estimated based on quantitation of sulfated glycosaminoglycan (sGAG) accumulation, immunohistochemistry of the presence and distribution of cartilage ECM proteins, and real-time RT-PCR analyis of mRNA expression of cartilage markers. Our results showed that COMP over-expression resulted in increased total sGAG content during the early phase of MSC chondrogenesis, and increased immuno-detectable levels of aggrecan and collagen type II in the ECM of COMP-transfected pellet and alginate cultures, indicating more abundant cartilaginous matrix. COMP transfection did not significantly increase the transcript levels of the early chondrogenic marker, Sox9, or aggrecan, suggesting that enhancement of MSC cartilage ECM was effected at post-transcriptional levels. These findings strongly suggest that COMP functions in mesenchymal chondrogenesis by enhancing cartilage ECM organization and assembly. The action of COMP is most likely mediated not via direct changes in cartilage matrix gene expression but via interactions of COMP with other cartilage ECM proteins, such as aggrecan and collagens, that result in enhanced assembly and retention. PMID:22095699

From single cells to tissues: interactions between the matrix and human breast cells in real time.

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Clifford Barnes

Full Text Available Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma--epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis.The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology.Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs.

Cell patterning without chemical surface modification: Cell cell interactions between printed bovine aortic endothelial cells (BAEC) on a homogeneous cell-adherent hydrogel

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Chen, C. Y.; Barron, J. A.; Ringeisen, B. R.

2006-10-01

Cell printing offers the unique ability to directly deposit one or multiple cell types directly onto a surface without the need to chemically pre-treat the surface with lithographic methods. We utilize biological laser printing (BioLP ™) to form patterns of bovine aortic endothelial cells (BAECs) onto a homogeneous cell adherent hydrogel surface. These normal cells are shown to retain near-100% viability post-printing. In order to determine whether BAECs encountered shear and/or heat stress during printing, immunocytochemical staining experiments were performed to detect potential expression of heat shock proteins (HSP) by the deposited cells. Printed BAECs expressed HSP at levels similar to negative control cells, indicating that the BioLP process does not expose cells to damaging levels of stress. However, HSP expression was slightly higher at the highest laser energy studied, suggesting more stress was present under these extreme conditions. Printed BAECs also showed preferential asymmetric growth and migration towards each other and away from the originally printed pattern, demonstrating a retained ability for the cells to communicate post-printing.

Effect of phosphate-based glass fibre surface properties on thermally produced poly(lactic acid) matrix composites.

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Mohammadi, Maziar Shah; Ahmed, Ifty; Muja, Naser; Rudd, Christopher D; Bureau, Martin N; Nazhat, Showan N

2011-12-01

Incorporation of soluble bioactive glass fibres into biodegradable polymers is an interesting approach for bone repair and regeneration. However, the glass composition and its surface properties significantly affect the nature of the fibre-matrix interface and composite properties. Herein, the effect of Si and Fe on the surface properties of calcium containing phosphate based glasses (PGs) in the system (50P(2)O(5)-40CaO-(10-x)SiO(2)-xFe(2)O(3), where x = 0, 5 and 10 mol.%) were investigated. Contact angle measurements revealed a higher surface energy, and surface polarity as well as increased hydrophilicity for Si doped PG which may account for the presence of surface hydroxyl groups. Two PG formulations, 50P(2)O(5)-40CaO-10Fe(2)O(3) (Fe10) and 50P(2)O(5)-40CaO-5Fe(2)O(3)-5SiO(2) (Fe5Si5), were melt drawn into fibres and randomly incorporated into poly(lactic acid) (PLA) produced by melt processing. The ageing in deionised water (DW), mechanical property changes in phosphate buffered saline (PBS) and cytocompatibility properties of these composites were investigated. In contrast to Fe10 and as a consequence of the higher surface energy and polarity of Fe5Si5, its incorporation into PLA led to increased inorganic/organic interaction indicated by a reduction in the carbonyl group of the matrix. PLA chain scission was confirmed by a greater reduction in its molecular weight in PLA-Fe5Si5 composites. In DW, the dissolution rate of PLA-Fe5Si5 was significantly higher than that of PLA-Fe10. Dissolution of the glass fibres resulted in the formation of channels within the matrix. Initial flexural strength was significantly increased through PGF incorporation. After PBS ageing, the reduction in mechanical properties was greater for PLA-Fe5Si5 compared to PLA-Fe10. MC3T3-E1 preosteoblasts seeded onto PG discs, PLA and PLA-PGF composites were evaluated for up to 7 days indicating that the materials were generally cytocompatible. In addition, cell alignment along the PGF

Retinal ganglion cells with distinct directional preferences differ in molecular identity, structure, and central projections.

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Kay, Jeremy N; De la Huerta, Irina; Kim, In-Jung; Zhang, Yifeng; Yamagata, Masahito; Chu, Monica W; Meister, Markus; Sanes, Joshua R

2011-05-25

The retina contains ganglion cells (RGCs) that respond selectively to objects moving in particular directions. Individual members of a group of ON-OFF direction-selective RGCs (ooDSGCs) detect stimuli moving in one of four directions: ventral, dorsal, nasal, or temporal. Despite this physiological diversity, little is known about subtype-specific differences in structure, molecular identity, and projections. To seek such differences, we characterized mouse transgenic lines that selectively mark ooDSGCs preferring ventral or nasal motion as well as a line that marks both ventral- and dorsal-preferring subsets. We then used the lines to identify cell surface molecules, including Cadherin 6, CollagenXXVα1, and Matrix metalloprotease 17, that are selectively expressed by distinct subsets of ooDSGCs. We also identify a neuropeptide, CART (cocaine- and amphetamine-regulated transcript), that distinguishes all ooDSGCs from other RGCs. Together, this panel of endogenous and transgenic markers distinguishes the four ooDSGC subsets. Patterns of molecular diversification occur before eye opening and are therefore experience independent. They may help to explain how the four subsets obtain distinct inputs. We also demonstrate differences among subsets in their dendritic patterns within the retina and their axonal projections to the brain. Differences in projections indicate that information about motion in different directions is sent to different destinations.

Aging of the skeletal muscle extracellular matrix drives a stem cell fibrogenic conversion.

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Stearns-Reider, Kristen M; D'Amore, Antonio; Beezhold, Kevin; Rothrauff, Benjamin; Cavalli, Loredana; Wagner, William R; Vorp, David A; Tsamis, Alkiviadis; Shinde, Sunita; Zhang, Changqing; Barchowsky, Aaron; Rando, Thomas A; Tuan, Rocky S; Ambrosio, Fabrisia

2017-06-01

Age-related declines in skeletal muscle regeneration have been attributed to muscle stem cell (MuSC) dysfunction. Aged MuSCs display a fibrogenic conversion, leading to fibrosis and impaired recovery after injury. Although studies have demonstrated the influence of in vitro substrate characteristics on stem cell fate, whether and how aging of the extracellular matrix (ECM) affects stem cell behavior has not been investigated. Here, we investigated the direct effect of the aged muscle ECM on MuSC lineage specification. Quantification of ECM topology and muscle mechanical properties reveals decreased collagen tortuosity and muscle stiffening with increasing age. Age-related ECM alterations directly disrupt MuSC responses, and MuSCs seeded ex vivo onto decellularized ECM constructs derived from aged muscle display increased expression of fibrogenic markers and decreased myogenicity, compared to MuSCs seeded onto young ECM. This fibrogenic conversion is recapitulated in vitro when MuSCs are seeded directly onto matrices elaborated by aged fibroblasts. When compared to young fibroblasts, fibroblasts isolated from aged muscle display increased nuclear levels of the mechanosensors, Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), consistent with exposure to a stiff microenvironment in vivo. Accordingly, preconditioning of young fibroblasts by seeding them onto a substrate engineered to mimic the stiffness of aged muscle increases YAP/TAZ nuclear translocation and promotes secretion of a matrix that favors MuSC fibrogenesis. The findings here suggest that an age-related increase in muscle stiffness drives YAP/TAZ-mediated pathogenic expression of matricellular proteins by fibroblasts, ultimately disrupting MuSC fate. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

The structure of cell-matrix adhesions: the new frontier.

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Hanein, Dorit; Horwitz, Alan Rick

2012-02-01

Adhesions between the cell and the extracellular matrix (ECM) are mechanosensitive multi-protein assemblies that transmit force across the cell membrane and regulate biochemical signals in response to the chemical and mechanical environment. These combined functions in force transduction, signaling and mechanosensing contribute to cellular phenotypes that span development, homeostasis and disease. These adhesions form, mature and disassemble in response to actin organization and physical forces that originate from endogenous myosin activity or external forces by the extracellular matrix. Despite advances in our understanding of the protein composition, interactions and regulation, our understanding of matrix adhesion structure and organization, how forces affect this organization, and how these changes dictate specific signaling events is limited. Insights across multiple structural levels are acutely needed to elucidate adhesion structure and ultimately the molecular basis of signaling and mechanotransduction. Here we describe the challenges and recent advances and prospects for unraveling the structure of cell-matrix adhesions and their response to force. Copyright © 2011 Elsevier Ltd. All rights reserved.

Self-Assembled Matrix by Umbilical Cord Stem Cells

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Biagio Saitta

2011-09-01

Full Text Available Corneal integrity is critical for vision. Corneal wounds frequently heal with scarring that impairs vision. Recently, human umbilical cord mesenchymal stem cells (cord stem cells have been investigated for tissue engineering and therapy due to their availability and differentiation potential. In this study, we used cord stem cells in a 3-dimensional (3D stroma-like model to observe extracellular matrix organization, with human corneal fibroblasts acting as a control. For 4 weeks, the cells were stimulated with a stable Vitamin C (VitC derivative ±TGF-b1. After 4 weeks, the mean thickness of the constructs was ~30 mm; however, cord stem cell constructs had 50% less cells per unit volume, indicating the formation of a dense matrix. We found minimal change in decorin and lumican mRNA, and a significant increase in perlecan mRNA in the presence of TGF-b1. Keratocan on the other hand decreased with TGF-b1 in both cell lineages. With both cell types, the constructs possessed aligned collagen fibrils and associated glycosaminoglycans. Fibril diameters did not change with TGF-b1 stimulation or cell lineage; however, highly sulfated glycosaminoglycans associated with the collagen fibrils significantly increased with TGF-b1. Overall, we have shown that cord stem cells can secrete their own extracellular matrix and promote the deposition and sulfation of various proteoglycans. Furthermore, these cells are at least comparable to commonly used corneal fibroblasts and present an alternative for the 3D in vitro tissue engineered model.

Delineation of in vitro chondrogenesis of human synovial stem cells following preconditioning using decellularized matrix

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Zhang, Ying; Li, Jingting; Davis, Mary E.; Pei, Ming

2015-01-01

As a tissue-specific stem cell for chondrogenesis, synovium-derived stem cells (SDSCs) are a promising cell source for cartilage repair. However, a small biopsy can only provide a limited number of cells. Cell senescence from both in vitro expansion and donor age presents a big challenge for stem cell based cartilage regeneration. Here we found that expansion on decellularized extracellular matrix (dECM) full of three-dimensional nanostructured fibers provided SDSCs with unique surface profiles, low elasticity but large volume as well as fibroblast-like shape. dECM expanded SDSCs yielded larger pellets with intensive staining of type II collagen and sulfated glycosaminoglycans compared to those grown on plastic flasks while SDSCs grown in ECM yielded 28-day pellets with minimal matrix as evidenced by pellet size and chondrogenic marker staining, which was confirmed by both biochemical data and real-time PCR data. Our results also found lower levels of inflammatory genes in dECM expanded SDSCs that might be responsible for enhanced chondrogenic differentiation. Despite an increase in type X collagen in chondrogenically induced cells, dECM expanded cells had significantly lower potential for endochondral bone formation. Wnt and MAPK signals were actively involved in both expansion and chondrogenic induction of dECM expanded cells. Since young and healthy people can be potential donors for this matrix expansion system and decellularization can minimize immune concerns, human SDSCs expanded on this future commercially available dECM could be a potential cell source for autologous cartilage repair. PMID:25861949

Extracellular-matrix-mediated osmotic pressure drives Vibrio cholerae biofilm expansion and cheater exclusion.

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Yan, Jing; Nadell, Carey D; Stone, Howard A; Wingreen, Ned S; Bassler, Bonnie L

2017-08-23

Biofilms, surface-attached communities of bacteria encased in an extracellular matrix, are a major mode of bacterial life. How the material properties of the matrix contribute to biofilm growth and robustness is largely unexplored, in particular in response to environmental perturbations such as changes in osmotic pressure. Here, using Vibrio cholerae as our model organism, we show that during active cell growth, matrix production enables biofilm-dwelling bacterial cells to establish an osmotic pressure difference between the biofilm and the external environment. This pressure difference promotes biofilm expansion on nutritious surfaces by physically swelling the colony, which enhances nutrient uptake, and enables matrix-producing cells to outcompete non-matrix-producing cheaters via physical exclusion. Osmotic pressure together with crosslinking of the matrix also controls the growth of submerged biofilms and their susceptibility to invasion by planktonic cells. As the basic physicochemical principles of matrix crosslinking and osmotic swelling are universal, our findings may have implications for other biofilm-forming bacterial species.Most bacteria live in biofilms, surface-attached communities encased in an extracellular matrix. Here, Yan et al. show that matrix production in Vibrio cholerae increases the osmotic pressure within the biofilm, promoting biofilm expansion and physical exclusion of non-matrix producing cheaters.

Nonleachable Imidazolium-Incorporated Composite for Disruption of Bacterial Clustering, Exopolysaccharide-Matrix Assembly, and Enhanced Biofilm Removal.

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Hwang, Geelsu; Koltisko, Bernard; Jin, Xiaoming; Koo, Hyun

2017-11-08

Surface-grown bacteria and production of an extracellular polymeric matrix modulate the assembly of highly cohesive and firmly attached biofilms, making them difficult to remove from solid surfaces. Inhibition of cell growth and inactivation of matrix-producing bacteria can impair biofilm formation and facilitate removal. Here, we developed a novel nonleachable antibacterial composite with potent antibiofilm activity by directly incorporating polymerizable imidazolium-containing resin (antibacterial resin with carbonate linkage; ABR-C) into a methacrylate-based scaffold (ABR-modified composite; ABR-MC) using an efficient yet simplified chemistry. Low-dose inclusion of imidazolium moiety (∼2 wt %) resulted in bioactivity with minimal cytotoxicity without compromising mechanical integrity of the restorative material. The antibiofilm properties of ABR-MC were assessed using an exopolysaccharide-matrix-producing (EPS-matrix-producing) oral pathogen (Streptococcus mutans) in an experimental biofilm model. Using high-resolution confocal fluorescence imaging and biophysical methods, we observed remarkable disruption of bacterial accumulation and defective 3D matrix structure on the surface of ABR-MC. Specifically, the antibacterial composite impaired the ability of S. mutans to form organized bacterial clusters on the surface, resulting in altered biofilm architecture with sparse cell accumulation and reduced amounts of EPS matrix (versus control composite). Biofilm topology analyses on the control composite revealed a highly organized and weblike EPS structure that tethers the bacterial clusters to each other and to the surface, forming a highly cohesive unit. In contrast, such a structured matrix was absent on the surface of ABR-MC with mostly sparse and amorphous EPS, indicating disruption in the biofilm physical stability. Consistent with lack of structural organization, the defective biofilm on the surface of ABR-MC was readily detached when subjected to low shear

Regulation of pituitary hormones and cell proliferation by components of the extracellular matrix

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M. Paez-Pereda

2005-10-01

Full Text Available The extracellular matrix is a three-dimensional network of proteins, glycosaminoglycans and other macromolecules. It has a structural support function as well as a role in cell adhesion, migration, proliferation, differentiation, and survival. The extracellular matrix conveys signals through membrane receptors called integrins and plays an important role in pituitary physiology and tumorigenesis. There is a differential expression of extracellular matrix components and integrins during the pituitary development in the embryo and during tumorigenesis in the adult. Different extracellular matrix components regulate adrenocorticotropin at the level of the proopiomelanocortin gene transcription. The extracellular matrix also controls the proliferation of adrenocorticotropin-secreting tumor cells. On the other hand, laminin regulates the production of prolactin. Laminin has a dynamic pattern of expression during prolactinoma development with lower levels in the early pituitary hyperplasia and a strong reduction in fully grown prolactinomas. Therefore, the expression of extracellular matrix components plays a role in pituitary tumorigenesis. On the other hand, the remodeling of the extracellular matrix affects pituitary cell proliferation. Matrix metalloproteinase activity is very high in all types of human pituitary adenomas. Matrix metalloproteinase secreted by pituitary cells can release growth factors from the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. In summary, the differential expression of extracellular matrix components, integrins and matrix metalloproteinase contributes to the control of pituitary hormone production and cell proliferation during tumorigenesis.

Direct integration of the S-matrix applied to rigorous diffraction

International Nuclear Information System (INIS)

Iff, W; Lindlein, N; Tishchenko, A V

2014-01-01

A novel Fourier method for rigorous diffraction computation at periodic structures is presented. The procedure is based on a differential equation for the S-matrix, which allows direct integration of the S-matrix blocks. This results in a new method in Fourier space, which can be considered as a numerically stable and well-parallelizable alternative to the conventional differential method based on T-matrix integration and subsequent conversions from the T-matrices to S-matrix blocks. Integration of the novel differential equation in implicit manner is expounded. The applicability of the new method is shown on the basis of 1D periodic structures. It is clear however, that the new technique can also be applied to arbitrary 2D periodic or periodized structures. The complexity of the new method is O(N 3 ) similar to the conventional differential method with N being the number of diffraction orders. (fast track communication)

Matrix rigidity regulates cancer cell growth and cellular phenotype.

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Robert W Tilghman

2010-09-01

Full Text Available The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness of the microenvironment and how this response varies among cancer cell lines.In this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: "rigidity dependent" (those which show an increase in cell growth as extracellular rigidity is increased, and "rigidity independent" (those which grow equally on both soft and stiff substrates. Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug.These observations suggest that the mechanical properties of the matrix environment play a significant role in regulating the proliferation and the morphological properties of cancer cells. Further, the multiwell format of the soft-plate assay is a useful and effective adjunct to established 3-dimensional cell culture models.

Matrix Rigidity Regulates Cancer Cell Growth and Cellular Phenotype

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Tilghman, Robert W.; Cowan, Catharine R.; Mih, Justin D.; Koryakina, Yulia; Gioeli, Daniel; Slack-Davis, Jill K.; Blackman, Brett R.; Tschumperlin, Daniel J.; Parsons, J. Thomas

2010-01-01

Background The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines. Methodology/Principal Findings In this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: “rigidity dependent” (those which show an increase in cell growth as extracellular rigidity is increased), and “rigidity independent” (those which grow equally on both soft and stiff substrates). Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug. Conclusions/Significance These observations suggest that the mechanical properties of the matrix environment play a significant role in regulating the proliferation and the morphological properties of cancer cells. Further, the multiwell format of the soft-plate assay is a useful and effective adjunct to established 3-dimensional cell culture models. PMID:20886123

Influence of Porcine Intervertebral Disc Matrix on Stem Cell Differentiation

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Hans-Lothar Fuchsbauer

2011-08-01

Full Text Available For back disorders, cell therapy is one approach for a real regeneration of a degenerated nucleus pulposus. Human mesenchymal stem cells (hMSC could be differentiated into nucleus pulposus (NP-like cells and used for cell therapy. Therefore it is necessary to find a suitable biocompatible matrix, which supports differentiation. It could be shown that a differentiation of hMSC in a microbial transglutaminase cross-linked gelatin matrix is possible, but resulted in a more chondrocyte-like cell type. The addition of porcine NP extract to the gelatin matrix caused a differentiation closer to the desired NP cell phenotype. This concludes that a hydrogel containing NP extract without any other supplements could be suitable for differentiation of hMSCs into NP cells. The NP extract itself can be cross-linked by transglutaminase to build a hydrogel free of NP atypical substrates. As shown by side-specific biotinylation, the NP extract contains molecules with free glutamine and lysine residues available for the transglutaminase.

Rapid method for direct identification of bacteria in urine and blood culture samples by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: intact cell vs. extraction method.

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Ferreira, L; Sánchez-Juanes, F; Muñoz-Bellido, J L; González-Buitrago, J M

2011-07-01

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast and reliable technology for the identification of microorganisms with proteomics approaches. Here, we compare an intact cell method and a protein extraction method before application on the MALDI plate for the direct identification of microorganisms in both urine and blood culture samples from clinical microbiology laboratories. The results show that the intact cell method provides excellent results for urine and is a good initial method for blood cultures. The extraction method complements the intact cell method, improving microorganism identification from blood culture. Thus, we consider that MALDI-TOF MS performed directly on urine and blood culture samples, with the protocols that we propose, is a suitable technique for microorganism identification, as compared with the routine methods used in the clinical microbiology laboratory. © 2010 The Authors. Clinical Microbiology and Infection © 2010 European Society of Clinical Microbiology and Infectious Diseases.

Surface Acoustic Waves Grant Superior Spatial Control of Cells Embedded in Hydrogel Fibers.

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Lata, James P; Guo, Feng; Guo, Jinshan; Huang, Po-Hsun; Yang, Jian; Huang, Tony Jun

2016-10-01

By exploiting surface acoustic waves and a coupling layer technique, cells are patterned within a photosensitive hydrogel fiber to mimic physiological cell arrangement in tissues. The aligned cell-polymer matrix is polymerized with short exposure to UV light and the fiber is extracted. These patterned cell fibers are manipulated into simple and complex architectures, demonstrating feasibility for tissue-engineering applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Aberrant DNA methylation of matrix remodeling and cell adhesion related genes in pterygium.

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Andri K Riau

Full Text Available BACKGROUND: Pterygium is a common ocular surface disease characterized by abnormal epithelial and fibrovascular proliferation, invasion, and matrix remodeling. This lesion, which migrates from the periphery to the center of the cornea, impairs vision and causes considerable irritation. The mechanism of pterygium formation remains ambiguous, and current treatment is solely surgical excision, with a significant risk of recurrence after surgery. Here, we investigate the role of methylation in DNA sequences that regulate matrix remodeling and cell adhesion in pterygium formation. METHODOLOGY/PRINCIPAL FINDINGS: Pterygium and uninvolved conjunctiva samples were obtained from the same eye of patients undergoing surgery. The EpiTYPER Sequenom technology, based on differential base cleavage and bisulfite sequencing was used to evaluate the extent of methylation of 29 matrix and adhesion related genes. In pterygium, three CpG sites at -268, -32 and -29 bp upstream of transglutaminase 2 (TGM-2 transcription initiation were significantly hypermethylated (p<0.05, whereas hypomethylation was detected at CpGs +484 and +602 bp downstream of matrix metalloproteinase 2 (MMP-2 transcription start site, and -809, -762, -631 and -629 bp upstream of the CD24 transcription start site. RT-qPCR, western blot and immunofluorescent staining showed that transcript and protein expression were reduced for TGM-2 and increased for MMP-2 and CD24. Inhibition of methylation in cultured conjunctival epithelial cells increased these transcripts. CONCLUSIONS/SIGNIFICANCE: We found regions of aberrant DNA methylation which were consistent with alteration of TGM-2, MMP-2, and CD24 transcript and protein expression, and that inhibition of methylation in cultured cells can increase the expression of these genes. Since these genes were related to cell adhesion and matrix remodeling, dysregulation may lead to fibroblastic and neovascular changes and pterygium formation. These results

Matrix-immobilized BMP-2 on microcontact printed fibronectin as in vitro tool to study BMP-mediated signaling and cell migration

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Kristin eHauff

2015-05-01

Full Text Available During development, bone morphogenetic proteins (BMPs exert important functions in several tissues by regulating signaling for cell differentiation and migration. In vivo the extracellular matrix (ECM not only provides a support for adherent cells, but also presents a reservoir of growth factors (GFs. Several constituents of the ECM provide adhesive cues, which serve as binding sites for cell transmembrane receptors, such as integrins, which convey adhesion-mediated signaling to the intracellular compartment. Integrins do not function alone but rather crosstalk and cooperate with other receptors, such as GF receptors, in regulating cell responses to extracellular signals. To this, we present here the immobilization of BMP-2 onto cellular fibronectin (cFN, a key protein of the ECM, to investigate their impact on GF-mediated signaling and migration.Following biotinylation, BMP-2 was linked to biotinylated cFN using NeutrAvidin (NA as cross-linker. Characterization with QCM-D and ELISA confirmed the efficient immobilization of BMP-2 on cFN over a period of 24 h.To validate the bioactivity of matrix-immobilized BMP-2 (iBMP-2 we investigated short- and long-term responses of C2C12 myoblasts in comparison to soluble BMP-2 (sBMP-2 or in absence of GFs. Similarly to sBMP-2, iBMP-2 triggered Smad 1/5 phosphorylation and translocation into the nucleus corresponding to the activation of BMP-mediated Smad-dependent pathway. Additionally, successful suppression of myotube formation was observed after six days.We next implemented this approach to fabricate cFN micro patterned stripes by soft lithography. These stripes only allowed cell-surface interaction on the pattern due to passivation of the surface in between, thus serving as platform for studies on directed cell migration. During a 10 h-period, cells showed an increased migratory activity upon BMP-2 exposure.Thus, this versatile tool retains the GF's bioactivity and allows the presentation of ECM

An immunofluorescence assay for extracellular matrix components highlights the role of epithelial cells in producing a stable, fibrillar extracellular matrix

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Omar S. Qureshi

2017-10-01

Full Text Available Activated fibroblasts are considered major drivers of fibrotic disease progression through the production of excessive extracellular matrix (ECM in response to signals from damaged epithelial and inflammatory cells. Nevertheless, epithelial cells are capable of expressing components of the ECM, cross-linking enzymes that increase its stability and are sensitive to factors involved in the early stages of fibrosis. We therefore wanted to test the hypothesis that epithelial cells can deposit ECM in response to stimulation in a comparable manner to fibroblasts. We performed immunofluorescence analysis of components of stable, mature extracellular matrix produced by primary human renal proximal tubular epithelial cells and renal fibroblasts in response to cytokine stimulation. Whilst fibroblasts produced a higher basal level of extracellular matrix components, epithelial cells were able to deposit significant levels of fibronectin, collagen I, III and IV in response to cytokine stimulation. In response to hypoxia, epithelial cells showed an increase in collagen IV deposition but not in response to the acute stress stimuli aristolochic acid or hydrogen peroxide. When epithelial cells were in co-culture with fibroblasts we observed significant increases in the level of matrix deposition which could be reduced by transforming growth factor beta (TGF-β blockade. Our results highlight the role of epithelial cells acting as efficient producers of stable extracellular matrix which could contribute to renal tubule thickening in fibrosis.

Cell adhesion-mediated radioresistance (CAM-RR). Extracellular matrix-dependent improvement of cell survival in human tumor and normal cells in vitro

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Cordes, N.; Meineke, V.

2003-01-01

Background: Cell-extracellular matrix (ECM) contact is thought to have great impact on cellular mechanisms resulting in increased cell survival upon exposure to ionizing radiation. Several human tumor cell lines and normal human fibroblastic cell strains of different origin, all of them expressing the wide-spread and important integrin subunit β1, were irradiated, and clonogenic cell survival, β1-integrin cell surface expression, and adhesive functionality were investigated. Material and Methods: Human tumor cell lines A172 (glioblastoma), PATU8902 (pancreas carcinoma), SKMES1 (lung carcinoma), A549 (lung carcinoma), and IPC298 (melanoma) as well as normal human skin (HSF1) and lung fibroblasts (CCD32) and human keratinocytes (HaCaT) were irradiated with 0-8 Gy. Besides colony formation assays, β1-integrin cell surface expression by flow cytometry and adhesive functionality by adhesion assays were analyzed. Results: All cell lines showed improved clonogenic survival after irradiation in the presence of fibronectin as compared to plastic. Irradiated cells exhibited a significant, dose-dependent increase in β1-integrin cell surface expression following irradiation. As a parameter of the adhesive functionality of the β1-integrin, a radiation-dependent elevation of cell adhesion to fibronectin in comparison with adhesion to plastic was demonstrated. Conclusion: The in vitro cellular radiosensitivity is highly influenced by fibronectin according to the phenomenon of cell adhesion-mediated radioresistance. Additionally, our emerging data question the results of former and current in vitro cytotoxicity studies performed in the absence of an ECM. These findings might also be important for the understanding of malignant transformation, anchorage-independent cell growth, optimization of radiotherapeutic regimes and the prevention of normal tissue side effects on the basis of experimental radiobiological data. (orig.)

Patterned layers of adsorbed extracellular matrix proteins: influence on mammalian cell adhesion.

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Dupont-Gillain, C C; Alaerts, J A; Dewez, J L; Rouxhet, P G

2004-01-01

Three patterned systems aiming at the control of mammalian cell behavior are presented. The determinant feature common to these systems is the spatial distribution of extracellular matrix (ECM) proteins (mainly collagen) on polymer substrates. This distribution differs from one system to another with respect to the scale at which it is affected, from the supracellular to the supramolecular scale, and with respect to the way it is produced. In the first system, the surface of polystyrene was oxidized selectively to form micrometer-scale patterns, using photolithography. Adsorption of ECM proteins in presence of a competitor was enhanced on the oxidized domains, allowing selective cell adhesion to be achieved. In the second system, electron beam lithography was used to engrave grooves (depth and width approximately 1 microm) on a poly(methyl methacrylate) (PMMA) substratum. No modification of the surface chemistry associated to the created topography could be detected. Cell orientation along the grooves was only observed when collagen was preadsorbed on the substratum. In the third system, collagen adsorbed on PMMA was dried in conditions ensuring the formation of a nanometer-scale pattern. Cell adhesion was enhanced on such patterned collagen layers compared to smooth collagen layers.

Hydroxyapatite growth induced by native extracellular matrix deposition on solid surfaces

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Pramatarova L.

2005-02-01

Full Text Available Biological systems have a remarkable capability to produce perfect fine structures such as seashells, pearls, bones, teeth and corals. These structures are composites of interacting inorganic (calcium phosphate or carbonate minerals and organic counterparts. It is difficult to say with certainty which part has the primary role. For example, the growth of molluscan shell crystals is thought to be initiated from a solution by the extracellular organic matrix (ECM. According to this theory, the matrix induces nucleation of calcium containing crystals. Recently, an alternative theory has been put forward, stating that a class of granulocytic hemocytes would be directly involved in shell crystal production in oysters. In the work presented here the surface of AISI 316 stainless steel was modified by deposition of ECM proteins. The ability of the modified substrates to induce nucleation and growth of hydroxyapatite (HA from simulated body fluid (SBF was examined by a kinetic study using two methods: (1 a simple soaking process in SBF and (2 a laser-liquid-solid interaction (LLSI process which allows interaction between a scanning laser beam and a solid substrate immersed in SBF. The deposited HA layers were investigated by Fourier transform infrared spectroscopy (FTIR and scanning electron microscopy (SEM. It was found that a coating of stainless steel surface with native ECM proteins induced nucleation and growth of HA and facilitated its crystallization. By the process of simple soaking of the samples, irrespective of their horizontal or vertical position in the solution, HA layers were grown due to the reactive ECM-coated stainless steel surface. It was shown that the process occurring in the first stages of the growth was not only a result of the force of gravity. The application of the LLSI process strongly influenced HA formation on the ECM-modified substrates by promoting and enhancing the HA nucleation and growth through a synergistic effect

Matrix metalloproteinase 9 (MMP-9) mediated release of MMP-9 resistant stromal cell-derived factor 1α (SDF-1α) from surface modified polymer films.

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Steinhagen, Max; Hoffmeister, Peter-Georg; Nordsieck, Karoline; Hötzel, Rudi; Baumann, Lars; Hacker, Michael C; Schulz-Siegmund, Michaela; Beck-Sickinger, Annette G

2014-04-23

Preparation of smart materials by coatings of established surfaces with biomolecules will lead to the next generation of functionalized biomaterials. Rejection of implants is still a major problem in medical applications but masking the implant material with protein coatings is a promising approach. These layers not only disguise the material but also equip it with a certain biological function. The anti-inflammatory chemokine stromal cell-derived factor 1α (SDF-1α) is well suited to take over this function, because it efficiently attracts stem cells and promotes their differentiation and proliferation. At least the initial stem cell homing requires the formation of a concentration gradient. Thus, a reliable and robust release mechanism of SDF-1α from the material is essential. Several proteases, most notably matrix metalloproteinases, are upregulated during inflammation, which, in principle, can be exploited for a tightly controlled release of SDF-1α. Herein, we present the covalent immobilization of M-[S4V]-SDF-1α on novel biodegradable polymer films, which consist of heterobifunctional poly(ethylene glycol) and oligolactide-based functionalized macromers. A peptidic linker with a trimeric matrix metalloproteinase 9 (MMP-9) cleavage site (MCS) was used as connection and the linkage between the three components was achieved by combination of expressed protein ligation and Cu(I) catalyzed azide/alkyne cycloaddition. The MCS was used for MMP-9 mediated release of M-[S4V]-SDF-1α from the biomaterial and the released SDF-1α derivative was biologically active and induced strong cell migration, which demonstrates the great potential of this system.

Biomedical Application of Low Molecular Weight Heparin/Protamine Nano/Micro Particles as Cell- and Growth Factor-Carriers and Coating Matrix

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Masayuki Ishihara

2015-05-01

Full Text Available Low molecular weight heparin (LMWH/protamine (P nano/micro particles (N/MPs (LMWH/P N/MPs were applied as carriers for heparin-binding growth factors (GFs and for adhesive cells including adipose-derived stromal cells (ADSCs and bone marrow-derived mesenchymal stem cells (BMSCs. A mixture of LMWH and P yields a dispersion of N/MPs (100 nm–3 μm in diameter. LMWH/P N/MPs can be immobilized onto cell surfaces or extracellular matrix, control the release, activate GFs and protect various GFs. Furthermore, LMWH/P N/MPs can also bind to adhesive cell surfaces, inducing cells and LMWH/P N/MPs-aggregate formation. Those aggregates substantially promoted cellular viability, and induced vascularization and fibrous tissue formation in vivo. The LMWH/P N/MPs, in combination with ADSCs or BMSCs, are effective cell-carriers and are potential promising novel therapeutic agents for inducing vascularization and fibrous tissue formation in ischemic disease by transplantation of the ADSCs and LMWH/P N/MPs-aggregates. LMWH/P N/MPs can also bind to tissue culture plates and adsorb exogenous GFs or GFs from those cells. The LMWH/P N/MPs-coated matrix in the presence of GFs may provide novel biomaterials that can control cellular activity such as growth and differentiation. Furthermore, three-dimensional (3D cultures of cells including ADSCs and BMSCs using plasma-medium gel with LMWH/P N/MPs exhibited efficient cell proliferation. Thus, LMWH/P N/MPs are an adequate carrier both for GFs and for stromal cells such as ADSCs and BMSCs, and are a functional coating matrix for their cultures.

A cellular Potts model for the MMP-dependent and -independent cancer cell migration in matrix microtracks of different dimensions

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Scianna, Marco; Preziosi, Luigi

2014-03-01

Cell migration is fundamental in a wide variety of physiological and pathological phenomena, among other in cancer invasion and development. In particular, the migratory/invasive capability of single metastatic cells is fundamental in determining the malignancy of a solid tumor. Specific cell migration phenotypes result for instance from the reciprocal interplay between the biophysical and biochemical properties of both the malignant cells themselves and of the surrounding environment. In particular, the extracellular matrices (ECMs) forming connective tissues can provide both loosely organized zones and densely packed barriers, which may impact cell invasion mode and efficiency. The critical processes involved in cell movement within confined spaces are (i) the proteolytic activity of matrix metalloproteinases (MMPs) and (ii) the deformation of the entire cell body, and in particular of the nucleus. We here present an extended cellular Potts model (CPM) to simulate a bio-engineered matrix system, which tests the active motile behavior of a single cancer cell into narrow channels of different widths. As distinct features of our approach, the cell is modeled as a compartmentalized discrete element, differentiated in the nucleus and in the cytosolic region, while a directional shape-dependent movement is explicitly driven by the evolution of its polarity vector. As outcomes, we find that, in a large track, the tumor cell is not able to maintain a directional movement. On the contrary, a structure of subcellular width behaves as a contact guidance sustaining cell persistent locomotion. In particular, a MMP-deprived cell is able to repolarize and follow the micropattern geometry, while a full MMP activity leads to a secondary track expansion by degrading the matrix structure. Finally, we confirm that cell movement within a subnuclear structure can be achieved either by pericellular proteolysis or by a significant deformation of cell nucleus.

Platelet-tumor cell interaction with the subendothelial extracellular matrix: relationship to cancer metastasis

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Yahalom, J; Biran, S; Fuks, Z; Vlodavsky, I [Hadassah University Hospital, Jerusalem (Israel). Dept. of Radiation and Clinical Oncology; Eldor, A [Hadassah University Hospital, Jerusalem (Israel). Dept. of Hematology

1985-04-01

Dissemination of neoplastic cells within the body involves invasion of blood vessels by tumor cells. This requires adhesion of blood-borne cells to the luminal surface of the vascular endothelium, invasion through the endothelial cell layer and local dissolution of the subendothelial basement membrane. The authors studied the interaction of platelets and tumor cells with cultured vascular endothelial cells and their secreted basement membrane-like extracellular matrix (ECM). Interaction of platelets with this ECM was associated with platelet activation, aggregation and degradation of heparan sulfate in the ECM by means of the platelet heparitinase. Biochemical and scanning electron microscopy (SEM) studies have demonstrated that platelets may detect even minor gaps between adjacent endothelial cells and degrade the ECM heparan sulfate. Platelets were also shown to recruit lymphoma cells into minor gaps in the vascular endothelium. It is suggested that the platelet heparitinase is involved in the impairment of the integrity of the vessel wall and thus play a role in tumor cell metastasis.

Cell adhesion control by ion implantation into extra-cellular matrix

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Suzuki, Yoshiaki; Kusakabe, Masahiro; Kaibara, Makoto; Iwaki, Masaya; Sasabe, Hiroyuki; Nishisaka, Tsuyoshi

1994-01-01

Cell adhesion control of polymer surfaces by ion implantation into polymers and extra-cellular matrix has been studied by means of in vitro adhesion measurements of the carcinoma of the cervix (HeLa cell). The specimens used were polystyrene (PS), oxygen plasma treated polystyrene (PS-O), extra-cellular matrix (Collagen: Type I) coated polystyrene (PS-C), and gelatin coated polystyrene (PS-G). Ne + , Na + , and Ar + implantations were performed with a fluence of 1x10 15 ions/cm 2 at energies of 50, 100 and 150 keV. The chemical and physical structures of ion implanted specimens have been investigated by Fourier transform infrared spectroscopy (FT-IR-ATR), X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy. Ion implanted PS demonstrated a dramatic improvement of adhesion of HeLa cell. HeLa cell adhered only to ion implanted circular domains of a diameter about 0.1 mm on PS. By contrast, ion implanted PS-C, PS-G and PS-O domains inhibited the cell adhesion. These phenomena were observed on Ne + , Na + , and Ar + implanted specimens at energies of 50, 100, and 150 keV. Ion implantation broke the original chemical bonds to form new radicals such as =C=O, condensed rings, C-C, C-O and OH radical. Ion implanted PS had a large amount of new radicals compared with that of PS-C, PS-G and PS-O. Ion implantation broke NH and NH 3 bonds originating from amino acid in PS-C and PS-G. OH and =C=O caused by oxygen treatment in PS-O were also destroyed by ion implantation. It is concluded that cell adhesion to ion implanted PS was caused by carbon structure and new radicals induced by ion implantation. The inhibition of HeLa cell adhesion on PS-C, PS-G and PS-O was caused by the destruction of cell adhesion properties of amino acid, OH and =C=O by radiation effects. ((orig.))

The role of extracellular matrix metalloproteinase inducer (EMMPRIN) in the regulation of bovine endometrial cell functions.

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Mishra, Birendra; Kizaki, Keiichiro; Sato, Takashi; Ito, Akira; Hashizume, Kazuyoshi

2012-06-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell surface glycoprotein that stimulates the production of several matrix metalloproteinases (MMPs) for tissue remodeling. Previously, we detected EMMPRIN in the bovine endometrium, and it is mainly expressed in the luminal and glandular epithelium whereas MMPs are expressed in the underlying stroma. From this expression pattern, we hypothesized that EMMPRIN may regulate stromal MMPs in endometrial cell functions. To test this hypothesis, a coculture of epithelial and stromal cells was performed using a transwell system. In the coculture, epithelial cells were cultured on the insert membrane and stromal cell on the surface of well plates. Expression of stromal MMP-2 and MMP-14 was significantly higher in coculture with epithelial cell. Further, with the addition of anti-EMMPRIN antibody into the epithelial cell compartment, the expression of stromal EMMPRIN and MMP-2 and MMP-14 was decreased. To identify the active site of EMMPRIN for the augmentation of MMPs, EMMPRIN synthetic peptides that correspond to the extracellular loop domain-I (EM1, EM2, EM3, and EM4) were added into the epithelial cell compartment, and only EM2 at a higher dose interfered with EMMPRIN-mediated expression of MMP-14. Next, we examined the effects of progesterone and/or estrogen on the expression of EMMPRIN, MMP-2, and MMP-14. Progesterone (300 nM) significantly stimulated the expression of EMMPRIN but had no effects on any of the MMPs. These results suggest that EMMPRIN derived from epithelial cells regulates MMPs in the endometrium under progesterone-rich conditions and may thereby modulate bovine endometrial cell functions during gestation.

Cell surface engineering of industrial microorganisms for biorefining applications.

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Tanaka, Tsutomu; Kondo, Akihiko

2015-11-15

In order to decrease carbon emissions and negative environmental impacts of various pollutants, biofuel/biochemical production should be promoted for replacing fossil-based industrial processes. Utilization of abundant lignocellulosic biomass as a feedstock has recently become an attractive option. In this review, we focus on recent efforts of cell surface display using industrial microorganisms such as Escherichia coli and yeast. Cell surface display is used primarily for endowing cellulolytic activity on the host cells, and enables direct fermentation to generate useful fuels and chemicals from lignocellulosic biomass. Cell surface display systems are systematically summarized, and the drawbacks/perspectives as well as successful application of surface display for industrial biotechnology are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

Illustration of extensive extracellular matrix at the epithelial-mesenchymal interface within the renal stem/progenitor cell niche

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Minuth Will W

2012-09-01

Full Text Available Abstract Background Stem/progenitor cells are promising candidates to treat diseased renal parenchyma. However, implanted stem/progenitor cells are exposed to a harmful atmosphere of degenerating parenchyma. To minimize hampering effects after an implantation investigations are in progress to administer these cells within an artificial polyester interstitum supporting survival. Learning from nature the renal stem/progenitor cell niche appears as a valuable model. At this site epithelial stem/progenitor cells within the collecting duct ampulla face mesenchymal stem/progenitor cells. Both cell types do not have close contact but are separated by a wide interstitium. Methods To analyze extracellular matrix in this particular interstitium, special contrasting for transmission electron microscopy was performed. Kidneys of neonatal rabbits were fixed in solutions containing glutaraldehyde (GA or in combination with cupromeronic blue, ruthenium red and tannic acid. Results GA revealed a basal lamina at the ampulla and a bright but inconspicuously looking interstitial space. In contrast, GA containing cupromeronic blue exhibits numerous proteoglycan braces lining from the ampulla towards the interstitial space. GA containing ruthenium red or tannic acid demonstrates clouds of extracellular matrix protruding from the basal lamina of the ampulla to the surface of mesenchymal stem/progenitor cells. Conclusions The actual data show that the interstitium between epithelial and mesenchymal stem/progenitor cells contains much more and up to date unknown extracellular matrix than earlier observed by classical GA fixation.

Synergistic interactions of blood-borne immune cells, fibroblasts and extracellular matrix drive repair in an in vitro peri-implant wound healing model

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Burkhardt, Melanie A.; Waser, Jasmin; Milleret, Vincent; Gerber, Isabel; Emmert, Maximilian Y.; Foolen, Jasper; Hoerstrup, Simon P.; Schlottig, Falko; Vogel, Viola

2016-02-01

Low correlations of cell culture data with clinical outcomes pose major medical challenges with costly consequences. While the majority of biomaterials are tested using in vitro cell monocultures, the importance of synergistic interactions between different cell types on paracrine signalling has recently been highlighted. In this proof-of-concept study, we asked whether the first contact of surfaces with whole human blood could steer the tissue healing response. This hypothesis was tested using alkali-treatment of rough titanium (Ti) surfaces since they have clinically been shown to improve early implant integration and stability, yet blood-free in vitro cell cultures poorly correlated with in vivo tissue healing. We show that alkali-treatment, compared to native Ti surfaces, increased blood clot thickness, including platelet adhesion. Strikingly, blood clots with entrapped blood cells in synergistic interactions with fibroblasts, but not fibroblasts alone, upregulated the secretion of major factors associated with fast healing. This includes matrix metalloproteinases (MMPs) to break down extracellular matrix and the growth factor VEGF, known for its angiogenic potential. Consequently, in vitro test platforms, which consider whole blood-implant interactions, might be superior in predicting wound healing in response to biomaterial properties.

Matrix production and organization by endothelial colony forming cells in mechanically strained engineered tissue constructs.

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Nicky de Jonge

Full Text Available AIMS: Tissue engineering is an innovative method to restore cardiovascular tissue function by implanting either an in vitro cultured tissue or a degradable, mechanically functional scaffold that gradually transforms into a living neo-tissue by recruiting tissue forming cells at the site of implantation. Circulating endothelial colony forming cells (ECFCs are capable of differentiating into endothelial cells as well as a mesenchymal ECM-producing phenotype, undergoing Endothelial-to-Mesenchymal-transition (EndoMT. We investigated the potential of ECFCs to produce and organize ECM under the influence of static and cyclic mechanical strain, as well as stimulation with transforming growth factor β1 (TGFβ1. METHODS AND RESULTS: A fibrin-based 3D tissue model was used to simulate neo-tissue formation. Extracellular matrix organization was monitored using confocal laser-scanning microscopy. ECFCs produced collagen and also elastin, but did not form an organized matrix, except when cultured with TGFβ1 under static strain. Here, collagen was aligned more parallel to the strain direction, similar to Human Vena Saphena Cell-seeded controls. Priming ECFC with TGFβ1 before exposing them to strain led to more homogenous matrix production. CONCLUSIONS: Biochemical and mechanical cues can induce extracellular matrix formation by ECFCs in tissue models that mimic early tissue formation. Our findings suggest that priming with bioactives may be required to optimize neo-tissue development with ECFCs and has important consequences for the timing of stimuli applied to scaffold designs for both in vitro and in situ cardiovascular tissue engineering. The results obtained with ECFCs differ from those obtained with other cell sources, such as vena saphena-derived myofibroblasts, underlining the need for experimental models like ours to test novel cell sources for cardiovascular tissue engineering.

CD147-targeting siRNA inhibits cell-matrix adhesion of human malignant melanoma cells by phosphorylating focal adhesion kinase.

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Nishibaba, Rie; Higashi, Yuko; Su, Juan; Furukawa, Tatsuhiko; Kawai, Kazuhiro; Kanekura, Takuro

2012-01-01

CD147/basigin, highly expressed on the surface of malignant tumor cells including malignant melanoma (MM) cells, plays a critical role in the invasiveness and metastasis of MM. Metastasis is an orchestrated process comprised of multiple steps including adhesion and invasion. Integrin, a major adhesion molecule, co-localizes with CD147/basigin on the cell surface. Using the human MM cell line A375 that highly expresses CD147/basigin, we investigated whether CD147/basigin is involved in adhesion in association with integrin. CD147/basigin was knocked-down using siRNA targeting CD147 to elucidate the role of CD147/basigin. Cell adhesion was evaluated by adhesion assay on matrix-coated plates. The localization of integrin was inspected under a confocal microscope and the expression and phosphorylation of focal adhesion kinase (FAK), a downstream kinase of integrin, were examined by western blot analysis. Silencing of CD147/basigin in A375 cells by siRNA induced the phosphorylation of FAK at Y397. Integrin identified on the surface of parental cells was distributed in a speckled fashion in the cytoplasm of CD147 knockdown cells, resulting in morphological changes from a round to a polygonal shape with pseudopodial protrusions. Silencing of CD147/basigin in A375 cells clearly weakened their adhesiveness to collagen I and IV. Our results suggest that CD147/basigin regulates the adhesion of MM cells to extracellular matrices and of integrin β1 signaling via the phosphorylation of FAK. © 2011 Japanese Dermatological Association.

Endocytosis of collagen by hepatic stellate cells regulates extracellular matrix dynamics.

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Bi, Yan; Mukhopadhyay, Dhriti; Drinane, Mary; Ji, Baoan; Li, Xing; Cao, Sheng; Shah, Vijay H

2014-10-01

Hepatic stellate cells (HSCs) generate matrix, which in turn may also regulate HSCs function during liver fibrosis. We hypothesized that HSCs may endocytose matrix proteins to sense and respond to changes in microenvironment. Primary human HSCs, LX2, or mouse embryonic fibroblasts (MEFs) [wild-type; c-abl(-/-); or Yes, Src, and Fyn knockout mice (YSF(-/-))] were incubated with fluorescent-labeled collagen or gelatin. Fluorescence-activated cell sorting analysis and confocal microscopy were used for measuring cellular internalization of matrix proteins. Targeted PCR array and quantitative real-time PCR were used to evaluate gene expression changes. HSCs and LX2 cells endocytose collagens in a concentration- and time-dependent manner. Endocytosed collagen colocalized with Dextran 10K, a marker of macropinocytosis, and 5-ethylisopropyl amiloride, an inhibitor of macropinocytosis, reduced collagen internalization by 46%. Cytochalasin D and ML7 blocked collagen internalization by 47% and 45%, respectively, indicating that actin and myosin are critical for collagen endocytosis. Wortmannin and AKT inhibitor blocked collagen internalization by 70% and 89%, respectively, indicating that matrix macropinocytosis requires phosphoinositide-3-kinase (PI3K)/AKT signaling. Overexpression of dominant-negative dynamin-2 K44A blocked matrix internalization by 77%, indicating a role for dynamin-2 in matrix macropinocytosis. Whereas c-abl(-/-) MEF showed impaired matrix endocytosis, YSF(-/-) MEF surprisingly showed increased matrix endocytosis. It was also associated with complex gene regulations that related with matrix dynamics, including increased matrix metalloproteinase 9 (MMP-9) mRNA levels and zymographic activity. HSCs endocytose matrix proteins through macropinocytosis that requires a signaling network composed of PI3K/AKT, dynamin-2, and c-abl. Interaction with extracellular matrix regulates matrix dynamics through modulating multiple gene expressions including MMP-9

Extracellular matrix collagen alters cell proliferation and cell cycle progression of human uterine leiomyoma smooth muscle cells.

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Koohestani, Faezeh; Braundmeier, Andrea G; Mahdian, Arash; Seo, Jane; Bi, JiaJia; Nowak, Romana A

2013-01-01

Uterine leiomyomas (ULs) are benign tumors occurring in the majority of reproductive aged women. Despite the high prevalence of these tumors, little is known about their etiology. A hallmark of ULs is the excessive deposition of extracellular matrix (ECM), primarily collagens. Collagens are known to modulate cell behavior and function singularly or through interactions with integrins and growth factor-mediated mitogenic pathways. To better understand the pathogenesis of ULs and the role of ECM collagens in their growth, we investigated the interaction of leiomyoma smooth muscle cells (LSMCs) with two different forms of collagen, non-polymerized collagen (monomeric) and polymerized collagen (fibrillar), in the absence or presence of platelet-derived growth factor (PDGF), an abundant growth factor in ULs. Primary cultures of human LSMCS from symptomatic patients were grown on these two different collagen matrices and their morphology, cytoskeletal organization, cellular proliferation, and signaling pathways were evaluated. Our results showed that LSMCs had distinct morphologies on the different collagen matrices and their basal as well as PDGF-stimulated proliferation varied on these matrices. These differences in proliferation were accompanied by changes in cell cycle progression and p21, an inhibitory cell cycle protein. In addition we found alterations in the phosphorylation of focal adhesion kinase, cytoskeletal reorganization, and activation of the mitogen activated protein kinase (MAPK) signaling pathway. In conclusion, our results demonstrate a direct effect of ECM on the proliferation of LSMCs through interplay between the collagen matrix and the PDGF-stimulated MAPK pathway. In addition, these findings will pave the way for identifying novel therapeutic approaches for ULs that target ECM proteins and their signaling pathways in ULs.

Laser surface modification of decellularized extracellular cartilage matrix for cartilage tissue engineering.

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Goldberg-Bockhorn, Eva; Schwarz, Silke; Subedi, Rachana; Elsässer, Alexander; Riepl, Ricarda; Walther, Paul; Körber, Ludwig; Breiter, Roman; Stock, Karl; Rotter, Nicole

2018-02-01

The implantation of autologous cartilage as the gold standard operative procedure for the reconstruction of cartilage defects in the head and neck region unfortunately implicates a variety of negative effects at the donor site. Tissue-engineered cartilage appears to be a promising alternative. However, due to the complex requirements, the optimal material is yet to be determined. As demonstrated previously, decellularized porcine cartilage (DECM) might be a good option to engineer vital cartilage. As the dense structure of DECM limits cellular infiltration, we investigated surface modifications of the scaffolds by carbon dioxide (CO 2 ) and Er:YAG laser application to facilitate the migration of chondrocytes inside the scaffold. After laser treatment, the scaffolds were seeded with human nasal septal chondrocytes and analyzed with respect to cell migration and formation of new extracellular matrix proteins. Histology, immunohistochemistry, SEM, and TEM examination revealed an increase of the scaffolds' surface area with proliferation of cell numbers on the scaffolds for both laser types. The lack of cytotoxic effects was demonstrated by standard cytotoxicity testing. However, a thermal denaturation area seemed to hinder the migration of the chondrocytes inside the scaffolds, even more so after CO 2 laser treatment. Therefore, the Er:YAG laser seemed to be better suitable. Further modifications of the laser adjustments or the use of alternative laser systems might be advantageous for surface enlargement and to facilitate migration of chondrocytes into the scaffold in one step.

Functional dynamics of cell surface membrane proteins.

Science.gov (United States)

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

Identification of changes in the inorganic matrix of dye sensitized solar cells during preparation

Energy Technology Data Exchange (ETDEWEB)

Graaf, Harald; Maedler, Carsten; Kehr, Mirko; Baumgaertel, Thomas [Institute of Physics, Chemnitz University of Technology, Reichenhainer Str. 70, 09126 Chemnitz (Germany); Oekermann, Torsten [Institute of Physical Chemistry and Electrochemistry, Leibniz University Hannover, 30167 Hannover (Germany)

2009-12-15

Nanoporous zinc oxide films can be prepared by electrochemical co-deposition with the dye eosin Y (EY) as template. A subsequent desorption of the dye with aqueous KOH is a key step for the increased efficiency of such dye sensitized solar cells (DSSC). In this contribution, the partial dissolving and reorganization of the zinc oxide film during the desorption step has been studied in detail by X-ray diffraction, Kelvin probe force microscopy, and atomic force microscopy (AFM) as well as scanning electron microscopy. It is found that the reorganization leads to an enhancement of crystal orientation and a reduction of the defect concentration at the surface of zinc oxide, being a possible reason for suppressed recombination of electrons in these films. Also, an increased defect concentration in ZnO was found when co-deposited with EY. For these co-deposited films, AFM investigations showed a coverage of the surface with dye aggregates. High resolution AFM investigations revealed the nanocrystalline and highly porous structure of the inorganic matrix after desorption of the dye. Photographs of the organic/inorganic hybrid thin film material were investigated in this study. Left side: film directly after deposition, right side: after desorption of the organic dye. (Abstract Copyright [2009], Wiley Periodicals, Inc.)

Oxidation behaviour of cast aluminium matrix composites with Ce surface coatings

International Nuclear Information System (INIS)

Pardo, A.; Merino, M.C.; Arrabal, R.; Feliu, S.; Viejo, F.

2007-01-01

The oxidation behaviour of SiC-reinforced aluminium matrix composites (A3xx.x/SiCp) has been studied after Ce-based treatments. Kinetics data of oxidation process were obtained from gravimetric tests performed at different temperatures (350, 425 and 500 o C). The nature of the oxidation layer was analyzed by scanning electron and atomic force microscopy, energy dispersive X-ray analysis, X-ray photoelectron spectroscopy and X-ray diffraction. The extent of oxidation degradation in untreated composites was preferentially localized in matrix/SiCp interfaces favouring the MgO formation. Ce coatings favoured a uniform oxidation of the composite surface with MgAl 2 O 4 spinel formation. This oxide increased the surface hardness of the materials

Measuring methods of matrix diffusion

International Nuclear Information System (INIS)

Muurinen, A.; Valkiainen, M.

1988-03-01

In Finland the spent nuclear fuel is planned to be disposed of at large depths in crystalline bedrock. The radionuclides which are dissolved in the groundwater may be able to diffuse into the micropores of the porous rock matrix and thus be withdrawn from the flowing water in the fractures. This phenomenon is called matrix diffusion. A review over matrix diffusion is presented in the study. The main interest is directed to the diffusion of non-sorbing species. The review covers diffusion experiments and measurements of porosity, pore size, specific surface area and water permeability

Fibroblast Cluster Formation on 3D Collagen Matrices Requires Cell Contraction-Dependent Fibronectin Matrix Organization

Science.gov (United States)

da Rocha-Azevedo, Bruno; Ho, Chin-Han; Grinnell, Frederick

2012-01-01

Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell-matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy. PMID:23117111

Fibroblast cluster formation on 3D collagen matrices requires cell contraction dependent fibronectin matrix organization.

Science.gov (United States)

da Rocha-Azevedo, Bruno; Ho, Chin-Han; Grinnell, Frederick

2013-02-15

Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin (FN) fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell-matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy. Copyright © 2012 Elsevier Inc. All rights reserved.

Coating extracellular matrix proteins on a (3-aminopropyl)triethoxysilane-treated glass substrate for improved cell culture.

Science.gov (United States)

Masuda, Hiro-taka; Ishihara, Seiichiro; Harada, Ichiro; Mizutani, Takeomi; Ishikawa, Masayori; Kawabata, Kazushige; Haga, Hisashi

2014-01-01

We demonstrate that a (3-aminopropyl)triethoxysilane-treated glass surface is superior to an untreated glass surface for coating with extracellular matrix (ECM) proteins when used as a cell culture substrate to observe cell physiology and behavior. We found that MDCK cells cultured on untreated glass coated with ECM removed the coated ECM protein and secreted different ECM proteins. In contrast, the cells did not remove the coated ECM protein when seeded on (3-aminopropyl)triethoxysilane-treated (i.e., silanized) glass coated with ECM. Furthermore, the morphology and motility of cells grown on silanized glass differed from those grown on non-treated glass, even when both types of glass were initially coated with laminin. We also found that cells on silanized glass coated with laminin had higher motility than those on silanized glass coated with fibronectin. Based on our results, we suggest that silanized glass is a more suitable cell culture substrate than conventional non-treated glass when coated by ECM for observations of ECM effects on cell physiology.

Bee venom induces apoptosis and suppresses matrix metaloprotease-2 expression in human glioblastoma cells

Directory of Open Access Journals (Sweden)

Mohsen Sisakht

Full Text Available Abstract Glioblastoma is the most common malignant brain tumor representing with poor prognosis, therapy resistance and high metastasis rate. Increased expression and activity of matrix metalloproteinase-2, a member of matrix metalloproteinase family proteins, has been reported in many cancers including glioblastoma. Inhibition of matrix metalloproteinase-2 expression has resulted in reduced aggression of glioblastoma tumors in several reports. In the present study, we evaluated effect of bee venom on expression and activity of matrix metalloproteinase-2 as well as potential toxicity and apoptogenic properties of bee venom on glioblastoma cells. Human A172 glioblastoma cells were treated with increasing concentrations of bee venom. Then, cell viability, apoptosis, matrix metalloproteinase-2 expression, and matrix metalloproteinase-2 activity were measured using MMT assay, propidium iodide staining, real time-PCR, and zymography, respectively. The IC50 value of bee venom was 28.5 µg/ml in which it leads to decrease of cell viability and induction of apoptosis. Incubation with bee venom also decreased the expression of matrix metalloproteinase-2 in this cell line (p < 0.05. In zymography, there was a reverse correlation between bee venom concentration and total matrix metalloproteinase-2 activity. Induction of apoptosis as well as inhibition of matrix metalloproteinase-2 activity and expression can be suggested as molecular mechanisms involved in cytotoxic and antimetastatic effects of bee venom against glioblastoma cells.

Wetting and dewetting of extracellular matrix and glycocalix models

International Nuclear Information System (INIS)

Tanaka, Motomu; Rehfeldt, Florian; Schneider, Matthias F; Mathe, Gerald; Albersdoerfer, Antero; Neumaier, Klaus R; Purrucker, Oliver; Sackmann, Erich

2005-01-01

In this paper, we study wetting and dewetting of hydrated biopolymer layers mediating cell-cell and cell-tissue contacts, called the extracellular matrix and cell surface glycocalix, by the combination of various physical techniques. Here, the sum of the net effects of the various interfacial forces, which is referred to as the disjoining pressure, is used as a semi-quantitative measure to describe the thermodynamics of hydrated interlayers. The disjoining pressure can be measured by applying external forces to maintain the equilibrium distance between two parallel surfaces (in biology, two neighbouring plasma membranes). Using artificial models of the extracellular matrix and glycocalix, we describe stable cell-cell contacts in terms of the wetting (or spreading) of complex fluids on polymer surfaces. In fact, the adjustment of the wetting interaction via thin hydrating layers enables us to transform three-dimensional cell membranes into quasi-two-dimensional films on macroscopically large surfaces. Fine-tuning of local wetting conditions at the interface further allows for the selective wetting of native cell membranes on microstructured polysaccharide films, which has a large potential for individual detection of biological functions in confined geometries

Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis

International Nuclear Information System (INIS)

Kaljot, K.T.; Shaw, R.D.; Greenberg, H.B.; Rubin, D.H.

1988-01-01

Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 [VP3]; 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. To determine whether trypsin treatment affected rotavirus internalization, the authors studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 mediated 51 Cr, [ 14 C]choline, and [ 3 H]inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration

Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis

Energy Technology Data Exchange (ETDEWEB)

Kaljot, K.T.; Shaw, R.D.; Greenberg, H.B. (Stanford Univ. School of Medicine, CA (USA) Palo Alto Veterans Administration Medical Center, CA (USA)); Rubin, D.H. (Univ. of Pennsylvania, Philadelphia (USA))

1988-04-01

Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 (VP3); 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. To determine whether trypsin treatment affected rotavirus internalization, the authors studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 mediated {sup 51}Cr, ({sup 14}C)choline, and ({sup 3}H)inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.

Inhibition of αvβ3 integrin induces loss of cell directionality of oral squamous carcinoma cells (OSCC.

Directory of Open Access Journals (Sweden)

Cyntia F Montenegro

Full Text Available The connective tissue formed by extracellular matrix (ECM rich in fibronectin and collagen consists a barrier that cancer cells have to overpass to reach blood vessels and then a metastatic site. Cell adhesion to fibronectin is mediated by αvβ3 and α5β1 integrins through an RGD motif present in this ECM protein, thus making these receptors key targets for cell migration studies. Here we investigated the effect of an RGD disintegrin, DisBa-01, on the migration of human fibroblasts (BJ and oral squamous cancer cells (OSCC, SCC25 on a fibronectin-rich environment. Time-lapse images were acquired on fibronectin-coated glass-bottomed dishes. Migration speed and directionality analysis indicated that OSCC cells, but not fibroblasts, showed significant decrease in both parameters in the presence of DisBa-01 (1μM and 2μM. Integrin expression levels of the α5, αv and β3 subunits were similar in both cell lines, while β1 subunit is present in lower levels on the cancer cells. Next, we examined whether the effects of DisBa-01 were related to changes in adhesion properties by using paxillin immunostaining and total internal reflection fluorescence TIRF microscopy. OSCCs in the presence of DisBa-01 showed increased adhesion sizes and number of maturing adhesion. The same parameters were analyzed usingβ3-GFP overexpressing cells and showed that β3 overexpression restored cell migration velocity and the number of maturing adhesion that were altered by DisBa-01. Surface plasmon resonance analysis showed that DisBa-01 has 100x higher affinity for αvβ3 integrin than forα5β1 integrin. In conclusion, our results suggest that the αvβ3 integrin is the main receptor involved in cell directionality and its blockage may be an interesting alternative against metastasis.

Fc-receptors and surface immunoglobulins in cells of the hairy cell leukemia

International Nuclear Information System (INIS)

Rieber, E.P.; Linke, R.P.; Riethmueller, G.; Heyden, H.W. von; Waller, H.D.

1976-01-01

Using 125 I-labelled aggregated IgG in a quantitative assay a strong expression of Fc-receptors was found on the leukemic cells of a patient with hairy cell leukemia. The Fc-receptor activity on these cells was much higher than that on monocytes and B-lymphocytes from normal blood. Surface immunoglobulins were detected by radioautography using radioactively labelled (Fab') 2 -fragments of monospecific antibodies directed against immunoglobulin heavy chains. Prior to radioautography the cells were stained for the tartrate resistant acid phosphatase. It is found that all cells containing this enzyme bore delta-chains on their surface. On more than 90% of these cells a simultaneous expression of μ-chains was detected. γ-chains could only be demonstrated on cells which were negative for the tartrate resistant acid phosphatase; part of these cells, however, were hairy cells by morphological criteria. (orig.) [de

Fc-receptors and surface immunoglobulins in cells of the hairy cell leukemia

Energy Technology Data Exchange (ETDEWEB)

Rieber, E P; Linke, R P; Riethmueller, G [Tuebingen Univ. (Germany, F.R.). Abt. fuer Experimentelle Chirurgie und Immunologie; Heyden, H.W. von; Waller, H D [Tuebingen Univ. (Germany, F.R.). Abt. Innere Medizin 2

1976-01-01

Using /sup 125/I-labelled aggregated IgG in a quantitative assay a strong expression of Fc-receptors was found on the leukemic cells of a patient with hairy cell leukemia. The Fc-receptor activity on these cells was much higher than that on monocytes and B-lymphocytes from normal blood. Surface immunoglobulins were detected by radioautography using radioactively labelled (Fab')/sub 2/-fragments of monospecific antibodies directed against immunoglobulin heavy chains. Prior to radioautography the cells were stained for the tartrate resistant acid phosphatase. It is found that all cells containing this enzyme bore delta-chains on their surface. On more than 90% of these cells a simultaneous expression of ..mu..-chains was detected. ..gamma..-chains could only be demonstrated on cells which were negative for the tartrate resistant acid phosphatase; part of these cells, however, were hairy cells by morphological criteria.

A direct derivation of the exact Fisther information matrix of Gaussian vector state space models

NARCIS (Netherlands)

Klein, A.A.B.; Neudecker, H.

2000-01-01

This paper deals with a direct derivation of Fisher's information matrix of vector state space models for the general case, by which is meant the establishment of the matrix as a whole and not element by element. The method to be used is matrix differentiation, see [4]. We assume the model to be

Polymer microfilters with nanostructured surfaces for the culture of circulating cancer cells

International Nuclear Information System (INIS)

Makarova, Olga V.; Adams, Daniel L.; Divan, Ralu; Rosenmann, Daniel; Zhu, Peixuan; Li, Shuhong; Amstutz, Platte; Tang, Cha-Mei

2016-01-01

There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and without an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma cancer cell lines after isolation on the various types of material modifications. Our data suggest that RIE modified polymer filters can isolate model cell lines while retaining ell viability, and that the RIE filter modification allows T24 monolayering cells to proliferate as a structured cluster. - Highlights: • Surface topographical effects the growth patterns and cell function of cancer cells • Nanoscale surface topography on polymer filters for circulating tumor cell culture • Membrane fabricated directly on polymer surfaces utilized for polymer etching • Nanotopography alters cell shape, phenotype and growth patterns of cancer cells • Nanoscale surface topography dictates monolayering or 3D structured cell culture

Polymer microfilters with nanostructured surfaces for the culture of circulating cancer cells

Energy Technology Data Exchange (ETDEWEB)

Makarova, Olga V. [Creatv MicroTech, Inc., 2242 West Harrison St., Chicago 60612, IL (United States); Adams, Daniel L., E-mail: dan@creatvmicrotech.com [Creatv MicroTech, Inc., 1 Deer Park Drive, Monmouth Junction, NJ 08852 (United States); Divan, Ralu; Rosenmann, Daniel [Center for Nanoscale Materials, Argonne National Laboratory, 9700 South Cass Ave., Argonne 60439, IL (United States); Zhu, Peixuan; Li, Shuhong; Amstutz, Platte; Tang, Cha-Mei [Creatv MicroTech, Inc., 11609 Lake Potomac Drive, Potomac 20854, MD (United States)

2016-09-01

There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and without an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma cancer cell lines after isolation on the various types of material modifications. Our data suggest that RIE modified polymer filters can isolate model cell lines while retaining ell viability, and that the RIE filter modification allows T24 monolayering cells to proliferate as a structured cluster. - Highlights: • Surface topographical effects the growth patterns and cell function of cancer cells • Nanoscale surface topography on polymer filters for circulating tumor cell culture • Membrane fabricated directly on polymer surfaces utilized for polymer etching • Nanotopography alters cell shape, phenotype and growth patterns of cancer cells • Nanoscale surface topography dictates monolayering or 3D structured cell culture.

Non-Muscle Myosin II Isoforms Have Different Functions in Matrix Rearrangement by MDA-MB-231 Cells.

Directory of Open Access Journals (Sweden)

Bridget Hindman

Full Text Available The role of a stiffening extra-cellular matrix (ECM in cancer progression is documented but poorly understood. Here we use a conditioning protocol to test the role of nonmuscle myosin II isoforms in cell mediated ECM arrangement using collagen constructs seeded with breast cancer cells expressing shRNA targeted to either the IIA or IIB heavy chain isoform. While there are several methods available to measure changes in the biophysical characteristics of the ECM, we wanted to use a method which allows for the measurement of global stiffness changes as well as a dynamic response from the sample over time. The conditioning protocol used allows the direct measurement of ECM stiffness. Using various treatments, it is possible to determine the contribution of various construct and cellular components to the overall construct stiffness. Using this assay, we show that both the IIA and IIB isoforms are necessary for efficient matrix remodeling by MDA-MB-231 breast cancer cells, as loss of either isoform changes the stiffness of the collagen constructs as measured using our conditioning protocol. Constructs containing only collagen had an elastic modulus of 0.40 Pascals (Pa, parental MDA-MB-231 constructs had an elastic modulus of 9.22 Pa, while IIA and IIB KD constructs had moduli of 3.42 and 7.20 Pa, respectively. We also calculated the cell and matrix contributions to the overall sample elastic modulus. Loss of either myosin isoform resulted in decreased cell stiffness, as well as a decrease in the stiffness of the cell-altered collagen matrices. While the total construct modulus for the IIB KD cells was lower than that of the parental cells, the IIB KD cell-altered matrices actually had a higher elastic modulus than the parental cell-altered matrices (4.73 versus 4.38 Pa. These results indicate that the IIA and IIB heavy chains play distinct and non-redundant roles in matrix remodeling.

Capillary network formation from dispersed endothelial cells: Influence of cell traction, cell adhesion, and extracellular matrix rigidity

Science.gov (United States)

Ramos, João R. D.; Travasso, Rui; Carvalho, João

2018-01-01

The formation of a functional vascular network depends on biological, chemical, and physical processes being extremely well coordinated. Among them, the mechanical properties of the extracellular matrix and cell adhesion are fundamental to achieve a functional network of endothelial cells, able to fully cover a required domain. By the use of a Cellular Potts Model and Finite Element Method it is shown that there exists a range of values of endothelial traction forces, cell-cell adhesion, and matrix rigidities where the network can spontaneously be formed, and its properties are characterized. We obtain the analytical relation that the minimum traction force required for cell network formation must obey. This minimum value for the traction force is approximately independent on the considered cell number and cell-cell adhesion. We quantify how these two parameters influence the morphology of the resulting networks (size and number of meshes).

Effect of Uniaxial Tensile Cyclic Loading Regimes on Matrix Organization and Tenogenic Differentiation of Adipose-Derived Stem Cells Encapsulated within 3D Collagen Scaffolds

Directory of Open Access Journals (Sweden)

Gayathri Subramanian

2017-01-01

Full Text Available Adipose-derived mesenchymal stem cells have become a popular cell choice for tendon repair strategies due to their relative abundance, ease of isolation, and ability to differentiate into tenocytes. In this study, we investigated the solo effect of different uniaxial tensile strains and loading frequencies on the matrix directionality and tenogenic differentiation of adipose-derived stem cells encapsulated within three-dimensional collagen scaffolds. Samples loaded at 0%, 2%, 4%, and 6% strains and 0.1 Hz and 1 Hz frequencies for 2 hours/day over a 7-day period using a custom-built uniaxial tensile strain bioreactor were characterized in terms of matrix organization, cell viability, and musculoskeletal gene expression profiles. The results displayed that the collagen fibers of the loaded samples exhibited increased matrix directionality with an increase in strain values. Gene expression analyses demonstrated that ASC-encapsulated collagen scaffolds loaded at 2% strain and 0.1 Hz frequency showed significant increases in extracellular matrix genes and tenogenic differentiation markers. Importantly, no cross-differentiation potential to osteogenic, chondrogenic, and myogenic lineages was observed at 2% strain and 0.1 Hz frequency loading condition. Thus, 2% strain and 0.1 Hz frequency were identified as the appropriate mechanical loading regime to induce tenogenic differentiation of adipose-derived stem cells cultured in a three-dimensional environment.

Effect of Uniaxial Tensile Cyclic Loading Regimes on Matrix Organization and Tenogenic Differentiation of Adipose-Derived Stem Cells Encapsulated within 3D Collagen Scaffolds.

Science.gov (United States)

Subramanian, Gayathri; Stasuk, Alexander; Elsaadany, Mostafa; Yildirim-Ayan, Eda

2017-01-01

Adipose-derived mesenchymal stem cells have become a popular cell choice for tendon repair strategies due to their relative abundance, ease of isolation, and ability to differentiate into tenocytes. In this study, we investigated the solo effect of different uniaxial tensile strains and loading frequencies on the matrix directionality and tenogenic differentiation of adipose-derived stem cells encapsulated within three-dimensional collagen scaffolds. Samples loaded at 0%, 2%, 4%, and 6% strains and 0.1 Hz and 1 Hz frequencies for 2 hours/day over a 7-day period using a custom-built uniaxial tensile strain bioreactor were characterized in terms of matrix organization, cell viability, and musculoskeletal gene expression profiles. The results displayed that the collagen fibers of the loaded samples exhibited increased matrix directionality with an increase in strain values. Gene expression analyses demonstrated that ASC-encapsulated collagen scaffolds loaded at 2% strain and 0.1 Hz frequency showed significant increases in extracellular matrix genes and tenogenic differentiation markers. Importantly, no cross-differentiation potential to osteogenic, chondrogenic, and myogenic lineages was observed at 2% strain and 0.1 Hz frequency loading condition. Thus, 2% strain and 0.1 Hz frequency were identified as the appropriate mechanical loading regime to induce tenogenic differentiation of adipose-derived stem cells cultured in a three-dimensional environment.

A dielectric matrix calculation of the surface-plasmon energy for the silicon (100) surface

International Nuclear Information System (INIS)

Forsyth, A.J.; Smith, A.E.; Josefsson, T.W.

1996-01-01

Full text: As an extension of previous work, we present preliminary calculations for the dielectric properties of the silicon (100) surface. In particular, the |q|→0 and |q|=2π/a(1,0,0) surface loss function, and corresponding surface plasmon energies have been calculated within a simple model for the silicon surface. The results have been obtained from the Adler and Wiser dielectric matrix (DM). The bandstructure used for the calculation was based on the highly successful empirical pseudopotential method of Cohen and Chelikovsky. We have used a 59 plane wave basis for the bandstructure, and have chosen a DM size of 59 x 59. Results are compared and contrasted with volume plasmon calculations, free electron calculations and experiment

Probes for anionic cell surface detection

Science.gov (United States)

Smith, Bradley D.

2013-03-05

Embodiments of the present invention are generally directed to compositions comprising a class of molecular probes for detecting the presence of anionic cell surfaces. Embodiments include compositions that are enriched for these compositions and preparations, particularly preparations suitable for use as laboratory/clinical reagents and diagnostic indicators, either alone or as part of a kit. An embodiment of the invention provides for a highly selective agent useful in the discernment and identification of dead or dying cells, such as apoptotic cells, in a relatively calcium-free environment. An embodiment of the invention provides a selective agent for the identification of bacteria in a mixed population of bacterial cells and nonbacterial cells.

A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion

DEFF Research Database (Denmark)

Faassen, A E; Schrager, J A; Klein, D J

1992-01-01

The metastatic spread of tumor cells occurs through a complex series of events, one of which involves the adhesion of tumor cells to extracellular matrix (ECM) components. Multiple interactions between cell surface receptors of an adherent tumor cell and the surrounding ECM contribute to cell...... collagen could also be inhibited by removing cell surface chondroitin sulfate with chondroitinase. In contrast, type I collagen-mediated melanoma cell adhesion and spreading were not affected by either beta-D-xyloside or chondroitinase treatments. These results suggest that mouse melanoma CSPG...... was shown to be mediated, at least in part, by chondroitin sulfate. Additionally we have determined that mouse melanoma CSPG is composed of a 110-kD core protein that is recognized by anti-CD44 antibodies on Western blots. Collectively, our data suggests that interactions between a cell surface CD44-related...

Multi-scale cell/surface interaction on modified titanium aluminum vanadium surfaces

Science.gov (United States)

Chen, Jianbo

ingrowth, pore coverage, cell adhesion and proliferation was observed to increase with decreasing pore size. It was found that fiber geometries provided guidance for cell spreading along the fiber directions. However, the larger gaps in fiber geometries made pore bridging difficult. Finally, this dissertation presents an in vivo study of the combined effects of laser microgrooving and RGD-coating on the osseointegration of implanted Ti-6Al-4V pins. Both histological and biomechanical results show that the combination of laser microgrooving and RGD-coating results in improved osseointegration over the control surfaces. All the above findings have important implications for future orthopedic and dental implant design.

Immobilization of microbial cells on cellulose-polymer surfaces by radiation polymerization

International Nuclear Information System (INIS)

Kumakura, M.; Kaetsu, I.

1983-01-01

Streptomyces phaeochromogens cells were immobilized on cellulose-polymer surfaces by radiation polymerization using hydrophilic monomers and paper. The enzyme activity of immobilized cell sheets was higher than that of immobilized cell composites obtained by the usual radiation polymerization technique. The enzyme activity of the sheets was affected by monomer concentration, the thickness of paper, and the degree of polymerization of paper. The copolymerization of hydroxyethyl methacrylate and methoxytetraethyleneglycol methacrylate in the sheets led to a further increase of the enzyme activity due to the increase of the hydrophilicity of the polymer matrix. The Michaelis constant of the sheets from low monomer concentration was close to that of intact cells

Differential expression pattern of extracellular matrix molecules during chondrogenesis of mesenchymal stem cells from bone marrow and adipose tissue

DEFF Research Database (Denmark)

Mehlhorn, A T; Niemeyer, P; Kaiser, S

2006-01-01

Adipose-derived adult stem cells (ADASCs) or bone marrow-derived mesenchymal stem cells (BMSCs) are considered as alternative cell sources for cell-based cartilage repair due to their ability to produce cartilage-specific matrix. This article addresses the differential expression pattern...... chondroinduction. TGF-beta1 induces alternative splicing of the alpha(1)-procollagen type II transcript in BMSCs, but not in ADASCs. These findings may direct the development of a cell-specific culture environment either to prevent hypertrophy in BMSCs or to promote chondrogenic maturation in ADASCs....

Two-way regulation between cells and aligned collagen fibrils: local 3D matrix formation and accelerated neural differentiation of human decidua parietalis placental stem cells.

Science.gov (United States)

Li, Wen; Zhu, Bofan; Strakova, Zuzana; Wang, Rong

2014-08-08

It has been well established that an aligned matrix provides structural and signaling cues to guide cell polarization and cell fate decision. However, the modulation role of cells in matrix remodeling and the feedforward effect on stem cell differentiation have not been studied extensively. In this study, we report on the concerted changes of human decidua parietalis placental stem cells (hdpPSCs) and the highly ordered collagen fibril matrix in response to cell-matrix interaction. With high-resolution imaging, we found the hdpPSCs interacted with the matrix by deforming the cell shape, harvesting the nearby collagen fibrils, and reorganizing the fibrils around the cell body to transform a 2D matrix to a localized 3D matrix. Such a unique 3D matrix prompted high expression of β-1 integrin around the cell body that mediates and facilitates the stem cell differentiation toward neural cells. The study offers insights into the coordinated, dynamic changes at the cell-matrix interface and elucidates cell modulation of its matrix to establish structural and biochemical cues for effective cell growth and differentiation. Copyright © 2014 Elsevier Inc. All rights reserved.

Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

2008-06-26

Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a 'cuboidal' epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-{beta}-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.

Independent Analysis of the Flagellum Surface and Matrix Proteomes Provides Insight into Flagellum Signaling in Mammalian-infectious Trypanosoma brucei*

Science.gov (United States)

Oberholzer, Michael; Langousis, Gerasimos; Nguyen, HoangKim T.; Saada, Edwin A.; Shimogawa, Michelle M.; Jonsson, Zophonias O.; Nguyen, Steven M.; Wohlschlegel, James A.; Hill, Kent L.

2011-01-01

The flagellum of African trypanosomes is an essential and multifunctional organelle that functions in motility, cell morphogenesis, and host-parasite interaction. Previous studies of the trypanosome flagellum have been limited by the inability to purify flagella without first removing the flagellar membrane. This limitation is particularly relevant in the context of studying flagellum signaling, as signaling requires surface-exposed proteins in the flagellar membrane and soluble signaling proteins in the flagellar matrix. Here we employ a combination of genetic and mechanical approaches to purify intact flagella from the African trypanosome, Trypanosoma brucei, in its mammalian-infectious stage. We combined flagellum purification with affinity-purification of surface-exposed proteins to conduct independent proteomic analyses of the flagellum surface and matrix fractions. The proteins identified encompass a broad range of molecular functionalities, including many predicted to function in signaling. Immunofluorescence and RNA interference studies demonstrate flagellum localization and function for proteins identified and provide insight into mechanisms of flagellum attachment and motility. The flagellum surface proteome includes many T. brucei-specific proteins and is enriched for proteins up-regulated in the mammalian-infectious stage of the parasite life-cycle. The combined results indicate that the flagellum surface presents a diverse and dynamic host-parasite interface that is well-suited for host-parasite signaling. PMID:21685506

Increased extracellular matrix density decreases MCF10A breast cell acinus formation in 3D culture conditions.

Science.gov (United States)

Lance, Amanda; Yang, Chih-Chao; Swamydas, Muthulekha; Dean, Delphine; Deitch, Sandy; Burg, Karen J L; Dréau, Didier

2016-01-01

The extracellular matrix (ECM) contributes to the generation and dynamic of normal breast tissue, in particular to the generation of polarized acinar and ductal structures. In vitro 3D culture conditions, including variations in the composition of the ECM, have been shown to directly influence the formation and organization of acinus-like and duct-like structures. Furthermore, the density of the ECM appears to also play a role in the normal mammary tissue and tumour formation. Here we show that the density of the ECM directly influences the number, organization and function of breast acini. Briefly, non-malignant human breast MCF10A cells were incubated in increasing densities of a Matrigel®-collagen I matrix. Elastic moduli near and distant to the acinus structures were measured by atomic force microscopy, and the number of acinus structures was determined. Immunochemistry was used to investigate the expression levels of E-cadherin, laminin, matrix metalloproteinase-14 and ß-casein in MCF10A cells. The modulus of the ECM was significantly increased near the acinus structures and the number of acinus structures decreased with the increase in Matrigel-collagen I density. As evaluated by the expression of laminin, the organization of the acinus structures present was altered as the density of the ECM increased. Increases in both E-cadherin and MMP14 expression by MCF10A cells as ECM density increased were also observed. In contrast, MCF10A cells expressed lower ß-casein levels as the ECM density increased. Taken together, these observations highlight the key role of ECM density in modulating the number, organization and function of breast acini. Copyright © 2013 John Wiley & Sons, Ltd.

Using cell-substrate impedance and live cell imaging to measure real-time changes in cellular adhesion and de-adhesion induced by matrix modification.

Science.gov (United States)

Rees, Martin D; Thomas, Shane R

2015-02-19

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Studies of cell biomechanics with surface micro-/nano-technology

International Nuclear Information System (INIS)

Wang Dong; Zhang Wei; Jiang Xingyu

2011-01-01

We report the recent progress in our studies of cell biology using micro-/nano-technology. Cells have a size of several to tens of microns, which makes them easily manipulated by micro-/nano-technology. The shape of the cell influences the alignment of the actin cytoskeleton, which bears the main forces of the cell, maintains the shape,and mediates a series of biochemical reactions. We invented a stretching device and studied the real-time actin filament dynamics under stretch. We found that one stretch cycle shortened the actin filaments and promoted their reassemble process. Cell migration is a complex mechanical process. We found that cell geometry determines the cell polarity and migration direction. We fabricated three-dimensional surfaces to mimic the topography in vivo, and further built a cell culture model by integrating the three-dimensional surface, microfluidics, cell patterning,and coculturing of multiple cell types. We also investigated the neuronal guidance by surface patterning. (authors)

Direct synthesis of II-VI compound nanocrystals in polymer matrix

International Nuclear Information System (INIS)

Antolini, F.; Di Luccio, T.; Laera, A.M.; Mirenghi, L.; Piscopiello, E.; Re, M.; Tapfer, L.

2007-01-01

The production of II-VI semiconductor compound - polymer matrix nanocomposites by a direct in-situ thermolysis process is described. Metal-thiolate precursor molecules embedded in a polymer matrix decompose by a thermal annealing and the nucleation of semiconductor nanocrystals occurs. It is shown that the nucleation of nanoparticles and the formation of the nanocomposite can be also achieved by laser beam irradiation; this opens the way towards a ''lithographic'' in-situ nanocomposite production process. A possible growth and nanocomposite formation mechanism, describing the structural and chemical transformation of the precursor molecules, their decomposition and the formation of the nanoparticles, is presented. (copyright 2007 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

Past matrix stiffness primes epithelial cells and regulates their future collective migration through a mechanical memory.

Science.gov (United States)

Nasrollahi, Samila; Walter, Christopher; Loza, Andrew J; Schimizzi, Gregory V; Longmore, Gregory D; Pathak, Amit

2017-11-01

During morphogenesis and cancer metastasis, grouped cells migrate through tissues of dissimilar stiffness. Although the influence of matrix stiffness on cellular mechanosensitivity and motility are well-recognized, it remains unknown whether these matrix-dependent cellular features persist after cells move to a new microenvironment. Here, we interrogate whether priming of epithelial cells by a given matrix stiffness influences their future collective migration on a different matrix - a property we refer to as the 'mechanical memory' of migratory cells. To prime cells on a defined matrix and track their collective migration onto an adjoining secondary matrix of dissimilar stiffness, we develop a modular polyacrylamide substrate through step-by-step polymerization of different PA compositions. We report that epithelial cells primed on a stiff matrix migrate faster, display higher actomyosin expression, form larger focal adhesions, and retain nuclear YAP even after arriving onto a soft secondary matrix, as compared to their control behavior on a homogeneously soft matrix. Priming on a soft ECM causes a reverse effect. The depletion of YAP dramatically reduces this memory-dependent migration. Our results present a previously unidentified regulation of mechanosensitive collective cell migration by past matrix stiffness, in which mechanical memory depends on YAP activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ulex europaeus I lectin induces activation of matrix-metalloproteinase-2 in endothelial cells.

Science.gov (United States)

Gomez, D E; Yoshiji, H; Kim, J C; Thorgeirsson, U P

1995-11-02

In this report, we show that the lectin Ulex europaeus agglutinin I (UEA I), which binds to alpha-linked fucose residues on the surface of endothelial cells, mediates activation of the 72-kDa matrix metalloproteinase-2 (MMP-2). A dose-dependent increase in the active 62-kDa form of MMP-2 was observed in conditioned medium from monkey aortic endothelial cells (MAEC) following incubation with concentrations of UEA I ranging from 2 to 100 micrograms/ml. The increase in the 62-kDa MMP-2 gelatinolytic activity was not reflected by a rise in MMP-2 gene expression. The UEA I-mediated activation of MMP-2 was blocked by L-fucose, which competes with UEA I for binding to alpha-fucose. These findings may suggest that a similar in vivo mechanism exists, whereby adhesive interactions between tumor cell lectins and endothelial cells can mediate MMP-2 activation.

Surface-modified gold nanorods for specific cell targeting

Science.gov (United States)

Wang, Chan-Ung; Arai, Yoshie; Kim, Insun; Jang, Wonhee; Lee, Seonghyun; Hafner, Jason H.; Jeoung, Eunhee; Jung, Deokho; Kwon, Youngeun

2012-05-01

Gold nanoparticles (GNPs) have unique properties that make them highly attractive materials for developing functional reagents for various biomedical applications including photothermal therapy, targeted drug delivery, and molecular imaging. For in vivo applications, GNPs need to be prepared with very little or negligible cytotoxicitiy. Most GNPs are, however, prepared using growth-directing surfactants such as cetyl trimethylammonium bromide (CTAB), which are known to have considerable cytotoxicity. In this paper, we describe an approach to remove CTAB to a non-toxic concentration. We optimized the conditions for surface modification with methoxypolyethylene glycol thiol (mPEG), which replaced CTAB and formed a protective layer on the surface of gold nanorods (GNRs). The cytotoxicities of pristine and surface-modified GNRs were measured in primary human umbilical vein endothelial cells and human cell lines derived from hepatic carcinoma cells, embryonic kidney cells, and thyroid papillary carcinoma cells. Cytotoxicity assays revealed that treating cells with GNRs did not significantly affect cell viability except for thyroid papillary carcinoma cells. Thyroid cancer cells were more susceptible to residual CTAB, so CTAB had to be further removed by dialysis in order to use GNRs for thyroid cell targeting. PEGylated GNRs are further modified to present monoclonal antibodies that recognize a specific surface marker, Na-I symporter, for thyroid cells. Antibody-conjugated GNRs specifically targeted human thyroid cells in vitro.

Autonomous molecular cascades for evaluation of cell surfaces

Science.gov (United States)

Rudchenko, Maria; Taylor, Steven; Pallavi, Payal; Dechkovskaia, Alesia; Khan, Safana; Butler, Vincent P., Jr.; Rudchenko, Sergei; Stojanovic, Milan N.

2013-08-01

Molecular automata are mixtures of molecules that undergo precisely defined structural changes in response to sequential interactions with inputs. Previously studied nucleic acid-based automata include game-playing molecular devices (MAYA automata) and finite-state automata for the analysis of nucleic acids, with the latter inspiring circuits for the analysis of RNA species inside cells. Here, we describe automata based on strand-displacement cascades directed by antibodies that can analyse cells by using their surface markers as inputs. The final output of a molecular automaton that successfully completes its analysis is the presence of a unique molecular tag on the cell surface of a specific subpopulation of lymphocytes within human blood cells.

Monitoring of anatabine release by methyl jasmonate elicited BY-2 cells using surface-enhanced Raman scattering.

Science.gov (United States)

De Bleye, C; Dumont, E; Dispas, A; Hubert, C; Sacré, P-Y; Netchacovitch, L; De Muyt, B; Kevers, C; Dommes, J; Hubert, Ph; Ziemons, E

2016-11-01

A new application of surface-enhanced Raman scattering (SERS) in the field of plant material analysis is proposed in this study. The aim was to monitor the release of anatabine by methyl jasmonate (MeJa) elicited Bright Yellow-2 (BY-2) cells. Gold nanoparticles (AuNps) were used as SERS substrate. The first step was to study the SERS activity of anatabine in a complex matrix comprising the culture medium and BY-2 cells. The second step was the calibration. This one was successfully performed directly in the culture medium in order to take into account the matrix effect, by spiking the medium with different concentrations of anatabine, leading to solutions ranging from 250 to 5000µgL(-1). A univariate analysis was performed, the intensity of a band situated at 1028cm(-1), related to anatabine, was plotted against the anatabine concentration. A linear relationship was observed with a R(2) of 0.9951. During the monitoring study, after the MeJa elicitation, samples were collected from the culture medium containing BY-2 cells at 0, 24h, 48h, 72h and 96h and were analysed using SERS. Finally, the amount of anatabine released in the culture medium was determined using the response function, reaching a plateau after 72h of 82µg of anatabine released/g of fresh weight (FW) MeJa elicited BY-2 cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Directive Nanophysical Cues for Regenerative Neural Cell Systems

Science.gov (United States)

Ayres, Virginia; Tiryaki, Volkan Mujdat; Ahmed, Ijaz; Shreiber, David

Until recently, implantables such as stents, probes, wafers and scaffolds have been viewed as passive vehicles for the delivery of physical, pharmacological and cellular interventions. Recent research, however, indicates that the physical environments that implantables present supply directive cues in their own right that work in conjunction with biochemical cues and produce a jointly-directed outcome. We will present our research in CNS repairs using advanced scanning probe microscopy, electron microscopies and contact angle measurements to quantitatively describe the nanoscale elasticity, surface roughness, work of adhesion and surface polarity for investigation of scaffold environments. We will also present our research using super-resolution immunocytochemistry and atomic force microscopy to evaluate neural cell morphological responses with associated micro filament, microtubule and intermediate filament expressions, along with results on how and which integrin-family receptors are possibly involved. Finally, we will present our novel application of k-means cluster analysis applied across multiple experimental modalities for quantification of synergistic scaffold properties and cell responses.

Laser Surface Treatment and Modification of Aluminum Alloy Matrix Composites

Science.gov (United States)

Abbass, Muna Khethier

2018-02-01

The present work aimed to study the laser surface treatment and modification of Al-4.0%Cu-1.0%Mg alloy matrix composite reinforced with 10%SiC particles produced by stir casting. The specimens of the base alloy and composite were irradiated with an Nd:YAG laser of 1000 mJ, 1064 nm and 3 Hz . Dry wear test using the pin-on -disc technique at different sliding times (5-30 min) at a constant applied load and sliding speed were performed before and after laser treatment. Micro hardness and wear resistance were increased for all samples after laser hardening treatment. The improvement of these properties is explained by microstructural homogenization and grain refinement of the laser treated surface. Modification and refinement of SiC particles and grain refinement in the microstructure of the aluminum alloy matrix (α-Al) were observed by optical and SEM micrographs. The highest increase in hardness was 21.4% and 26.2% for the base alloy and composite sample respectively.

Implementation of the CCGM approximation for surface diffraction using Wigner R-matrix theory

International Nuclear Information System (INIS)

Lauderdale, J.G.; McCurdy, C.W.

1983-01-01

The CCGM approximation for surface scattering proposed by Cabrera, Celli, Goodman, and Manson [Surf. Sci. 19, 67 (1970)] is implemented for realistic surface interaction potentials using Wigner R-matrix theory. The resulting procedure is highly efficient computationally and is in no way limited to hard wall or purely repulsive potentials. Comparison is made with the results of close-coupling calculations of other workers which include the same diffraction channels in order to fairly evaluate the CCGM approximation which is an approximation to the coupled channels Lippman--Schwinger equation for the T matrix. The shapes of selective adsorption features, whether maxima or minima, in the scattered intensity are well represented in this approach for cases in which the surface corrugation is not too strong

Organic composite-mediated surface coating of human acellular bone matrix with strontium.

Science.gov (United States)

Huang, Yi-Zhou; Wang, Jing-Jing; Huang, Yong-Can; Wu, Cheng-Guang; Zhang, Yi; Zhang, Chao-Liang; Bai, Lin; Xie, Hui-Qi; Li, Zhao-Yang; Deng, Li

2018-03-01

Acellular bone matrix (ACBM) provides an osteoconductive scaffold for bone repair, but its osteoinductivity is poor. Strontium (Sr) improves the osteoinductivity of bone implants. In this study, we developed an organic composite-mediated strontium coating strategy for ACBM scaffolds by using the ion chelating ability of carboxymethyl cellulose (CMC) and the surface adhesion ability of dopamine (DOPA). The organic coating composite, termed the CMC-DOPA-Sr composite, was synthesized under a mild condition, and its chemical structure and strontium ion chelating ability were then determined. After surface decoration, the physicochemical properties of the strontium-coated ACBM (ACBM-Sr) scaffolds were characterized, and their biocompatibility and osteoinductivity were determined in vitro and in vivo. The results showed that the CMC-DOPA-Sr composite facilitated strontium coating on the surface of ACBM scaffolds. The ACBM-Sr scaffolds possessed a sustained strontium ion release profile, exhibited good cytocompatibility, and enhanced the osteogenic differentiation of mesenchymal stem cells in vitro. Furthermore, the ACBM-Sr scaffolds showed good histocompatibility after subcutaneous implantation in nude mice. Taken together, this study provided a simple and mild strategy to realize strontium coating for ACBM scaffolds, which resulted in good biocompatibility and improved osteoinductivity. Copyright © 2017 Elsevier B.V. All rights reserved.

Cell Adhesions: Actin-Based Modules that Mediate Cell-Extracellular Matrix and Cell-Cell Interactions

Science.gov (United States)

Bachir, Alexia; Horwitz, Alan Rick; Nelson, W. James; Bianchini, Julie M.

2018-01-01

Cell adhesions link cells to the extracellular matrix (ECM) and to each other, and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping functional modules. These modules establish physical association with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as sense and translate the mechanical properties of the cellular environment to changes in cell organization and behavior. In this chapter we discuss the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions, and how adhesion molecules mediate crosstalk between cell-ECM and cell-cell adhesion sites. PMID:28679638

A physiologic three-dimensional cell culture system to investigate the role of decorin in matrix organisation and cell survival

International Nuclear Information System (INIS)

Seidler, Daniela G.; Schaefer, Liliana; Robenek, Horst; Iozzo, Renato V.; Kresse, Hans; Schoenherr, Elke

2005-01-01

In vivo cells exist in a three-dimensional environment generated and maintained by multiple cell-cell and cell-matrix interactions. Proteoglycans, like decorin, affect these complex interactions. Thus, we sought to investigate the role of decorin in a three-dimensional environment where the matrix was generated over time by decorin-deficient fibroblasts in the presence of L-ascorbic acid 2-phosphate. The cells were viable and proliferated in response to FGF2. Decorin was incorporated in the matrix and caused a ∼2 nm shift in the average diameter of the collagen fibrils, and the range and distribution of the fibrils became narrower and more uniform. Although there were no appreciable changes in collagen composition, we found that exogenous decorin induced the de novo synthesis of collagen I and V and cross-linked β (I). In the early phases of the three-dimensional culture, decorin reduced apoptosis. However, following the establishment of a three-dimensional matrix, the cells did not require decorin for their survival

Human epithelial cells increase their rigidity with ageing in vitro: direct measurements

International Nuclear Information System (INIS)

Berdyyeva, Tamara K; Woodworth, Craig D; Sokolov, Igor

2005-01-01

The decrease in elasticity of epithelial tissues with ageing contributes to many human diseases. This change was previously attributed to increased crosslinking of extracellular matrix proteins. Here we show that individual human epithelial cells also become significantly more rigid during ageing in vitro. Using atomic force microscopy (AFM), we found that the Young's modulus of viable cells was consistently increased two- to four-fold in older versus younger cells. Direct visualization of the cytoskeleton using a novel method involving the AFM suggested that increased rigidity of ageing cells was due to a higher density of cytoskeletal fibres. Our results identify a unique mechanism that might contribute to the age-related loss of elasticity in epithelial tissues

Osteoblastic response to pectin nanocoating on titanium surfaces

Energy Technology Data Exchange (ETDEWEB)

Gurzawska, Katarzyna, E-mail: kagu@sund.ku.dk [Research Center for Ageing and Osteoporosis, Departments of Medicine and Diagnostics, Copenhagen University Hospital Glostrup, Ndr. Ringvej 57, 2600 Glostrup (Denmark); Institute of Odontology, Faculty of Health and Medical Sciences, University of Copenhagen, Nørre Allé 20, 2200 Copenhagen N (Denmark); Svava, Rikke [Department of Plant Environment Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Copenhagen Center for Glycomics, Institute for Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N (Denmark); Yihua, Yu; Haugshøj, Kenneth Brian [Microtechnology and Surface Analysis, Danish Technological Institute, Gregersensvej 8, 2630 Taastrup (Denmark); Dirscherl, Kai [Dansk Fundamental Metrologi A/S, Matematiktorvet 307, 2800 Lyngby (Denmark); Levery, Steven B. [Copenhagen Center for Glycomics, Institute for Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N (Denmark); Byg, Inge [Department of Plant Environment Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Damager, Iben [Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd (Denmark); Nielsen, Martin W. [Department of Systems Biology, Technical University of Denmark, Matematiktorvet, Building 301, Kgs. Lyngby DK-2800 (Denmark); Jørgensen, Bodil [Department of Plant Environment Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark); Jørgensen, Niklas Rye [Research Center for Ageing and Osteoporosis, Departments of Medicine and Diagnostics, Copenhagen University Hospital Glostrup, Ndr. Ringvej 57, 2600 Glostrup (Denmark); and others

2014-10-01

Osseointegration of titanium implants can be improved by organic and inorganic nanocoating of the surface. The aim of our study was to evaluate the effect of organic nanocoating of titanium surface with unmodified and modified pectin Rhamnogalacturonan-Is (RG-Is) isolated from potato and apple with respect to surface properties and osteogenic response in osteoblastic cells. Nanocoatings on titanium surfaces were evaluated by scanning electron microscopy, contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy. The effect of coated RG-Is on cell adhesion, cell viability, bone matrix formation and mineralization was tested using SaOS-2 cells. Nanocoating with pectin RG-Is affected surface properties and in consequence changed the environment for cellular response. The cells cultured on surfaces coated with RG-Is from potato with high content of linear 1.4-linked galactose produced higher level of mineralized matrix compared with control surfaces and surfaces coated with RG-I with low content of linear 1.4-linked galactose. The study showed that the pectin RG-Is nanocoating not only changed chemical and physical titanium surface properties, but also specific coating with RG-Is containing high amount of galactan increased mineralized matrix formation of osteoblastic cells in vitro. - Highlights: • Surface nanocoating with plant-derived Rhamnogalacturonan-I (RG-I) is proposed. • Titanium surface became more hydrophilic after RG-Is nanocoating. • RG-Is with high galactose content resulted in high level of mineralized matrix. • RG-I is a new candidate for improvement of bone healing and osseointegration.

Carbide-reinforced metal matrix composite by direct metal deposition

Science.gov (United States)

Novichenko, D.; Thivillon, L.; Bertrand, Ph.; Smurov, I.

Direct metal deposition (DMD) is an automated 3D laser cladding technology with co-axial powder injection for industrial applications. The actual objective is to demonstrate the possibility to produce metal matrix composite objects in a single-step process. Powders of Fe-based alloy (16NCD13) and titanium carbide (TiC) are premixed before cladding. Volume content of the carbide-reinforced phase is varied. Relationships between the main laser cladding parameters and the geometry of the built-up objects (single track, 2D coating) are discussed. On the base of parametric study, a laser cladding process map for the deposition of individual tracks was established. Microstructure and composition of the laser-fabricated metal matrix composite objects are examined. Two different types of structures: (a) with the presence of undissolved and (b) precipitated titanium carbides are observed. Mechanism of formation of diverse precipitated titanium carbides is studied.

Ultrastructural analysis of bone nodules formed in vitro by isolated fetal rat calvaria cells

International Nuclear Information System (INIS)

Bhargava, U.; Bar-Lev, M.; Bellows, C.G.; Aubin, J.E.

1988-01-01

When cells enzymatically digested from 21 d fetal rat calvaria are grown in ascorbic acid and Na beta-glycerophosphate, they form discrete three-dimensional nodular structures with the histological and immunohistochemical appearance of woven bone. The present investigation was undertaken to verify that bone-like features were identifiable at the ultrastructural level. The nodules formed on top of a fibroblast-like multilayer of cells. The upper surface of the nodules was lined by a continuous layer of cuboidal osteoblastic cells often seen to be joined by adherens junctions. Numerous microvilli, membrane protrusions, and coated pits could be seen on the upper surface of these cells, their cytoplasm contained prominent RER and Golgi membranes, and processes extended from their lower surfaces into a dense, highly organized collagenous matrix. Some osteocyte-like cells were completely embedded within this matrix; they also displayed RER and prominent processes which extended through the matrix and often made both adherens and gap junctional contacts with the processes of other cells. The fibroblastic cells not participating in nodule formation were surrounded by a less dense collagenous matrix and, in contrast to the matrix of the nodules, it did not mineralize. An unmineralized osteoid-like layer was seen directly below the cuboidal top layer of cells. A mineralization front was detectable below this in which small, discrete structures resembling matrix vesicles and feathery mineral crystals were evident and frequently associated with the collagen fibrils. More heavily mineralized areas were seen further into the nodule. Electron microprobe and electron and X-ray diffraction analysis confirmed the mineral to be hydroxyapatite

Modification of surface/neuron interfaces for neural cell-type specific responses: a review

International Nuclear Information System (INIS)

Chen, Cen; Kong, Xiangdong; Lee, In-Seop

2016-01-01

Surface/neuron interfaces have played an important role in neural repair including neural prostheses and tissue engineered scaffolds. This comprehensive literature review covers recent studies on the modification of surface/neuron interfaces. These interfaces are identified in cases both where the surfaces of substrates or scaffolds were in direct contact with cells and where the surfaces were modified to facilitate cell adhesion and controlling cell-type specific responses. Different sources of cells for neural repair are described, such as pheochromocytoma neuronal-like cell, neural stem cell (NSC), embryonic stem cell (ESC), mesenchymal stem cell (MSC) and induced pluripotent stem cell (iPS). Commonly modified methods are discussed including patterned surfaces at micro- or nano-scale, surface modification with conducting coatings, and functionalized surfaces with immobilized bioactive molecules. These approaches to control cell-type specific responses have enormous potential implications in neural repair. (paper)

Direct calculation of resonance energies and widths using an R-matrix approach

International Nuclear Information System (INIS)

Schneider, B.I.

1981-01-01

A modified R-matrix technique is presented which determines the eigenvalues and widths of resonant states by the direct diagonalization of a complex, non-Hermitian matrix. The method utilizes only real basis sets and requires a minimum of complex arithmetic. The method is applied to two problems, a set of coupled square wells and the Pi/sub g/ resonance of N 2 in the static-exchange approximation. The results of the calculation are in good agreement with other methods and converge very quickly with basis-set size

Osteogenic Matrix Cell Sheets Facilitate Osteogenesis in Irradiated Rat Bone

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Yoshinobu Uchihara

2015-01-01

Full Text Available Reconstruction of large bone defects after resection of malignant musculoskeletal tumors is a significant challenge in orthopedic surgery. Extracorporeal autogenous irradiated bone grafting is a treatment option for bone reconstruction. However, nonunion often occurs because the osteogenic capacity is lost by irradiation. In the present study, we established an autogenous irradiated bone graft model in the rat femur to assess whether osteogenic matrix cell sheets improve osteogenesis of the irradiated bone. Osteogenic matrix cell sheets were prepared from bone marrow-derived stromal cells and co-transplanted with irradiated bone. X-ray images at 4 weeks after transplantation showed bridging callus formation around the irradiated bone. Micro-computed tomography images at 12 weeks postoperatively showed abundant callus formation in the whole circumference of the irradiated bone. Histology showed bone union between the irradiated bone and host femur. Mechanical testing showed that the failure force at the irradiated bone site was significantly higher than in the control group. Our study indicates that osteogenic matrix cell sheet transplantation might be a powerful method to facilitate osteogenesis in irradiated bones, which may become a treatment option for reconstruction of bone defects after resection of malignant musculoskeletal tumors.

Periodontal Bioengineering: A Discourse in Surface Topographies, Progenitor Cells and Molecular Profiles

Science.gov (United States)

Dangaria, Smit J.

2011-12-01

Stem/progenitor cells are a population of cells capable of providing replacement cells for a given differentiated cell type. We have applied progenitor cell-based technologies to generate novel tissue-engineered implants that use biomimetic strategies with the ultimate goal of achieving full regeneration of lost periodontal tissues. Mesenchymal periodontal tissues such as cementum, alveolar bone (AB), and periodontal ligament (PDL) are neural crest-derived entities that emerge from the dental follicle (DF) at the onset of tooth root formation. Using a systems biology approach we have identified key differences between these periodontal progenitors on the basis of global gene expression profiles, gene cohort expression levels, and epigenetic modifications, in addition to differences in cellular morphologies. On an epigenetic level, DF progenitors featured high levels of the euchromatin marker H3K4me3, whereas PDL cells, AB osteoblasts, and cementoblasts contained high levels of the transcriptional repressor H3K9me3. Secondly, we have tested the influence of natural extracellular hydroxyapatite matrices on periodontal progenitor differentiation. Dimension and structure of extracellular matrix surfaces have powerful influences on cell shape, adhesion, and gene expression. Here we show that natural tooth root topographies induce integrin-mediated extracellular matrix signaling cascades in tandem with cell elongation and polarization to generate physiological periodontium-like tissues. In this study we replanted surface topography instructed periodontal ligament progenitors (PDLPs) into rat alveolar bone sockets for 8 and 16 weeks, resulting in complete attachment of tooth roots to the surrounding alveolar bone with a periodontal ligament fiber apparatus closely matching physiological controls along the entire root surface. Displacement studies and biochemical analyses confirmed that progenitor-based engineered periodontal tissues were similar to control teeth and

Novel image analysis methods for quantification of in situ 3-D tendon cell and matrix strain.

Science.gov (United States)

Fung, Ashley K; Paredes, J J; Andarawis-Puri, Nelly

2018-01-23

Macroscopic tendon loads modulate the cellular microenvironment leading to biological outcomes such as degeneration or repair. Previous studies have shown that damage accumulation and the phases of tendon healing are marked by significant changes in the extracellular matrix, but it remains unknown how mechanical forces of the extracellular matrix are translated to mechanotransduction pathways that ultimately drive the biological response. Our overarching hypothesis is that the unique relationship between extracellular matrix strain and cell deformation will dictate biological outcomes, prompting the need for quantitative methods to characterize the local strain environment. While 2-D methods have successfully calculated matrix strain and cell deformation, 3-D methods are necessary to capture the increased complexity that can arise due to high levels of anisotropy and out-of-plane motion, particularly in the disorganized, highly cellular, injured state. In this study, we validated the use of digital volume correlation methods to quantify 3-D matrix strain using images of naïve tendon cells, the collagen fiber matrix, and injured tendon cells. Additionally, naïve tendon cell images were used to develop novel methods for 3-D cell deformation and 3-D cell-matrix strain, which is defined as a quantitative measure of the relationship between matrix strain and cell deformation. The results support that these methods can be used to detect strains with high accuracy and can be further extended to an in vivo setting for observing temporal changes in cell and matrix mechanics during degeneration and healing. Copyright © 2017. Published by Elsevier Ltd.

A novel functional site of extracellular matrix metalloproteinase inducer (EMMPRIN) that limits the migration of human uterine cervical carcinoma cells.

Science.gov (United States)

Sato, Takashi; Watanabe, Mami; Hashimoto, Kei; Ota, Tomoko; Akimoto, Noriko; Imada, Keisuke; Nomizu, Motoyoshi; Ito, Akira

2012-01-01

EMMPRIN (extracellular matrix metalloproteinase inducer)/CD147, a membrane-bound glycoprotein with two extracellular loop domains (termed loops I and II), progresses tumor invasion and metastasis by increasing the production of matrix metalloproteinase (MMP) in peritumoral stoma cells. EMMPRIN has also been associated with the control of migration activity in some tumor cells, but little is known about how EMMPRIN regulates tumor cell migration. In the present study, EMMPRIN siRNA suppressed the gene expression and production of EMMPRIN in human uterine cervical carcinoma SKG-II cells. An in vitro scratch wound assay showed enhancement of migration of EMMPRIN-knockdown SKG-II cells. In addition, the SKG-II cell migration was augmented by adding an E. coli-expressed human EMMPRIN mutant with two extracellular loop domains (eEMP-I/II), which bound to the cell surface of SKG-II cells. However, eEMP-I/II suppressed the native EMMPRIN-mediated augmentation of proMMP-1/procollagenase-1 production in a co-culture of the SKG-II cells and human uterine cervical fibroblasts, indicating that the augmentation of SKG-II cell migration resulted from the interference of native EMMPRIN functions by eEMP-I/II on the cell surface. Furthermore, a systematic peptide screening method using nine synthetic EMMPRIN peptides coding the loop I and II domains (termed EM1-9) revealed that EM9 (170HIENLNMEADPGQYR184) facilitated SKG-II cell migration. Moreover, SKG-II cell migration was enhanced by administration of an antibody against EM9, but not EM1 which is a crucial site for the MMP inducible activity of EMMPRIN. Therefore, these results provide novel evidence that EMMPRIN on the cell surface limits the cell migration of human uterine cervical carcinoma cells through 170HIENLNMEADPGQYR184 in the loop II domain. Finally, these results should provide an increased understanding of the functions of EMMPRIN in malignant cervical carcinoma cells, and could contribute to the development of

Downregulation of Connective Tissue Growth Factor by Three-Dimensional Matrix Enhances Ovarian Carcinoma Cell Invasion

Science.gov (United States)

Barbolina, Maria V.; Adley, Brian P.; Kelly, David L.; Shepard, Jaclyn; Fought, Angela J.; Scholtens, Denise; Penzes, Peter; Shea, Lonnie D.; Sharon Stack, M

2010-01-01

Epithelial ovarian carcinoma (EOC) is a leading cause of death from gynecologic malignancy, due mainly to the prevalence of undetected metastatic disease. The process of cell invasion during intra-peritoneal anchoring of metastatic lesions requires concerted regulation of many processes, including modulation of adhesion to the extracellular matrix and localized invasion. Exploratory cDNA microarray analysis of early response genes (altered after 4 hours of 3-dimensional collagen culture) coupled with confirmatory real-time RT-PCR, multiple three-dimensional cell culture matrices, Western blot, immunostaining, adhesion, migration, and invasion assays were used to identify modulators of adhesion pertinent to EOC progression and metastasis. cDNA microarray analysis indicated a dramatic downregulation of connective tissue growth factor (CTGF) in EOC cells placed in invasion-mimicking conditions (3-dimensional type I collagen). Examination of human EOC specimens revealed that CTGF expression was absent in 46% of the tested samples (n=41), but was present in 100% of normal ovarian epithelium samples (n=7). Reduced CTGF expression occurs in many types of cells and may be a general phenomenon displayed by cells encountering a 3D environment. CTGF levels were inversely correlated with invasion such that downregulation of CTGF increased, while its upregulation reduced, collagen invasion. Cells adhered preferentially to a surface comprised of both collagen I and CTGF relative to either component alone using α6β1 and α3β1 integrins. Together these data suggest that downregulation of CTGF in EOC cells may be important for cell invasion through modulation of cell-matrix adhesion. PMID:19382180

Metal Matrix Composite Solar Cell Metallization

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Wilt David M.

2017-01-01

Full Text Available Advanced solar cells are moving to ever thinner formats in order to save mass and in some cases improve performance. As cells are thinned, the possibility that they may fracture or cleave due to mechanical stresses is increased. Fractures of the cell can degrade the overall device performance if the fracture propagates through the contact metallization, which frequently occurs. To address this problem, a novel semiconductor metallization system based on multi-walled carbon nanotube (CNT reinforcement, termed metal matrix composite (MMC metallization is under investigation. Electro-mechanical characterization of MMC films demonstrate their ability to provide electrical conductivity over >40 micron wide cracks in the underlying semiconductor, with the carbon nanotubes bridging the gap. In addition, these materials show a “self-healing” behaviour, electrically reconnecting at ~30 microns when strained past failure. Triple junction (TJ space cells with MMC metallization demonstrated no loss in Jsc after intentional fracture, whereas TJ cells with conventional metallization suffer up to 50% Jsc loss.

1,8-Bis(dimethylamino)naphthalene/9-aminoacridine: A new binary matrix for lipid fingerprinting of intact bacteria by matrix assisted laser desorption ionization mass spectrometry

Energy Technology Data Exchange (ETDEWEB)

Calvano, C.D., E-mail: cosimadamiana.calvano@uniba.it [Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Via Orabona, 4, 70126 Bari (Italy); Monopoli, A.; Ditaranto, N. [Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Via Orabona, 4, 70126 Bari (Italy); Palmisano, F. [Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Via Orabona, 4, 70126 Bari (Italy); Centro Interdipartimentale di Ricerca S.M.A.R.T., Università degli Studi di Bari Aldo Moro, Via Orabona, 4, 70126 Bari (Italy)

2013-10-10

Graphical abstract: -- Highlights: •New binary matrix for less ionizable lipid analysis with no interfering peaks. •Combined MALDI and X-ray photoelectron spectroscopy (XPS) analyses. •Fast lipid fingerprint on Gram positive and Gram negative bacteria by MALDI MS. •Mapping of phospholipids by XPS imaging. •Very fast membrane lipid extraction procedure. -- Abstract: The effectiveness of a novel binary matrix composed of 1,8-bis(dimethylamino)naphthalene (DMAN; proton sponge) and 9-aminoacridine (9AA) for the direct lipid analysis of whole bacterial cells by matrix assisted laser desorption ionization mass spectrometry (MALDI MS) is demonstrated. Deprotonated analyte signals nearly free of matrix-related ions were observed in negative ion mode. The effect of the most important factors (laser energy, pulse voltage, DMAN/9AA ratio, analyte/matrix ratio) was investigated using a Box–Behnken response surface design followed by multi-response optimization in order to simultaneously maximize signal-to-noise (S/N) ratio and resolution. The chemical surface composition of single or mixed matrices was explored by X-ray photoelectron spectroscopy (XPS). Moreover, XPS imaging was used to map the spatial distribution of a model phospholipid in single or binary matrices. The DMAN/9AA binary matrix was then successfully applied to the analysis of intact Gram positive (Lactobacillus sanfranciscensis) or Gram negative (Escherichia coli) microorganisms. About fifty major membrane components (free fatty acids, mono-, di- and tri-glycerides, phospholipids, glycolipids and cardiolipins) were quickly and easily detected over a mass range spanning from ca. 200 to ca. 1600 m/z. Moreover, mass spectra with improved S/N ratio (compared to single matrices), reduced chemical noise and no formation of matrix-clusters were invariably obtained demonstrating the potential of this binary matrix to improve sensitivity.

1,8-Bis(dimethylamino)naphthalene/9-aminoacridine: A new binary matrix for lipid fingerprinting of intact bacteria by matrix assisted laser desorption ionization mass spectrometry

International Nuclear Information System (INIS)

Calvano, C.D.; Monopoli, A.; Ditaranto, N.; Palmisano, F.

2013-01-01

Graphical abstract: -- Highlights: •New binary matrix for less ionizable lipid analysis with no interfering peaks. •Combined MALDI and X-ray photoelectron spectroscopy (XPS) analyses. •Fast lipid fingerprint on Gram positive and Gram negative bacteria by MALDI MS. •Mapping of phospholipids by XPS imaging. •Very fast membrane lipid extraction procedure. -- Abstract: The effectiveness of a novel binary matrix composed of 1,8-bis(dimethylamino)naphthalene (DMAN; proton sponge) and 9-aminoacridine (9AA) for the direct lipid analysis of whole bacterial cells by matrix assisted laser desorption ionization mass spectrometry (MALDI MS) is demonstrated. Deprotonated analyte signals nearly free of matrix-related ions were observed in negative ion mode. The effect of the most important factors (laser energy, pulse voltage, DMAN/9AA ratio, analyte/matrix ratio) was investigated using a Box–Behnken response surface design followed by multi-response optimization in order to simultaneously maximize signal-to-noise (S/N) ratio and resolution. The chemical surface composition of single or mixed matrices was explored by X-ray photoelectron spectroscopy (XPS). Moreover, XPS imaging was used to map the spatial distribution of a model phospholipid in single or binary matrices. The DMAN/9AA binary matrix was then successfully applied to the analysis of intact Gram positive (Lactobacillus sanfranciscensis) or Gram negative (Escherichia coli) microorganisms. About fifty major membrane components (free fatty acids, mono-, di- and tri-glycerides, phospholipids, glycolipids and cardiolipins) were quickly and easily detected over a mass range spanning from ca. 200 to ca. 1600 m/z. Moreover, mass spectra with improved S/N ratio (compared to single matrices), reduced chemical noise and no formation of matrix-clusters were invariably obtained demonstrating the potential of this binary matrix to improve sensitivity

Investigation of in vitro bone cell adhesion and proliferation on Ti using direct current stimulation

International Nuclear Information System (INIS)

Bodhak, Subhadip; Bose, Susmita; Kinsel, William C.; Bandyopadhyay, Amit

2012-01-01

Our objective was to establish an in vitro cell culture protocol to improve bone cell attachment and proliferation on Ti substrate using direct current stimulation. For this purpose, a custom made electrical stimulator was developed and a varying range of direct currents, from 5 to 25 μA, was used to study the current stimulation effect on bone cells cultured on conducting Ti samples in vitro. Cell–material interaction was studied for a maximum of 5 days by culturing with human fetal osteoblast cells (hFOB). The direct current was applied in every 8 h time interval and the duration of electrical stimulation was kept constant at 15 min for all cases. In vitro results showed that direct current stimulation significantly favored bone cell attachment and proliferation in comparison to nonstimulated Ti surface. Immunochemistry and confocal microscopy results confirmed that the cell adhesion was most pronounced on 25 μA direct current stimulated Ti surfaces as hFOB cells expressed higher vinculin protein with increasing amount of direct current. Furthermore, MTT assay results established that cells grew 30% higher in number under 25 μA electrical stimulation as compared to nonstimulated Ti surface after 5 days of culture period. In this work we have successfully established a simple and cost effective in vitro protocol offering easy and rapid analysis of bone cell–material interaction which can be used in promotion of bone cell attachment and growth on Ti substrate using direct current electrical stimulation in an in vitro model. - Highlights: ► D.C. stimulation was used to enhance in vitro bone cell adhesion and proliferation. ► Cells cultured on Ti were stimulated by using a custom made electrical stimulator. ► Optimization was performed by using a varying range of direct currents ∼ 5 to 25 μA. ► 25 μA stimulation was found most beneficial for promotion of cell adhesion/growth.

Dynamic 3D cell rearrangements guided by a fibronectin matrix underlie somitogenesis.

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Gabriel G Martins

Full Text Available Somites are transient segments formed in a rostro-caudal progression during vertebrate development. In chick embryos, segmentation of a new pair of somites occurs every 90 minutes and involves a mesenchyme-to-epithelium transition of cells from the presomitic mesoderm. Little is known about the cellular rearrangements involved, and, although it is known that the fibronectin extracellular matrix is required, its actual role remains elusive. Using 3D and 4D imaging of somite formation we discovered that somitogenesis consists of a complex choreography of individual cell movements. Epithelialization starts medially with the formation of a transient epithelium of cuboidal cells, followed by cell elongation and reorganization into a pseudostratified epithelium of spindle-shaped epitheloid cells. Mesenchymal cells are then recruited to this medial epithelium through accretion, a phenomenon that spreads to all sides, except the lateral side of the forming somite, which epithelializes by cell elongation and intercalation. Surprisingly, an important contribution to the somite epithelium also comes from the continuous egression of mesenchymal cells from the core into the epithelium via its apical side. Inhibition of fibronectin matrix assembly first slows down the rate, and then halts somite formation, without affecting pseudopodial activity or cell body movements. Rather, cell elongation, centripetal alignment, N-cadherin polarization and egression are impaired, showing that the fibronectin matrix plays a role in polarizing and guiding the exploratory behavior of somitic cells. To our knowledge, this is the first 4D in vivo recording of a full mesenchyme-to-epithelium transition. This approach brought new insights into this event and highlighted the importance of the extracellular matrix as a guiding cue during morphogenesis.

Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.

Science.gov (United States)

Thievessen, Ingo; Fakhri, Nikta; Steinwachs, Julian; Kraus, Viola; McIsaac, R Scott; Gao, Liang; Chen, Bi-Chang; Baird, Michelle A; Davidson, Michael W; Betzig, Eric; Oldenbourg, Rudolf; Waterman, Clare M; Fabry, Ben

2015-11-01

Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation. © FASEB.

Novel Direction Of Arrival Estimation Method Based on Coherent Accumulation Matrix Reconstruction

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Li Lei

2015-04-01

Full Text Available Based on coherent accumulation matrix reconstruction, a novel Direction Of Arrival (DOA estimation decorrelation method of coherent signals is proposed using a small sample. First, the Signal to Noise Ratio (SNR is improved by performing coherent accumulation operation on an array of observed data. Then, according to the structure characteristics of the accumulated snapshot vector, the equivalent covariance matrix, whose rank is the same as the number of array elements, is constructed. The rank of this matrix is proved to be determined just by the number of incident signals, which realize the decorrelation of coherent signals. Compared with spatial smoothing method, the proposed method performs better by effectively avoiding aperture loss with high-resolution characteristics and low computational complexity. Simulation results demonstrate the efficiency of the proposed method.

α2 Integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells

International Nuclear Information System (INIS)

Ozeki, Nobuaki; Kawai, Rie; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

2015-01-01

We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blot and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3, integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells. - Highlights: • Odontoblast differentiation requires activation of α2 integrin, Emmprin and MMP-3. • α2 integrin, Emmprin and MMP-3 form a sequential signaling cascade. • β1 integrin acts a specific trigger for odontoblast differentiation. • The role of these effectors is highly novel and unanticipated

α2 Integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells

Energy Technology Data Exchange (ETDEWEB)

Ozeki, Nobuaki; Kawai, Rie; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki [Department of Endodontics, School of Dentistry, Aichi Gakuin University, Nagoya, Aichi 464-8651 (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650 (Japan); Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, Nagoya, Aichi 464-8651 (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650 (Japan)

2015-02-01

We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blot and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3, integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells. - Highlights: • Odontoblast differentiation requires activation of α2 integrin, Emmprin and MMP-3. • α2 integrin, Emmprin and MMP-3 form a sequential signaling cascade. • β1 integrin acts a specific trigger for odontoblast differentiation. • The role of these effectors is highly novel and unanticipated.

Calcium alginate gels as stem cell matrix-making paracrine stem cell activity available for enhanced healing after surgery.

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Andreas Schmitt

Full Text Available Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs.

Inverting adherent cells for visualizing ECM interactions at the basal cell side

Energy Technology Data Exchange (ETDEWEB)

Gudzenko, Tetyana [DFG-Center for Functional Nanostructures, Karlsruhe Institute of Technology (KIT), Wolfgang-Gaede-Strasse 1a, 76131 Karlsruhe (Germany); Franz, Clemens M., E-mail: clemens.franz@kit.edu [DFG-Center for Functional Nanostructures, Karlsruhe Institute of Technology (KIT), Wolfgang-Gaede-Strasse 1a, 76131 Karlsruhe (Germany)

2013-05-15

Interactions with the extracellular matrix (ECM) govern a wide range of cellular functions, including survival, migration and invasion. However, in adherent cells these interactions occur primarily on the basal cell side, making them inaccessible to high-resolution, surface-scanning imaging techniques such as atomic force microscopy (AFM) or scanning electron microscopy (SEM). Here we describe a fast and reliable method for inverting adherent cells, exposing the basal cell membrane for direct analysis by AFM or SEM in combination with fluorescence microscopy. Cells including their matrix adhesion sites remain intact during the inversion process and are transferred together with the complete array of basally associated ECM proteins. Molecular features of ECM proteins, such as the characteristic 67 nm collagen D-periodicity, are well preserved after inversion. To demonstrate the versatility of the method, we compared basal interactions of fibroblasts with fibrillar collagen I and fibronectin matrices. While fibroblasts remodel the fibronectin layer exclusively from above, they actively invade even thin collagen layers by contacting individual collagen nanofibrils both basally and apically through a network of cellular extensions. Cell–matrix entanglement coincides with enhanced cell spreading and flattening, indicating that nanoscale ECM interactions govern macroscopic changes in cell morphology. The presented cell inversion technique can thus provide novel insight into nanoscale cell–matrix interactions at the basal cell side. - Highlights: ► We present a novel method for inverting adherent cells to expose the basal cell side. ► Basal cell sides can be imaged at high resolution by AFM and SEM. ► Cells can be inverted together with the underlying extracellular matrix. ► AFM images of inverted cells provide a nanoscale look at basal cell–ECM interactions.

Endothelial cell-derived matrix promotes the metabolic functional maturation of hepatocyte via integrin-Src signalling.

Science.gov (United States)

Guo, Xinyue; Li, Weihong; Ma, Minghui; Lu, Xin; Zhang, Haiyan

2017-11-01

The extracellular matrix (ECM) microenvironment is involved in the regulation of hepatocyte phenotype and function. Recently, the cell-derived extracellular matrix has been proposed to represent the bioactive and biocompatible materials of the native ECM. Here, we show that the endothelial cell-derived matrix (EC matrix) promotes the metabolic maturation of human adipose stem cell-derived hepatocyte-like cells (hASC-HLCs) through the activation of the transcription factor forkhead box protein A2 (FOXA2) and the nuclear receptors hepatocyte nuclear factor 4 alpha (HNF4α) and pregnane X receptor (PXR). Reducing the fibronectin content in the EC matrix or silencing the expression of α5 integrin in the hASC-HLCs inhibited the effect of the EC matrix on Src phosphorylation and hepatocyte maturation. The inhibition of Src phosphorylation using the inhibitor PP2 or silencing the expression of Src in hASC-HLCs also attenuated the up-regulation of the metabolic function of hASC-HLCs in a nuclear receptor-dependent manner. These data elucidate integrin-Src signalling linking the extrinsic EC matrix signals and metabolic functional maturation of hepatocyte. This study provides a model for studying the interaction between hepatocytes and non-parenchymal cell-derived matrix. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Biomolecular strategies for cell surface engineering

Science.gov (United States)

Wilson, John Tanner

backbone molecular weight, PEG chain length, and grafting ratio, PLL-g-PEG copolymers were rendered cytocompatible and used to initiate and propagate the growth of cell surface-supported PEM films. Planar characterization of this novel class of PEM films indicated that film thickness and composition may be tailored through appropriate control of layer number and copolymer properties. Furthermore, these investigations have helped establish a conceptual framework for the rational design of cell surface-supported thin films, with the objective of translating the diverse biomedical and biotechnological applications of PEM films to cellular interfaces. Important to the development of effective conformal islet coatings is an inherent strategy through which to incorporate bioactive molecules for directing desired biochemical or cellular responses. Towards this end, PLL-g-PEG copolymers functionalized with biotin, azide, and hydrazide moieties were synthesized and used, either alone or in combination, to capture streptavidin-, triphenylphosphine-, and aldehyde-labeled probes, respectively, on the islet surface. Additionally, PEM films assembled using alginate chemically modified to contain aldehyde groups could be used to introduce hydrazide-functionalized molecules to the islet surface. Hence, modified film constituents may be used as modular elements for controlling the chemical composition cell and tissue surfaces. Finally, we report a strategy for tethering thrombomodulin (TM) to the islet surface. Through site-specific, C-terminal biotinylation of TM and optimization of cell surface biotinylation, TM could be integrated with the islet surface. Re-engineering of islet surfaces with TM resulted in an increased catalytic capacity of islets to generate the powerful anti-inflammatory agent, activated protein C (APC), thereby providing a facile strategy for increasing the local concentration of APC at the site of transplantation.

Promising Noninvasive Cellular Phenotype in Prostate Cancer Cells Knockdown of Matrix Metalloproteinase 9

Directory of Open Access Journals (Sweden)

Aditi Gupta

2013-01-01

Full Text Available Cell surface interaction of CD44 and MMP9 increases migration and invasion of PC3 cells. We show here that stable knockdown of MMP9 in PC3 cells switches CD44 isoform expression from CD44s to CD44v6 which is more glycosylated. These cells showed highly adhesive morphology with extensive cell spreading which is due to the formation of focal adhesions and well organized actin-stress fibers. MMP9 knockdown blocks invadopodia formation and matrix degradation activity as well. However, CD44 knockdown PC3 cells failed to develop focal adhesions and stress fibers; hence these cells make unstable adhesions. A part of the reason for these changes could be caused by silencing of CD44v6 as well. Immunostaining of prostate tissue microarray sections illustrated significantly lower levels of CD44v6 in adenocarcinoma than normal tissue. Our results suggest that interaction between CD44 and MMP9 is a potential mechanism of invadopodia formation. CD44v6 expression may be essential for the protection of non-invasive cellular phenotype. CD44v6 decrease may be a potential marker for prognosis and therapeutics.

Nuclear matrix and structural and functional compartmentalization of the eucaryotic cell nucleus.

Science.gov (United States)

Razin, S V; Borunova, V V; Iarovaia, O V; Vassetzky, Y S

2014-07-01

Becoming popular at the end of the 20th century, the concept of the nuclear matrix implies the existence of a nuclear skeleton that organizes functional elements in the cell nucleus. This review presents a critical analysis of the results obtained in the study of nuclear matrix in the light of current views on the organization of the cell nucleus. Numerous studies of nuclear matrix have failed to provide evidence of the existence of such a structure. Moreover, the existence of a filamentous structure that supports the nuclear compartmentalization appears to be unnecessary, since this function is performed by the folded genome itself.

Dependence of the specific surface area of the nuclear fuel with the matrix oxidation

International Nuclear Information System (INIS)

Gomez, F.; Quinones, J.; Iglesias, E.; Rodriguez, N.

2008-01-01

This paper is focused on the study of the changes in the specific surface area measured using BET techniques. The objective is to obtain a relation between this parameter and the change in the matrix stoichiometry (i.e., oxidation increase). None of the actual models used for extrapolating the behaviour of the spent fuel matrix under repository conditions have included this dependence yet. In this work the specific surface area of different uranium oxide were measured using N 2 (g) and Kr(g). The starting material was UO 2+x (s) with a size powder distribution lower than 20 μm. The results included in this paper shown a strong dependence on specific surface area with the matrix stoichiometry, i.e., and increase of more than one order of magnitude (SUO 2 = 6 m 2 *g -1 and SU 3 O 8 = 16.07 m 2 *g -1 ). Furthermore, the particle size distribution measured as a function of the thermal treatment done shows changes on the powder size related to the changes observed in the uranium oxide stoichiometry. (authors)

Non-supersymmetric matrix strings from generalized Yang-Mills theory on arbitrary Riemann surfaces

International Nuclear Information System (INIS)

Billo, M.; D'Adda, A.; Provero, P.

2000-01-01

We quantize pure 2d Yang-Mills theory on an arbitrary Riemann surface in the gauge where the field strength is diagonal. Twisted sectors originate, as in Matrix string theory, from permutations of the eigenvalues around homotopically non-trivial loops. These sectors, that must be discarded in the usual quantization due to divergences occurring when two eigenvalues coincide, can be consistently kept if one modifies the action by introducing a coupling of the field strength to the space-time curvature. This leads to a generalized Yang-Mills theory whose action reduces to the usual one in the limit of zero curvature. After integrating over the non-diagonal components of the gauge fields, the theory becomes a free string theory (sum over unbranched coverings) with a U(1) gauge theory on the world-sheet. This is shown to be equivalent to a lattice theory with a gauge group which is the semi-direct product of S N and U(1) N . By using well known results on the statistics of coverings, the partition function on arbitrary Riemann surfaces and the kernel functions on surfaces with boundaries are calculated. Extensions to include branch points and non-abelian groups on the world-sheet are briefly commented upon

Nanofiber density determines endothelial cell behavior on hydrogel matrix

Energy Technology Data Exchange (ETDEWEB)

Berti, Fernanda V., E-mail: fernanda@intelab.ufsc.br [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Rambo, Carlos R. [Department of Electrical Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Dias, Paulo F. [Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Porto, Luismar M. [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil)

2013-12-01

When cultured under static conditions, bacterial cellulose pellicles, by the nature of the polymer synthesis that involves molecular oxygen, are characterized by two distinct surface sides. The upper surface is denser in fibers (entangled) than the lower surface that shows greater surface porosity. Human umbilical vein endothelial cells (HUVECs) were used to exploit how the microarchitecture (i.e., surface porosity, fiber network structure, surface topology, and fiber density) of bacterial cellulose pellicle surfaces influence cell–biomaterial interaction and therefore cell behavior. Adhesion, cell ingrowth, proliferation, viability and cell death mechanisms were evaluated on the two pellicle surface sides. Cell behavior, including secondary necrosis, is influenced only by the microarchitecture of the surface, since the biomaterial is extremely pure (constituted of cellulose and water only). Cell–cellulose fiber interaction is the determinant signal in the cell–biomaterial responses, isolated from other frequently present interferences such as protein and other chemical traces usually present in cell culture matrices. Our results suggest that microarchitecture of hydrogel materials might determine the performance of biomedical products, such as bacterial cellulose tissue engineering constructs (BCTECs). - Highlights: • Topography of BC pellicle is relevant to determine endothelial cells' fate. • Cell–biomaterial response is affected by the topography of BC-pellicle surface. • Endothelial cells exhibit different behavior depending on the BC topography. • Apoptosis and necrosis of endothelial cells were affected by the BC topography.

Nanofiber density determines endothelial cell behavior on hydrogel matrix

International Nuclear Information System (INIS)

Berti, Fernanda V.; Rambo, Carlos R.; Dias, Paulo F.; Porto, Luismar M.

2013-01-01

When cultured under static conditions, bacterial cellulose pellicles, by the nature of the polymer synthesis that involves molecular oxygen, are characterized by two distinct surface sides. The upper surface is denser in fibers (entangled) than the lower surface that shows greater surface porosity. Human umbilical vein endothelial cells (HUVECs) were used to exploit how the microarchitecture (i.e., surface porosity, fiber network structure, surface topology, and fiber density) of bacterial cellulose pellicle surfaces influence cell–biomaterial interaction and therefore cell behavior. Adhesion, cell ingrowth, proliferation, viability and cell death mechanisms were evaluated on the two pellicle surface sides. Cell behavior, including secondary necrosis, is influenced only by the microarchitecture of the surface, since the biomaterial is extremely pure (constituted of cellulose and water only). Cell–cellulose fiber interaction is the determinant signal in the cell–biomaterial responses, isolated from other frequently present interferences such as protein and other chemical traces usually present in cell culture matrices. Our results suggest that microarchitecture of hydrogel materials might determine the performance of biomedical products, such as bacterial cellulose tissue engineering constructs (BCTECs). - Highlights: • Topography of BC pellicle is relevant to determine endothelial cells' fate. • Cell–biomaterial response is affected by the topography of BC-pellicle surface. • Endothelial cells exhibit different behavior depending on the BC topography. • Apoptosis and necrosis of endothelial cells were affected by the BC topography

Interaction of Munc18c and syntaxin4 facilitates invadopodium formation and extracellular matrix invasion of tumor cells.

Science.gov (United States)

Brasher, Megan I; Martynowicz, David M; Grafinger, Olivia R; Hucik, Andrea; Shanks-Skinner, Emma; Uniacke, James; Coppolino, Marc G

2017-09-29

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

GLYCAN-DIRECTED CAR-T CELLS.

Science.gov (United States)

Steentoft, Catharina; Migliorini, Denis; King, Tiffany R; Mandel, Ulla; June, Carl H; Posey, Avery D

2018-01-23

Cancer immunotherapy is rapidly advancing in the treatment of a variety of hematopoietic cancers, including pediatric acute lymphoblastic leukemia and diffuse large B cell lymphoma, with chimeric antigen receptor (CAR)-T cells. CARs are genetically encoded artificial T cell receptors that combine the antigen specificity of an antibody with the machinery of T cell activation. However, implementation of CAR technology in the treatment of solid tumors has been progressing much slower. Solid tumors are characterized by a number of challenges that need to be overcome, including cellular heterogeneity, immunosuppressive tumor microenvironment (TME), and, in particular, few known cancer-specific targets. Post-translational modifications that differentially occur in malignant cells generate valid cell surface, cancer-specific targets for CAR-T cells. We previously demonstrated that CAR-T cells targeting an aberrant O-glycosylation of MUC1, a common cancer marker associated with changes in cell adhesion, tumor growth, and poor prognosis, could control malignant growth in mouse models. Here, we discuss the field of glycan-directed CAR-T cells and review the different classes of antibodies specific for glycan-targeting, including the generation of high affinity O-glycopeptide antibodies. Finally, we discuss historic and recently investigated glycan targets for CAR-T cells and provide our perspective on how targeting the tumor glycoproteome and/or glycome will improve CAR-T immunotherapy. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Proteoglycans, ion channels and cell-matrix adhesion

DEFF Research Database (Denmark)

Mitsou, Ioli; Multhaupt, Hinke A.B.; Couchman, John R.

2017-01-01

, maintenance, repair and disease.The cytoplasmic domains of syndecans, while having no intrinsic kinase activity, can nevertheless signal through binding proteins.All syndecans appear to be connected to the actin cytoskeleton and can therefore contribute to cell adhesion, notably to the ECM and migration.......Recent data now suggest that syndecans can regulate stretchactivated ion channels.The structure and function of the syndecans and the ion channels are reviewed here, along with an analysis of ion channel functions in cell-matrix adhesion.This area sheds new light on the syndecans, not least since evidence...

Aminopeptidase N is directly sorted to the apical domain in MDCK cells

DEFF Research Database (Denmark)

Wessels, H P; Hansen, Gert Helge; Fuhrer, C

1990-01-01

In different epithelial cell types, integral membrane proteins appear to follow different sorting pathways to the apical surface. In hepatocytes, several apical proteins were shown to be transported there indirectly via the basolateral membrane, whereas in MDCK cells a direct sorting pathway from...

Topographical cues of direct metal laser sintering titanium surfaces facilitate osteogenic differentiation of bone marrow mesenchymal stem cells through epigenetic regulation.

Science.gov (United States)

Zheng, Guoying; Guan, Binbin; Hu, Penghui; Qi, Xingying; Wang, Pingting; Kong, Yu; Liu, Zihao; Gao, Ping; Li, Rui; Zhang, Xu; Wu, Xudong; Sui, Lei

2018-04-27

To investigate the role of hierarchical micro/nanoscale topography of direct metal laser sintering (DMLS) titanium surfaces in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), as well as the possible underlying epigenetic mechanism. Three groups of titanium specimens were prepared, including DMLS group, sandblasted, large-grit, acid-etched (SLA) group and smooth titanium (Ti) group. BMSCs were cultured on discs followed by surface characterization. Cell adhesion and proliferation were examined by SEM and CCK-8 assay, while osteogenic-related gene expression was detected by real-time RT-PCR. Immunofluorescence, western blotting and in vivo study were also performed to evaluate the potential for osteogenic induction of materials. In addition, to investigate the underlying epigenetic mechanisms, immunofluorescence and western blotting were performed to evaluate the global level of H3K4me3 during osteogenesis. The H3K4me3 and H3K27me3 levels at the promoter area of the osteogenic gene Runx2 were detected by ChIP assay. The DMLS surface exhibits greater protein adsorption ability and shows better cell adhesion performance than SLA and Ti surfaces. Moreover, both in vitro and in vivo studies demonstrated that the DMLS surface is more favourable for the osteogenic differentiation of BMSCs than SLA and Ti surfaces. Accordingly, osteogenesis-associated gene expression in BMSCs is efficiently induced by a rapid H3K27 demethylation and increase in H3K4me3 levels at gene promoters upon osteogenic differentiation on DMLS titanium surface. Topographical cues of DMLS surfaces have greater potential for the induction of osteogenic differentiation of BMSCs than SLA and Ti surfaces both in vitro and in vivo. A potential epigenetic mechanism is that the appropriate topography allows rapid H3K27 demethylation and an increased H3K4me3 level at the promoter region of osteogenesis-associated genes during the osteogenic differentiation of BMSCs. © 2018 John Wiley

Cell Surface Enzymatic Engineering-Based Approaches to Improve Cellular Therapies

KAUST Repository

AbuElela, Ayman

2014-06-06

The cell surface represents the interface between the cell and its environment. As such, the cell surface controls cell–cell interactions and functions such as adhesion and migration, and will transfer external cues to regulate processes such as survival, death, and differentiation. Redefining the cell surface by temporarily (or permanently) modifying the molecular landscape of the plasma membrane affects the way in which the cell interacts with its environment and influences the information that is relayed into the cell along downstream signaling pathways. This chapter outlines the role of key enzymes, the glycosyltransferases, in posttranslationally modifying proteins and lipids to fine-tune cells, ability to migrate. These enzymes are critical in controlling the formation of a platform structure, sialyl Lewis x (sLex), on circulating cells that plays a central role in the recognition and recruitment by selectin counter receptors on endothelial cells that line blood vessels of tissues throughout the body. By developing methods to manipulate the activity of these enzymes and hence the cell surface structures that result, treatments can be envisioned that direct the migration of therapeutic cells to specific locations throughout the body and also to inhibit metastasis of detrimental cells such as circulating tumor cells.

Calculation of the Cholesky factor directly from the stiffness matrix of the structural element

International Nuclear Information System (INIS)

Prates, C.L.M.; Soriano, H.L.

1978-01-01

The analysis of the structures of nuclear power plants requires the evaluation of the internal forces. This is attained by the solution of a system of equations. This solution takes most of the computing time and memory. One of the ways it can be achieved is based on the Cholesky factor. The structural matrix of the coeficients is transformed into an upper triangular matrix by the Cholesky decomposition. Cholesky factor can be obtained directly from the stiffness matrix of the structural element. The result can thus be obtained in a more precise and quick way. (Author)

Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation

Directory of Open Access Journals (Sweden)

Hanley Edward N

2000-10-01

Full Text Available Abstract Background The relationship between cell shape, proliferation, and extracellular matrix (ECM production, important aspects of cell behavior, is examined in a little-studied cell type, the human annulus cell from the intervertebral disc, during monolayer vs three-dimensional (3D culture. Results Three experimental studies showed that cells respond specifically to culture microenvironments by changes in cell shape, mitosis and ECM production: 1 Cell passages showed extensive immunohistochemical evidence of Type I and II collagens only in 3D culture. Chondroitin sulfate and keratan sulfate were abundant in both monolayer and 3D cultures. 2 Cells showed significantly greater proliferation in monolayer in the presence of platelet-derived growth factor compared to cells in 3D. 3 Cells on Matrigel™-coated monolayer substrates became rounded and formed nodular colonies, a finding absent during monolayer growth. Conclusions The cell's in vivo interactions with the ECM can regulate shape, gene expression and other cell functions. The shape of the annulus cell changes markedly during life: the young, healthy disc contains spindle shaped cells and abundant collagen. With aging and degeneration, many cells assume a strikingly different appearance, become rounded and are surrounded by unusual accumulations of ECM products. In vitro manipulation of disc cells provides an experimental window for testing how disc cells from given individuals respond when they are grown in environments which direct cells to have either spindle- or rounded-shapes. In vitro assessment of the response of such cells to platelet-derived growth factor and to Matrigel™ showed a continued influence of cell shape even in the presence of a growth factor stimulus. These findings contribute new information to the important issue of the influence of cell shape on cell behavior.

Extracellular-matrix-mediated osmotic pressure drives Vibrio cholerae biofilm expansion and cheater exclusion

OpenAIRE

Yan, Jing; Nadell, Carey D.; Stone, Howard A.; Wingreen, Ned S.; Bassler, Bonnie L.

2017-01-01

Biofilms, surface-attached communities of bacteria encased in an extracellular matrix, are a major mode of bacterial life. How the material properties of the matrix contribute to biofilm growth and robustness is largely unexplored, in particular in response to environmental perturbations such as changes in osmotic pressure. Here, using Vibrio cholerae as our model organism, we show that during active cell growth, matrix production enables biofilm-dwelling bacterial cells to establish an osmot...

Concentric artificial impedance surface for directional sound beamforming

Directory of Open Access Journals (Sweden)

Kyungjun Song

2017-03-01

Full Text Available Utilizing acoustic metasurfaces consisting of subwavelength resonant textures, we design an artificial impedance surface by creating a new boundary condition. We demonstrate a circular artificial impedance surface with surface impedance modulation for directional sound beamforming in three-dimensional space. This artificial impedance surface is implemented by revolving two-dimensional Helmholtz resonators with varying internal coiled path. Physically, the textured surface has inductive surface impedance on its inner circular patterns and capacitive surface impedance on its outer circular patterns. Directional receive beamforming can be achieved using an omnidirectional microphone located at the focal point formed by the gradient-impeding surface. In addition, the uniaxial surface impedance patterning inside the circular aperture can be used for steering the direction of the main lobe of the radiation pattern.

Depressed immune surveillance against cancer: role of deficient T cell: extracellular matrix interactions.

Science.gov (United States)

Górski, A; Castronovo, V; Stepień-Sopniewska, B; Grieb, P; Ryba, M; Mrowiec, T; Korczak-Kowalska, G; Wierzbicki, P; Matysiak, W; Dybowska, B

1994-07-01

Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.

The sintered microsphere matrix for bone tissue engineering: in vitro osteoconductivity studies.

Science.gov (United States)

Borden, Mark; Attawia, Mohamed; Laurencin, Cato T

2002-09-05

A tissue engineering approach has been used to design three-dimensional synthetic matrices for bone repair. The osteoconductivity and degradation profile of a novel polymeric bone-graft substitute was evaluated in an in vitro setting. Using the copolymer poly(lactide-co-glycolide) [PLAGA], a sintering technique based on microsphere technology was used to fabricate three-dimensional porous scaffolds for bone regeneration. Osteoblasts and fibroblasts were seeded onto a 50:50 PLAGA scaffold. Morphologic evaluation through scanning electron microscopy demonstrated that both cell types attached and spread over the scaffold. Cells migrated through the matrix using cytoplasmic extensions to bridge the structure. Cross-sectional images indicated that cellular proliferation had penetrated into the matrix approximately 700 microm from the surface. Examination of the surfaces of cell/matrix constructs demonstrated that cellular proliferation had encompassed the pores of the matrix by 14 days of cell culture. With the aim of optimizing polymer composition and polymer molecular weight, a degradation study was conducted utilizing the matrix. The results demonstrate that degradation of the sintered matrix is dependent on molecular weight, copolymer ratio, and pore volume. From this data, it was determined that 75:25 PLAGA with an initial molecular weight of 100,000 has an optimal degradation profile. These studies show that the sintered microsphere matrix has an osteoconductive structure capable of functioning as a cellular scaffold with a degradation profile suitable for bone regeneration. Copyright 2002 Wiley Periodicals, Inc.

Response of MG63 osteoblast-like cells onto polycarbonate membrane surfaces with different micropore sizes.

Science.gov (United States)

Lee, Sang Jin; Choi, Jin San; Park, Ki Suk; Khang, Gilson; Lee, Young Moo; Lee, Hai Bang

2004-08-01

Response of different types of cells on materials is important for the applications of tissue engineering and regenerative medicine. It is recognized that the behavior of the cell adhesion, proliferation, and differentiation on materials depends largely on surface characteristics such as wettability, chemistry, charge, rigidity, and roughness. In this study, we examined the behavior of MG63 osteoblast-like cells cultured on a polycarbonate (PC) membrane surfaces with different micropore sizes (0.2-8.0 microm in diameter). Cell adhesion and proliferation to the PC membrane surfaces were determined by cell counting and MTT assay. The effect of surface micropore on the MG63 cells was evaluated by cell morphology, protein content, and alkaline phosphatase (ALP) specific activity. It seems that the cell adhesion and proliferation were progressively inhibited as the PC membranes had micropores with increasing size, probably due to surface discontinuities produced by track-etched pores. Increasing micropore size of the PC membrane results in improved protein synthesis and ALP specific activity in isolated cells. There was a statistically significant difference (Pmicropore sizes. The MG63 cells also maintained their phenotype under conditions that support a round cell shape. RT-PCR analysis further confirmed the osteogenic phenotype of the MG63 cells onto the PC membranes with different micropore sizes. In results, as micropore size is getting larger, cell number is reduced and cell differentiation and matrix production is increased. This study demonstrated that the surface topography plays an important role for phenotypic expression of the MG63 osteoblast-like cells.

Kinetic Monte Carlo simulation of growth of Ge quantum dot multilayers with amorphous matrix

Energy Technology Data Exchange (ETDEWEB)

Endres, Jan, E-mail: endres.jan@gmail.com; Holý, Václav; Daniš, Stanislav [Charles University, Faculty of Mathematics and Physics (Czech Republic); Buljan, Maja [Ruđer Bošković Institute (Croatia)

2017-04-15

Kinetic Monte Carlo method is used to simulate the growth of germanium quantum dot multilayers with amorphous matrix. We modified a model for self-assembled growth of quantum dots in crystalline matrix for the case of the amorphous one. The surface morphology given as hills above the buried dots is the main driving force for the ordering of the quantum dots. In the simulations, we observed a short-range self-ordering in the lateral direction. The ordering in lateral and vertical direction depends strongly on the surface morphology, mostly on the strength how the deposited material replicates previous surfaces.

Transfer matrix treatment of atomic chemisorption on transition metal surface

International Nuclear Information System (INIS)

Mariz, A.M.; Koiller, B.

1980-05-01

The atomic adsorption of hydrogen on paramagnetic nickel 100 surface is studied, using the Green's function formalism and the transfer matrix technique, which allows the treatment of the geometry of the system in a simple manner. Electronic correlation at the adatom orbital in a self consistent Hartree-Fock approach is incorporated. The adsorption energy, local density of states and charge transfer between the solid and the adatom are calculated for different crystal structures (sc and fcc) and adatom positions at the surface. The results are discussed in comparison with other theories and with available experimental data, with satisfactory agreement. (Author) [pt

Introducing catalyst in alkaline membrane for improved performance direct borohydride fuel cells

Science.gov (United States)

Qin, Haiying; Lin, Longxia; Chu, Wen; Jiang, Wei; He, Yan; Shi, Qiao; Deng, Yonghong; Ji, Zhenguo; Liu, Jiabin; Tao, Shanwen

2018-01-01

A catalytic material is introduced into the polymer matrix to prepare a novel polymeric alkaline electrolyte membrane (AEM) which simultaneously increases ionic conductivity, reduces the fuel cross-over. In this work, the hydroxide anion exchange membrane is mainly composed of poly(vinylalcohol) and alkaline exchange resin. CoCl2 is added into the poly(vinylalcohol) and alkaline exchange resin gel before casting the membrane to introduce catalytic materials. CoCl2 is converted into CoOOH after the reaction with KOH solution. The crystallinity of the polymer matrix decreases and the ionic conductivity of the composite membrane is notably improved by the introduction of Co-species. A direct borohydride fuel cell using the composite membrane exhibits an open circuit voltage of 1.11 V at 30 °C, which is notably higher than that of cells using other AEMs. The cell using the composite membrane achieves a maximum power density of 283 mW cm-2 at 60 °C while the cell using the membrane without Co-species only reaches 117 mW cm-2 at the same conditions. The outstanding performance of the cell using the composite membrane benefits from impregnation of the catalytic Co-species in the membrane, which not only increases the ionic conductivity but also reduces electrode polarization thus improves the fuel cell performance. This work provides a new approach to develop high-performance fuel cells through adding catalysts in the electrolyte membrane.

Self assembled temperature responsive surfaces for generation of cell patches for bone tissue engineering

International Nuclear Information System (INIS)

Valmikinathan, Chandra M; ChangWei; Xu Jiahua; Yu Xiaojun

2012-01-01

One of the major challenges in the fabrication of tissue engineered scaffolds is the ability of the scaffold to biologically mimic autograft-like tissues. One of the alternate approaches to achieve this is by the application of cell seeded scaffolds with optimal porosity and mechanical properties. However, the current approaches for seeding cells on scaffolds are not optimal in terms of seeding efficiencies, cell penetration into the scaffold and more importantly uniform distribution of cells on the scaffold. Also, recent developments in scaffold geometries to enhance surface areas, pore sizes and porosities tend to further complicate the scenario. Cell sheet-based approaches for cell seeding have demonstrated a successful approach to generate scaffold-free tissue engineering approaches. However, the method of generating the temperature responsive surface is quite challenging and requires carcinogenic reagents and gamma rays. Therefore, here, we have developed temperature responsive substrates by layer-by-layer self assembly of smart polymers. Multilayer thin films prepared from tannic acid and poly N-isopropylacrylamide were fabricated based on their electrostatic and hydrogen bonding interactions. Cell attachment and proliferation studies on these thin films showed uniform cell attachment on the substrate, matching tissue culture plates. Also, the cells could be harvested as cell patches and sheets from the scaffolds, by reducing the temperature for a short period of time, and seeded onto porous scaffolds for tissue engineering applications. An enhanced cell seeding efficiency on scaffolds was observed using the cell patch-based technique as compared to seeding cells in suspension. Owing to the already pre-existent cell–cell and cell–extracellular matrix interactions, the cell patch showed the ability to reattach rapidly onto scaffolds and showed enhanced ability to proliferate and differentiate into a bone-like matrix. (paper)

Balancing Cell Migration with Matrix Degradation Enhances Gene Delivery to Cells Cultured Three-Dimensionally Within Hydrogels

Science.gov (United States)

Shepard, Jaclyn A.; Huang, Alyssa; Shikanova, Ariella; Shea, Lonnie D.

2010-01-01

In regenerative medicine, hydrogels are employed to fill defects and support the infiltration of cells that can ultimately regenerate tissue. Gene delivery within hydrogels targeting infiltrating cells has the potential to promote tissue formation, but the delivery efficiency of nonviral vectors within hydrogels is low hindering their applicability in tissue regeneration. To improve their functionality, we have conducted a mechanistic study to investigate the contribution of cell migration and matrix degradation on gene delivery. In this report, lipoplexes were entrapped within hydrogels based on poly(ethylene glycol) (PEG) crosslinked with peptides containing matrix metalloproteinase degradable sequences. The mesh size of these hydrogels is substantially less than the size of the entrapped lipoplexes, which can function to retain vectors. Cell migration and transfection were simultaneously measured within hydrogels with varying density of cell adhesion sites (Arg-Gly-Asp peptides) and solids content. Increasing RGD density increased expression levels up to 100-fold, while greater solids content sustained expression levels for 16 days. Increasing RGD density and decreasing solids content increased cell migration, which indicates expression levels increase with increased cell migration. Initially exposing cells to vector resulted in transient expression that declined after 2 days, verifying the requirement of migration to sustain expression. Transfected cells were predominantly located within the population of migrating cells for hydrogels that supported cell migration. Although the small mesh size retained at least 70% of the lipoplexes in the absence of cells after 32 days, the presence of cells decreased retention to 10% after 16 days. These results indicate that vectors retained within hydrogels contact migrating cells, and that persistent cell migration can maintain elevated expression levels. Thus matrix degradation and cell migration are fundamental design

Obtaining muonic density estimates via application of matrix formalism to proposed surface detector upgrade at the Pierre Auger Observatory

Energy Technology Data Exchange (ETDEWEB)

Schmidt, David; Engel, Ralph; Roth, Markus [Karlsruhe Institute of Technology, Karlsruhe (Germany); Collaboration: Pierre Auger-Collaboration

2015-07-01

Event-by-event identification of cosmic ray primary composition lends itself to enhanced event selection in the search for anisotropic arrival directions. Principally, the number of muons reaching Earth's surface in an extensive air shower is indicative of composition. The Pierre Auger Observatory seeks to capitalize on this axiom by improving reconstructed muonic density estimates via an upgrade to its surface detector array. This upgrade, consisting of placing a scintillator on top of each existing water Cherenkov detector, exploits the differing response of two detectors to muonic and electromagnetic particles. Exploitation of this difference may be expressed in a matrix formalism whose application to simulated proton and iron showers is presented here.

Improvement of the Convergence of the Invariant Imbedding T-Matrix Method

Science.gov (United States)

Zhai, S.; Panetta, R. L.; Yang, P.

2017-12-01

The invariant imbedding T-matrix method (IITM) is based on an electromagnetic volume integral equation to compute the T-matrix of an arbitrary scattering particle. A free-space Green's function is chosen as the integral kernel and thus each source point is placed in an imaginary vacuum spherical shell extending from the center to that source point. The final T-matrix (of the largest circumscribing sphere) is obtained through an iterative relation that, layer by layer, computes the T-matrix from the particle center to the outermost shell. On each spherical shell surface, an integration of the product of the refractive index 𝜀(𝜃, 𝜑) and vector spherical harmonics must be performed, resulting in the so-called U-matrix, which directly leads to the T-matrix on the spherical surface. Our observations indicate that the matrix size and sparseness are determined by the particular refractive index function 𝜀(𝜃, 𝜑). If 𝜀(𝜃, 𝜑) is an analytic function on the surface, then the matrix elements resulting from the integration decay rapidly, leading to sparse matrix; if 𝜀(𝜃, 𝜑) is not (for example, contains jump discontinuities), then the matrix elements decay slowly, leading to a large dense matrix. The intersection between an irregular scatterer and each spherical shell can leave jump discontinuities in 𝜀(𝜃, 𝜑) distributed over the shell surface. The aforementioned feature is analogous to the Gibbs phenomenon appearing in the orthogonal expansion of non-smooth functions with Hermitian eigenfunctions (complex exponential, Legendre, Bessel,...) where poor convergence speed is a direct consequence of the slow decay rate of the expansion coefficients. Various methods have been developed to deal with this slow convergence in the presence of discontinuities. Among the different approaches the most practical one may be a spectral filter: a filter is applied on the

Synthetic surface for expansion of human mesenchymal stem cells in xeno-free, chemically defined culture conditions.

Directory of Open Access Journals (Sweden)

Paula J Dolley-Sonneville

Full Text Available Human mesenchymal stem cells (HMSCS possess three properties of great interest for the development of cell therapies and tissue engineering: multilineage differentiation, immunomodulation, and production of trophic factors. Efficient ex vivo expansion of hMSCs is a challenging requirement for large scale production of clinical grade cells. Low-cost, robust, scalable culture methods using chemically defined materials need to be developed to address this need. This study describes the use of a xeno-free synthetic peptide acrylate surface, the Corning® Synthemax® Surface, for culture of hMSCs in serum-free, defined medium. Cell performance on the Corning Synthemax Surface was compared to cells cultured on biological extracellular matrix (ECM coatings in xeno-free defined medium and in traditional conditions on tissue culture treated (TCT plastic in fetal bovine serum (FBS supplemented medium. Our results show successful maintenance of hMSCs on Corning Synthemax Surface for eight passages, with cell expansion rate comparable to cells cultured on ECM and significantly higher than for cells in TCT/FBS condition. Importantly, on the Corning Synthemax Surface, cells maintained elongated, spindle-like morphology, typical hMSC marker profile and in vitro multilineage differentiation potential. We believe the Corning Synthemax Surface, in combination with defined media, provides a complete synthetic, xeno-free, cell culture system for scalable production of hMSCs.

The immunodominant influenza matrix t cell epitope recognized in human induces influenza protection in HLA-A2/Kb transgenic mice

International Nuclear Information System (INIS)

Plotnicky, H.; Cyblat-Chanal, D.; Aubry, J.-P.; Derouet, F.; Klinguer-Hamour, C.; Beck, A.; Bonnefoy, J.-Y.; Corvaiea, N.

2003-01-01

The protective efficacy of the influenza matrix protein epitope 58-66 (called M1), recognized in the context of human HLA-A2 molecules, was evaluated in a HLA-A2/K b transgenic mouse model of lethal influenza infection. Repeated subcutaneous immunizations with M1 increased the percentage of survival. This effect was mediated by T cells since protection was abolished following in vivo depletion of all T lymphocytes, CD8 + , or CD4 + T cells. The survival correlated with the detection of memory CD8 + splenocytes able to proliferate in vitro upon stimulation with M1 and to bind M1-loaded HLA-A2 dimers, as well as with M1-specific T cells in the lungs, which were directly cytotoxic to influenza-infected cells following influenza challenge. These results demonstrated for the first time that HLA-A2-restricted cytotoxic T cells specific for the major immunodominant influenza matrix epitope are protective against the infection. They encourage further in vivo evaluation of T cell epitopes recognized in the context of human MHC molecules

Biomimetic and enzyme-responsive dynamic hydrogels for studying cell-matrix interactions in pancreatic ductal adenocarcinoma.

Science.gov (United States)

Liu, Hung-Yi; Korc, Murray; Lin, Chien-Chi

2018-04-01

The tumor microenvironment (TME) governs all aspects of cancer progression and in vitro 3D cell culture platforms are increasingly developed to emulate the interactions between components of the stromal tissues and cancer cells. However, conventional cell culture platforms are inadequate in recapitulating the TME, which has complex compositions and dynamically changing matrix mechanics. In this study, we developed a dynamic gelatin-hyaluronic acid hybrid hydrogel system through integrating modular thiol-norbornene photopolymerization and enzyme-triggered on-demand matrix stiffening. In particular, gelatin was dually modified with norbornene and 4-hydroxyphenylacetic acid to render this bioactive protein photo-crosslinkable (through thiol-norbornene gelation) and responsive to tyrosinase-triggered on-demand stiffening (through HPA dimerization). In addition to the modified gelatin that provides basic cell adhesive motifs and protease cleavable sequences, hyaluronic acid (HA), an essential tumor matrix, was modularly and covalently incorporated into the cell-laden gel network. We systematically characterized macromer modification, gel crosslinking, as well as enzyme-triggered stiffening and degradation. We also evaluated the influence of matrix composition and dynamic stiffening on pancreatic ductal adenocarcinoma (PDAC) cell fate in 3D. We found that either HA-containing matrix or a dynamically stiffened microenvironment inhibited PDAC cell growth. Interestingly, these two factors synergistically induced cell phenotypic changes that resembled cell migration and/or invasion in 3D. Additional mRNA expression array analyses revealed changes unique to the presence of HA, to a stiffened microenvironment, or to the combination of both. Finally, we presented immunostaining and mRNA expression data to demonstrate that these irregular PDAC cell phenotypes were a result of matrix-induced epithelial-mesenchymal transition (EMT). Copyright © 2018 Elsevier Ltd. All rights

Transfer plate radioassay using cell monolayers to detect anti-cell surface antibodies synthesized by lymphocyte hybridomas

International Nuclear Information System (INIS)

Schneider, M.D.; Eisenbarth, G.S.

1979-01-01

A solid phase [ 125 I] Protein A radioassay for anti-cell surface antibodies is described, which employs target cell monolayers cultured on fenestrated polyvinyl chloride 96-well plates ('transfer plates'). The calibrated aperture in the bottom of each well is small enough to retain fluid contents by surface tension during monolayer growth, but also permits fluid to enter the wells when transfer plate are lowered into receptacles containing washing buffer on test sera. To assay for antibodies directed against target cell surface antigens, transfer plates bearing monolayers are inserted into microculture plates with corresponding 96-well geometry, thereby simultaneously sampling 96 wells. This assay allows rapid screening of hundreds of hybrid cell colonies for production of antibodies with desired tissue specificity. (Auth.)

Effect of rare earth oxide addition on microstructures of ultra-fine WC-Co particulate reinforced Cu matrix composites prepared by direct laser sintering

International Nuclear Information System (INIS)

Gu Dongdong; Shen Yifu; Zhao Long; Xiao Jun; Wu Peng; Zhu Yongbing

2007-01-01

This paper presents a detailed investigation into the influence of the rare earth (RE) oxide (La 2 O 3 ) addition upon the densification and the resultant microstructural characteristics of the submicron WC-Co particulate reinforced Cu matrix composites prepared by direct laser sintering. It is found that the relative density of the laser sintered sample with 1 wt.% La 2 O 3 addition increased by 11.5% as compared with the sample without RE addition. The addition of RE element favored the microstructural refinement and improved the particulate dispersion homogeneity and the particulate/matrix interfacial coherence. The metallurgical functions of the RE element in improving the sinterability were also addressed. It shows that due to the unique properties of RE element such as high surface activity and large atomic radius, the addition of trace RE element can decrease the surface tension of the melt, resist the grain growth coarsening and increase the heterogeneous nucleation rate during laser sintering

A stromal cell-derived factor-1 releasing matrix enhances the progenitor cell response and blood vessel growth in ischaemic skeletal muscle

Directory of Open Access Journals (Sweden)

D Kuraitis

2011-09-01

Full Text Available Although many regenerative cell therapies are being developed to replace or regenerate ischaemic muscle, the lack of vasculature and poor persistence of the therapeutic cells represent major limiting factors to successful tissue restoration. In response to ischaemia, stromal cell-derived factor-1 (SDF-1 is up-regulated by the affected tissue to stimulate stem cell-mediated regenerative responses. Therefore, we encapsulated SDF-1 into alginate microspheres and further incorporated these into an injectable collagen-based matrix in order to improve local delivery. Microsphere-matrix impregnation reduced the time for matrix thermogelation, and also increased the viscosity reached. This double-incorporation prolonged the release of SDF-1, which maintained adhesive and migratory bioactivity, attributed to chemotaxis in response to SDF-1. In vivo, treatment of ischaemic hindlimb muscle with microsphere-matrix led to increased mobilisation of bone marrow-derived progenitor cells, and also improved recruitment of angiogenic cells expressing the SDF-1 receptor (CXCR4 from bone marrow and local tissues. Both matrix and SDF-1-releasing matrix were successful at restoring perfusion, but SDF-1 treatment appeared to play an earlier role, as evidenced by arterioles that are phenotypically older and by increased angiogenic cytokine production, stimulating the generation of a qualitative microenvironment for a rapid and therefore more efficient regeneration. These results support the release of implanted SDF-1 as a promising method for enhancing progenitor cell responses and restoring perfusion to ischaemic tissues via neovascularisation.

High-content analysis of single cells directly assembled on CMOS sensor based on color imaging.

Science.gov (United States)

Tanaka, Tsuyoshi; Saeki, Tatsuya; Sunaga, Yoshihiko; Matsunaga, Tadashi

2010-12-15

A complementary metal oxide semiconductor (CMOS) image sensor was applied to high-content analysis of single cells which were assembled closely or directly onto the CMOS sensor surface. The direct assembling of cell groups on CMOS sensor surface allows large-field (6.66 mm×5.32 mm in entire active area of CMOS sensor) imaging within a second. Trypan blue-stained and non-stained cells in the same field area on the CMOS sensor were successfully distinguished as white- and blue-colored images under white LED light irradiation. Furthermore, the chemiluminescent signals of each cell were successfully visualized as blue-colored images on CMOS sensor only when HeLa cells were placed directly on the micro-lens array of the CMOS sensor. Our proposed approach will be a promising technique for real-time and high-content analysis of single cells in a large-field area based on color imaging. Copyright © 2010 Elsevier B.V. All rights reserved.

Direct laser writing of nanorough cell microbarriers on anatase/Si and graphite/Si

Energy Technology Data Exchange (ETDEWEB)

Xiang, Y.C. [Departamento de Física Aplicada and Instituto Nicolás Cabrera, Universidad Autónoma de Madrid, Madrid (Spain); Department of Physics, Blackett Laboratory, Imperial College London, Prince Consort Road, London SW7 2BW (United Kingdom); Martínez-Martínez, R.M. [Departamento de Física Aplicada and Instituto Nicolás Cabrera, Universidad Autónoma de Madrid, Madrid (Spain); Torres-Costa, V. [Departamento de Física Aplicada and Instituto Nicolás Cabrera, Universidad Autónoma de Madrid, Madrid (Spain); Centro de Microanálisis de Materiales, Universidad Autónoma de Madrid, Madrid (Spain); Agulló-Rueda, F. [Instituto de Ciencia de Materiales de Madrid (ICMM-CSIC), 28049 Madrid (Spain); García-Ruiz, J.P. [Departamento de Biología Molecular, Universidad Autónoma de Madrid, 28049 Madrid (Spain); Manso-Silván, M., E-mail: miguel.manso@uam.es [Departamento de Física Aplicada and Instituto Nicolás Cabrera, Universidad Autónoma de Madrid, Madrid (Spain)

2016-09-01

The formation of hierarchical structures consisting of microstripe barriers decorated with nanorough ablated materials prepared by direct laser writing is described. Linear features of circa 25 μm width and 12 μm height are achieved on amorphous and crystalline titania and graphitic carbon films deposited on silicon. Ablated protrusions build up barriers decorated by nanoscale Si-film reconstructions, as indicated by EDX maps and micro-Raman spectroscopy. Wettability tests show a dramatic change in water contact angle, which leads to almost full wetting after irradiation, irrespective of the original film composition. Fluorescence microscopy images of human mesenchymal stem cells cultured on 1D and 2D structures demonstrate the short term biocompatibility of the ablated surfaces. It is shown that cells adhere, extend and polarize on feature edges, independently of the type of surface, thus suggesting that the created nanoroughness is at the origin of the antifouling behavior. In particular, irradiated anatase and graphite surfaces demonstrate an increased performance of crystalline films for the creation of cell guiding and trapping devices. The results suggest that such laser processing of films may serve as a time-and-cost-efficient method for the design of few-cells analytical surfaces. - Graphical abstract: Response of human mesenchymal stem cells to the microbarriers grown by direct laser writing on anatase/Si. Microbarriers show low colonization and high induction of cellular polarization on the feature edges. Display Omitted - Highlights: • Hierarchical microbarriers by direct IR laser writing on thin films. • Complex reconstruction of irradiated materials includes nanorough features. • Ablated areas become fully wettable. • Short term biocompatibility of the grown hierarchical structures confirmed. • Particular designs functional for cell guiding or trapping.

Direct laser writing of nanorough cell microbarriers on anatase/Si and graphite/Si

International Nuclear Information System (INIS)

Xiang, Y.C.; Martínez-Martínez, R.M.; Torres-Costa, V.; Agulló-Rueda, F.; García-Ruiz, J.P.; Manso-Silván, M.

2016-01-01

The formation of hierarchical structures consisting of microstripe barriers decorated with nanorough ablated materials prepared by direct laser writing is described. Linear features of circa 25 μm width and 12 μm height are achieved on amorphous and crystalline titania and graphitic carbon films deposited on silicon. Ablated protrusions build up barriers decorated by nanoscale Si-film reconstructions, as indicated by EDX maps and micro-Raman spectroscopy. Wettability tests show a dramatic change in water contact angle, which leads to almost full wetting after irradiation, irrespective of the original film composition. Fluorescence microscopy images of human mesenchymal stem cells cultured on 1D and 2D structures demonstrate the short term biocompatibility of the ablated surfaces. It is shown that cells adhere, extend and polarize on feature edges, independently of the type of surface, thus suggesting that the created nanoroughness is at the origin of the antifouling behavior. In particular, irradiated anatase and graphite surfaces demonstrate an increased performance of crystalline films for the creation of cell guiding and trapping devices. The results suggest that such laser processing of films may serve as a time-and-cost-efficient method for the design of few-cells analytical surfaces. - Graphical abstract: Response of human mesenchymal stem cells to the microbarriers grown by direct laser writing on anatase/Si. Microbarriers show low colonization and high induction of cellular polarization on the feature edges. Display Omitted - Highlights: • Hierarchical microbarriers by direct IR laser writing on thin films. • Complex reconstruction of irradiated materials includes nanorough features. • Ablated areas become fully wettable. • Short term biocompatibility of the grown hierarchical structures confirmed. • Particular designs functional for cell guiding or trapping.

Conversion Matrix Analysis of SiGe HBT Gilbert Cell Mixers

DEFF Research Database (Denmark)

Johansen, Tom Keinicke; Vidkjær, Jens; Krozer, Viktor

2004-01-01

The frequency response of SiGe HBT active mixers based on the Gilbert cell topology is analyzed theoretically. The time-varying operation of the Gilbert cell mixer is taken into account by applying conversion matrix analysis. The main bandwidth limiting mechanisms experienced in SiGe HBT Gilbert ...

Glycoprotein on cell surfaces

International Nuclear Information System (INIS)

Muramatsu, T.

1975-01-01

There are conjugated polysaccharides in cell membranes and outside of animal cells, and they play important role in the control of cell behavior. In this paper, the studies on the glycoprotein on cell surfaces are reported. It was found that the glycoprotein on cell surfaces have both N-glycoside type and O-glycoside type saccharic chains. Therefore it can be concluded that the basic structure of the saccharic chains in the glycoprotein on cell surfaces is similar to that of blood serum and body fluid. The main glycoprotein in the membranes of red blood corpuscles has been studied most in detail, and it also has both types of saccharic chains. The glycoprotein in liver cell membranes was found to have only the saccharic chains of acid type and to be in different pattern from that in endoplasmic reticula and nuclear membranes, which also has the saccharic chains of neutral type. The structure of the saccharic chains of H-2 antigen, i.e. the peculiar glycoprotein on the surfaces of lymph system cells, has been studied, and it is similar to the saccharic chains of glycoprotein in blood serum. The saccharic chain structures of H-2 antigen and TL antigen are different. TL, H-2 (D), Lna and H-2 (K) are the glycoprotein on cell surfaces, and are independent molecules. The analysis of the saccharic chain patterns on cell surfaces was carried out, and it was shown that the acid type saccharic chains were similar to those of ordinary glycoprotein, because the enzyme of pneumococci hydrolyzed most of the acid type saccharic chains. The change of the saccharic chain patterns of glycoprotein on cell surfaces owing to canceration and multiplication is complex matter. (Kako, I.)

High hydrostatic pressure inactivation of Lactobacillus plantarum cells in (O/W)-emulsions is independent from cell surface hydrophobicity and lipid phase parameters

Science.gov (United States)

Kafka, T. A.; Reitermayer, D.; Lenz, C. A.; Vogel, R. F.

2017-07-01

Inactivation efficiency of high hydrostatic pressure (HHP) processing of food is strongly affected by food matrix composition. We investigated effects of fat on HHP inactivation of spoilage-associated Lactobacillus (L.) plantarum strains using defined oil-in-water (O/W)-emulsion model systems. Since fat-mediated effects on HHP inactivation could be dependent on interactions between lipid phase and microbial cells, three major factors possibly influencing such interactions were considered, that is, cell surface hydrophobicity, presence and type of surfactants, and oil droplet size. Pressure tolerance varied noticeably among L. plantarum strains and was independent of cell surface hydrophobicity. We showed that HHP inactivation of all strains tended to be more effective in presence of fat. The observation in both, surfactant-stabilized and surfactant-free (O/W)-emulsion, indicates that cell surface hydrophobicity is no intrinsic pressure resistance factor. In contrast to the presence of fat per se, surfactant type and oil droplet size did not affect inactivation efficiency.

Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells

Science.gov (United States)

Ortega, Fabian E.; Rengarajan, Michelle; Chavez, Natalie; Radhakrishnan, Prathima; Gloerich, Martijn; Bianchini, Julie; Siemers, Kathleen; Luckett, William S.; Lauer, Peter; Nelson, W. James; Theriot, Julie A.

2017-01-01

The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell–cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin–mediated coupling of the bacterium to F-actin is not required. PMID:28877987

The use of abrasive polishing and laser processing for developing polyurethane surfaces for controlling fibroblast cell behaviour

Energy Technology Data Exchange (ETDEWEB)

Irving, Michael; Murphy, Mark F; Lilley, Francis; French, Paul W; Burton, David R [General Engineering Research Institute, Liverpool John Moores University, Liverpool, L3 3AF (United Kingdom); Dixon, Simon [Biomer Technology LTD, 10 Seymour Court, Tudor Road, Manor Park, Runcorn, Cheshire, WA7 1SY (United Kingdom); Sharp, Martin C [General Engineering Research Institute, Liverpool John Moores University, Liverpool, L3 3AF (United Kingdom)

2017-02-01

Studies have shown that surfaces having micro and nano-scale features can be used to control cell behaviours including; cell proliferation, migration and adhesion. The aim of this work was to compare the use of laser processing and abrasive polishing to develop micro/nano-patterned polyurethane substrates for controlling fibroblast cell adhesion, migration and proliferation. Laser processing in a directional manner resulted in polyurethane surfaces having a ploughed field effect with micron-scale features. In contrast, abrasive polishing in a directional and random manner resulted in polyurethane surfaces having sub-micron scale features orientated in a linear or random manner. Results show that when compared with flat (non-patterned) polymer, both the laser processed and abrasive polished surface having randomly organised features, promoted significantly greater cell adhesion, while also enhancing cell proliferation after 72 h. In contrast, the abrasive polished surface having linear features did not enhance cell adhesion or proliferation when compared to the flat surface. For cell migration, the cells growing on the laser processed and abrasively polished random surface showed decreased levels of migration when compared to the flat surface. This study shows that both abrasive polishing and laser processing can be used to produce surfaces having features on the nano-scale and micron-scale, respectively. Surfaces produced using both techniques can be used to promote fibroblast cell adhesion and proliferation. Thus both methods offer a viable alternative to using lithographic techniques for developing patterned surfaces. In particular, abrasive polishing is an attractive method due to it being a simple, rapid and inexpensive method that can be used to produce surfaces having features on a comparable scale to more expensive, multi-step methods. - Highlights: • Abrasive polishing can generate nano-scratches on stainless steel to cast polymer films for cell

The use of abrasive polishing and laser processing for developing polyurethane surfaces for controlling fibroblast cell behaviour

International Nuclear Information System (INIS)

Irving, Michael; Murphy, Mark F; Lilley, Francis; French, Paul W; Burton, David R; Dixon, Simon; Sharp, Martin C

2017-01-01

Studies have shown that surfaces having micro and nano-scale features can be used to control cell behaviours including; cell proliferation, migration and adhesion. The aim of this work was to compare the use of laser processing and abrasive polishing to develop micro/nano-patterned polyurethane substrates for controlling fibroblast cell adhesion, migration and proliferation. Laser processing in a directional manner resulted in polyurethane surfaces having a ploughed field effect with micron-scale features. In contrast, abrasive polishing in a directional and random manner resulted in polyurethane surfaces having sub-micron scale features orientated in a linear or random manner. Results show that when compared with flat (non-patterned) polymer, both the laser processed and abrasive polished surface having randomly organised features, promoted significantly greater cell adhesion, while also enhancing cell proliferation after 72 h. In contrast, the abrasive polished surface having linear features did not enhance cell adhesion or proliferation when compared to the flat surface. For cell migration, the cells growing on the laser processed and abrasively polished random surface showed decreased levels of migration when compared to the flat surface. This study shows that both abrasive polishing and laser processing can be used to produce surfaces having features on the nano-scale and micron-scale, respectively. Surfaces produced using both techniques can be used to promote fibroblast cell adhesion and proliferation. Thus both methods offer a viable alternative to using lithographic techniques for developing patterned surfaces. In particular, abrasive polishing is an attractive method due to it being a simple, rapid and inexpensive method that can be used to produce surfaces having features on a comparable scale to more expensive, multi-step methods. - Highlights: • Abrasive polishing can generate nano-scratches on stainless steel to cast polymer films for cell

Connexin 43-mediated modulation of polarized cell movement and the directional migration of cardiac neural crest cells.

Science.gov (United States)

Xu, Xin; Francis, Richard; Wei, Chih Jen; Linask, Kaari L; Lo, Cecilia W

2006-09-01

Connexin 43 knockout (Cx43alpha1KO) mice have conotruncal heart defects that are associated with a reduction in the abundance of cardiac neural crest cells (CNCs) targeted to the heart. In this study, we show CNCs can respond to changing fibronectin matrix density by adjusting their migratory behavior, with directionality increasing and speed decreasing with increasing fibronectin density. However, compared with wild-type CNCs, Cx43alpha1KO CNCs show reduced directionality and speed, while CNCs overexpressing Cx43alpha1 from the CMV43 transgenic mice show increased directionality and speed. Altered integrin signaling was indicated by changes in the distribution of vinculin containing focal contacts, and altered temporal response of Cx43alpha1KO and CMV43 CNCs to beta1 integrin function blocking antibody treatment. High resolution motion analysis showed Cx43alpha1KO CNCs have increased cell protrusive activity accompanied by the loss of polarized cell movement. They exhibited an unusual polygonal arrangement of actin stress fibers that indicated a profound change in cytoskeletal organization. Semaphorin 3A, a chemorepellent known to inhibit integrin activation, was found to inhibit CNC motility, but in the Cx43alpha1KO and CMV43 CNCs, cell processes failed to retract with semaphorin 3A treatment. Immunohistochemical and biochemical analyses suggested close interactions between Cx43alpha1, vinculin and other actin-binding proteins. However, dye coupling analysis showed no correlation between gap junction communication level and fibronectin plating density. Overall, these findings indicate Cx43alpha1 may have a novel function in mediating crosstalk with cell signaling pathways that regulate polarized cell movement essential for the directional migration of CNCs.

Headspace versus direct immersion solid phase microextraction in complex matrixes: investigation of analyte behavior in multicomponent mixtures.

Science.gov (United States)

Gionfriddo, Emanuela; Souza-Silva, Érica A; Pawliszyn, Janusz

2015-08-18

This work aims to investigate the behavior of analytes in complex mixtures and matrixes with the use of solid-phase microextraction (SPME). Various factors that influence analyte uptake such as coating chemistry, extraction mode, the physicochemical properties of analytes, and matrix complexity were considered. At first, an aqueous system containing analytes bearing different hydrophobicities, molecular weights, and chemical functionalities was investigated by using commercially available liquid and solid porous coatings. The differences in the mass transfer mechanisms resulted in a more pronounced occurrence of coating saturation in headspace mode. Contrariwise, direct immersion extraction minimizes the occurrence of artifacts related to coating saturation and provides enhanced extraction of polar compounds. In addition, matrix-compatible PDMS-modified solid coatings, characterized by a new morphology that avoids coating fouling, were compared to their nonmodified analogues. The obtained results indicate that PDMS-modified coatings reduce artifacts associated with coating saturation, even in headspace mode. This factor, coupled to their matrix compatibility, make the use of direct SPME very practical as a quantification approach and the best choice for metabolomics studies where wide coverage is intended. To further understand the influence on analyte uptake on a system where additional interactions occur due to matrix components, ex vivo and in vivo sampling conditions were simulated using a starch matrix model, with the aim of mimicking plant-derived materials. Our results corroborate the fact that matrix handling can affect analyte/matrix equilibria, with consequent release of high concentrations of previously bound hydrophobic compounds, potentially leading to coating saturation. Direct immersion SPME limited the occurrence of the artifacts, which confirms the suitability of SPME for in vivo applications. These findings shed light into the implementation of in

Studying of Perovskite Nanoparticles in PMMA Matrix Used As Light Converter for Silicon Solar Cell

Directory of Open Access Journals (Sweden)

Lipiński M.

2017-09-01

Full Text Available The nanoparticles of CH3NH3PbBr3 hybrid perovskites were synthesized. These perovskite nanoparticles we embedded in polymethyl methacrylate (PMMA in order to obtain the composite, which we used as light converter for silicon solar cells. It was shown that the composite emit the light with the intensity maximum at about 527 nm when exited by a short wavelength (300÷450 nm of light. The silicon solar cells were used to examine the effect of down-conversion (DC process by perovskite nanoparticles embedded in PMMA. For experiments, two groups of monocrystalline silicon solar cells were used. The first one included the solar cells without surface texturization and antireflection coating. The second one included the commercial cells with surface texturization and antireflection coating. In every series of the cells one part of the cells were covered by composite (CH3NH3PbBr3 in PMMA layer and second part of cells by pure PMMA for comparison. It was shown that External Quantum Efficiency EQE of the photovoltaic cells covered by composite (CH3NH3PbBr3 in PMMA layer was improved in both group of the cells but unfortunately the Internal Quantum Efficiency was reduced. This reduction was caused by high absorption of the short wavelength light and reabsorption of the luminescence light. Therefore, the CH3NH3PbBr3 perovskite nanoparticles embedded in PMMA matrix were unable to increase silicon solar cell efficiency in the tested systems.

Transfer matrix representation for periodic planar media

Science.gov (United States)

Parrinello, A.; Ghiringhelli, G. L.

2016-06-01

Sound transmission through infinite planar media characterized by in-plane periodicity is faced by exploiting the free wave propagation on the related unit cells. An appropriate through-thickness transfer matrix, relating a proper set of variables describing the acoustic field at the two external surfaces of the medium, is derived by manipulating the dynamic stiffness matrix related to a finite element model of the unit cell. The adoption of finite element models avoids analytical modeling or the simplification on geometry or materials. The obtained matrix is then used in a transfer matrix method context, making it possible to combine the periodic medium with layers of different nature and to treat both hard-wall and semi-infinite fluid termination conditions. A finite sequence of identical sub-layers through the thickness of the medium can be handled within the transfer matrix method, significantly decreasing the computational burden. Transfer matrices obtained by means of the proposed method are compared with analytical or equivalent models, in terms of sound transmission through barriers of different nature.

Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: Implications for capillary-like tube formation in a fibrin matrix

NARCIS (Netherlands)

Kroon, M.E.; Koolwijk, P.; Vecht, B. van der; Hinsbergh, V.W.M. van

2000-01-01

Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix, hMVECs stimulated with fibroblast growth

Connective matrix organization in chronic granulomas of experimental paracoccidioidomycosis.

Science.gov (United States)

Kerr, I B; de Oliveira, P C; Lenzi, H L

1988-07-01

The histological and ultrastructural aspects of chronic granulomas from rats infected intraperitoneally with Paracoccidioides brasiliensis are described with special emphasis on the composition of the extracellular matrix. The granulomas were structurally arranged in two zones, one central containing fungi, and the other peripheral. The extracellular matrix was composed of collagen types I and III, proteoglycans, glycoprotein, and an undefined amorphous substance. The main cellular population was represented by macrophages, epithelioid cells, and giant cells in the central zone, and fibroblasts in the peripheral zone. The fibrotic process was a critical event in this stage of the infection, and showed a centrifugal direction. This might be provoked by direct stimulus from the fungi or by macrophage-fibroblastic interaction.

Transmembrane neural cell-adhesion molecule (NCAM), but not glycosyl-phosphatidylinositol-anchored NCAM, down-regulates secretion of matrix metalloproteinases

DEFF Research Database (Denmark)

Edvardsen, K; Chen, W; Rucklidge, G

1993-01-01

proteinases, and proteinase inhibitors all participate in the construction, maintenance, and remodeling of extracellular matrix by cells. The neural cell-adhesion molecule (NCAM)-negative rat glioma cell line BT4Cn secretes substantial amounts of metalloproteinases, as compared with its NCAM-positive mother......During embryogenesis interactions between cells and extracellular matrix play a central role in the modulation of cell motility, growth, and differentiation. Modulation of matrix structure is therefore crucial during development; extracellular matrix ligands, their receptors, extracellular...... cell line BT4C. We have transfected the BT4Cn cell line with cDNAs encoding the human NCAM-B and -C isoforms. We report here that the expression of transmembrane NCAM-B, but not of glycosyl-phosphatidylinositol-linked NCAM-C, induces a down-regulation of 92-kDa gelatinase (matrix metalloproteinase 9...

Endometrial stromal cell attachment and matrix homeostasis in abdominal wall endometriomas.

Science.gov (United States)

Itoh, Hiroko; Mogami, Haruta; Bou Nemer, Laurice; Word, Larry; Rogers, David; Miller, Rodney; Word, R Ann

2018-02-01

How does progesterone alter matrix remodeling in abdominal wall endometriomas compared with normal endometrium? Progesterone may prevent attachment of endometrial cells to the abdominal wall, but does not ameliorate abnormal stromal cell responses of abdominal wall endometriomas. Menstruation is a tightly orchestrated physiologic event in which steroid hormones and inflammatory cells cooperatively initiate shedding of the endometrium. Abdominal wall endometriomas represent a unique form of endometriosis in which endometrial cells inoculate fascia or dermis at the time of obstetrical or gynecologic surgery. Invasion of endometrium into ectopic sites requires matrix metalloproteinases (MMPs) for tissue remodeling but endometrium is not shed externally. Observational study in 14 cases and 19 controls. Tissues and stromal cells isolated from 14 abdominal wall endometriomas were compared with 19 normal cycling endometrium using immunohistochemistry, quantitative PCR, gelatin zymography and cell attachment assays. P values cell preps to provide scientific rigor to the conclusions. The results indicate that MMP2 and MMP9 are not increased by TGFβ1 in endometrioma stromal cells. Although progesterone prevents attachment of endometrioma cells to matrix components of the abdominal wall, it does not ameliorate these abnormal stromal cell responses to TGFβ1. N/A. Endometriomas were collected from women identified pre-operatively. Not all endometriomas were collected. Stromal cells from normal endometrium were from different patients, not women undergoing endometrioma resection. This work provides insight into the mechanisms by which progesterone may prevent abdominal wall endometriomas but, once established, are refractory to progesterone treatment. Tissue acquisition was supported by NIH P01HD087150. Authors have no competing interests. © The Author(s) 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All

Generating Chondromimetic Mesenchymal Stem Cell Spheroids by Regulating Media Composition and Surface Coating.

Science.gov (United States)

Sridharan, BanuPriya; Laflin, Amy D; Detamore, Michael S

2018-04-01

Spheroids of mesenchymal stem cells (MSCs) in cartilage tissue engineering have been shown to enhance regenerative potential owing to their 3D structure. In this study, we explored the possibility of priming spheroids under different media to replace the use of inductive surface coatings for chondrogenic differentiation. Rat bone marrow-derived MSCs were organized into cell spheroids by the hanging drop technique and subsequently cultured on hyaluronic acid (HA) coated or non-coated well plates under different cell media conditions. Endpoint analysis included cell viability, DNA and Glycosaminoglycan (GAG) and collagen content, gene expression and immunohistochemistry. For chondrogenic applications, MSC spheroids derived on non-coated surfaces outperformed the spheroids derived from HA-coated surfaces in matrix synthesis and collagen II gene expression. Spheroids on non-coated surfaces gave rise to the highest collagen and GAG when primed with medium containing insulin-like growth factor (IGF) for 1 week during spheroid formation. Spheroids that were grown in chondroinductive raw material-inclusive media such as aggrecan or chondroitin sulfate exhibited the highest Collagen II gene expression in the non-coated surface at 1 week. Media priming by growth factors and raw materials might be a more predictive influencer of chondrogenesis compared to inductive-surfaces. Such tailored bioactivity of the stem cell spheroids in the stage of the spheroid formation may give rise to a platform technology that may eventually produce spheroids capable of chondrogenesis achieved by mere media manipulation, skipping the need for additional culture on a modified surface, that paves the way for cost-effective technologies.

Glucose oxidase anode for biofuel cell based on direct electron transfer

Energy Technology Data Exchange (ETDEWEB)

Ivnitski, Dmitri; Branch, Brittany; Atanassov, Plamen [Department of Chemical and Nuclear Engineering, University of New Mexico, 209 Farris Engineering Center, Room 150, Albuquerque, NM 87131-0001 (United States); Apblett, Christopher [Sandia National Laboratories, Albuquerque, NM 87185 (United States)

2006-08-15

This paper presents a new design concept of a glucose oxidase (GO{sub x}) electrode as an anode for the biofuel cell based on direct electron transfer (DET) between the active site of an enzyme and the multi-walled carbon nanotube (MWNT)-modified electrode surface. Toray{sup (R)} carbon paper (TP) with a porous three-dimensional network (78% porosity) was used as a matrix for selectively growing multi-walled carbon nanotubes. The incorporation of MWCNTs into TP was provided by the chemical vapor deposition technique after an electrochemical transition of cobalt metal seeds. This approach has the ability to efficiently promote DET reactions. The morphologies and electrochemical characteristics of the GO{sub x} modified electrodes were investigated by scanning electron microscopy, cyclic voltammetry, and potentiometric methods. The combination of poly-cation polyethylenimine (PEI) with negatively charged glucose oxidase provides formation of circa 100nm thick films on the TP/MWCNT surface. The tetrabutylammonium bromide salt-treated Nafion{sup (R)} was used as GO{sub x} binder and proton-conducting medium. The TP/MWCNT/PEI/GO{sub x}/Nafion{sup (R)} modified electrode operates at 25{sup o}C in 0.02M phosphate buffer solution (pH 6.9) containing 0.1M KCl in the presence of 20mM glucose. The open circuit potential of GO{sub x} anode was between -0.38V and -0.4V vs. Ag/AgCl, which is closer to the redox potential of the FAD/FADH{sub 2} cofactor in the enzyme itself. The GO{sub x} electrode has a potential to work in vivo by using endogenous substances, such as glucose and oxygen. Such a glucose anode allows for the development of a new generation of miniaturized membrane-less biofuel cells. (author)

Calcium phosphate thin films enhance the response of human mesenchymal stem cells to nanostructured titanium surfaces

Directory of Open Access Journals (Sweden)

Mura M McCafferty

2014-05-01

Full Text Available The development of biomaterial surfaces possessing the topographical cues that can promote mesenchymal stem cell recruitment and, in particular, those capable of subsequently directing osteogenic differentiation is of increasing importance for the advancement of tissue engineering. While it is accepted that it is the interaction with specific nanoscale topography that induces mesenchymal stem cell differentiation, the potential for an attendant bioactive chemistry working in tandem with such nanoscale features to enhance this effect has not been considered to any great extent. This article presents a study of mesenchymal stem cell response to conformal bioactive calcium phosphate thin films sputter deposited onto a polycrystalline titanium nanostructured surface with proven capability to directly induce osteogenic differentiation in human bone marrow–derived mesenchymal stem cells. The sputter deposited surfaces supported high levels of human bone marrow–derived mesenchymal stem cell adherence and proliferation, as determined by DNA quantification. Furthermore, they were also found to be capable of directly promoting significant levels of osteogenic differentiation. Specifically, alkaline phosphatase activity, gene expression and immunocytochemical localisation of key osteogenic markers revealed that the nanostructured titanium surfaces and the bioactive calcium phosphate coatings could direct the differentiation towards an osteogenic lineage. Moreover, the addition of the calcium phosphate chemistry to the topographical profile of the titanium was found to induce increased human bone marrow–derived mesenchymal stem cell differentiation compared to that observed for either the titanium or calcium phosphate coating without an underlying nanostructure. Hence, the results presented here highlight that a clear benefit can be achieved from a surface engineering strategy that combines a defined surface topography with an attendant, conformal

Fatigue and frictional heating in ceramic matrix composites

DEFF Research Database (Denmark)

Jacobsen, T.K.; Sørensen, B.F.; Brøndsted, P.

1997-01-01

This paper describes an experimental technique for monitoring the damage evolution in ceramic matrix composites during cyclic testing. The damage is related to heat dissipation, which may be measured as radiated heat from the surface of the test specimen. In the present experimental set-up an iso......This paper describes an experimental technique for monitoring the damage evolution in ceramic matrix composites during cyclic testing. The damage is related to heat dissipation, which may be measured as radiated heat from the surface of the test specimen. In the present experimental set...... with a high spatial and temperature resolution and changes in the heat dissipation can be measured almost instantaneously. The technique has been tested on uni-directional ceramic matrix composites. Experimental results are shown and the possibilities and the limitations of the technique are discussed....

Immunophenotypic characterization of human T cells after in vitro exposure to different silicone breast implant surfaces.

Directory of Open Access Journals (Sweden)

Giuseppe Cappellano

Full Text Available The most common complication of silicone breast implants is capsular contracture (massive scar formation around the implant. We postulate that capsular contracture is always a sequel to inflammatory processes, with both innate and adaptive immune mechanisms participating. In general, fibroblasts and macrophages have been used as cell types to evaluate in vitro the biocompatibility of breast implant surfaces. Moreover, also T cells have been found at the implant site at the initial stage of fibrous capsule formation. However, only few studies have addressed the influence of surfaces with different textures on T-cell responses. The aim of the present study was to investigate the immune response of human peripheral blood mononuclear cells (PBMC to commercially available silicone breast implants in vitro. PBMC from healthy female blood donors were cultured on each silicone surface for 4 days. Proliferation and phenotype of cultured cells were assessed by flow cytometry. Cytokine levels were determined by multiplex and real-time assay. We found that silicone surfaces do not induce T-cell proliferation, nor do they extensively alter the proportion of T cell subsets (CD4, CD8, naïve, effector memory. Interestingly, cytokine profiling identified matrix specific differences, especially for IL-6 and TNF-α on certain surface topographies that could lead to increased fibrosis.

An Artificial Turf-Based Surrogate Surface Collector for the Direct Measurement of Atmospheric Mercury Dry Deposition

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Naima L. Hall

2017-02-01

Full Text Available This paper describes the development of a new artificial turf surrogate surface (ATSS sampler for use in the measurement of mercury (Hg dry deposition. In contrast to many existing surrogate surface designs, the ATSS utilizes a three-dimensional deposition surface that may more closely mimic the physical structure of many natural surfaces than traditional flat surrogate surface designs (water, filter, greased Mylar film. The ATSS has been designed to overcome several complicating factors that can impact the integrity of samples with other direct measurement approaches by providing a passive system which can be deployed for both short and extended periods of time (days to weeks, and is not contaminated by precipitation and/or invalidated by strong winds. Performance characteristics including collocated precision, in-field procedural and laboratory blanks were evaluated. The results of these performance evaluations included a mean collocated precision of 9%, low blanks (0.8 ng, high extraction efficiency (97%–103%, and a quantitative matrix spike recovery (100%.

Direct observation of nanoparticle-cancer cell nucleus interactions.

Science.gov (United States)

Dam, Duncan Hieu M; Lee, Jung Heon; Sisco, Patrick N; Co, Dick T; Zhang, Ming; Wasielewski, Michael R; Odom, Teri W

2012-04-24

We report the direct visualization of interactions between drug-loaded nanoparticles and the cancer cell nucleus. Nanoconstructs composed of nucleolin-specific aptamers and gold nanostars were actively transported to the nucleus and induced major changes to the nuclear phenotype via nuclear envelope invaginations near the site of the construct. The number of local deformations could be increased by ultrafast, light-triggered release of the aptamers from the surface of the gold nanostars. Cancer cells with more nuclear envelope folding showed increased caspase 3 and 7 activity (apoptosis) as well as decreased cell viability. This newly revealed correlation between drug-induced changes in nuclear phenotype and increased therapeutic efficacy could provide new insight for nuclear-targeted cancer therapy.

Switchable directional excitation surface plasmon polaritons with dielectric nanoantennas

DEFF Research Database (Denmark)

Sinev, I.; Komissarenko, F.; Bogdanov, A.

We demonstrate directional launching of surface plasmon polaritons on thin goldfilm with a single silicon nanosphere. The directivity pattern of the excited surface waves exhibits rapid switching from forward to backward excitation, which is driven by the mutual interference of magnetic and elect......We demonstrate directional launching of surface plasmon polaritons on thin goldfilm with a single silicon nanosphere. The directivity pattern of the excited surface waves exhibits rapid switching from forward to backward excitation, which is driven by the mutual interference of magnetic...

Blood vessel endothelium-directed tumor cell streaming in breast tumors requires the HGF/C-Met signaling pathway.

Science.gov (United States)

Leung, E; Xue, A; Wang, Y; Rougerie, P; Sharma, V P; Eddy, R; Cox, D; Condeelis, J

2017-05-11

During metastasis to distant sites, tumor cells migrate to blood vessels. In vivo, breast tumor cells utilize a specialized mode of migration known as streaming, where a linear assembly of tumor cells migrate directionally towards blood vessels on fibronectin-collagen I-containing extracellular matrix (ECM) fibers in response to chemotactic signals. We have successfully reconstructed tumor cell streaming in vitro by co-plating tumors cells, macrophages and endothelial cells on 2.5 μm thick ECM-coated micro-patterned substrates. We found that tumor cells and macrophages, when plated together on the micro-patterned substrates, do not demonstrate sustained directional migration in only one direction (sustained directionality) but show random bi-directional walking. Sustained directionality of tumor cells as seen in vivo was established in vitro when beads coated with human umbilical vein endothelial cells were placed at one end of the micro-patterned 'ECM fibers' within the assay. We demonstrated that these endothelial cells supply the hepatocyte growth factor (HGF) required for the chemotactic gradient responsible for sustained directionality. Using this in vitro reconstituted streaming system, we found that directional streaming is dependent on, and most effectively blocked, by inhibiting the HGF/C-Met signaling pathway between endothelial cells and tumor cells. Key observations made with the in vitro reconstituted system implicating C-Met signaling were confirmed in vivo in mammary tumors using the in vivo invasion assay and intravital multiphoton imaging of tumor cell streaming. These results establish HGF/C-Met as a central organizing signal in blood vessel-directed tumor cell migration in vivo and highlight a promising role for C-Met inhibitors in blocking tumor cell streaming and metastasis in vivo, and for use in human trials.

Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application.

Science.gov (United States)

Schüürmann, Jan; Quehl, Paul; Festel, Gunter; Jose, Joachim

2014-10-01

Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes.

Perfusion directed 3D mineral formation within cell-laden hydrogels.

Science.gov (United States)

Sawyer, Stephen William; Shridhar, Shivkumar Vishnempet; Zhang, Kairui; Albrecht, Lucas; Filip, Alex; Horton, Jason; Soman, Pranav

2018-06-08

Despite the promise of stem cell engineering and the new advances in bioprinting technologies, one of the major challenges in the manufacturing of large scale bone tissue scaffolds is the inability to perfuse nutrients throughout thick constructs. Here, we report a scalable method to create thick, perfusable bone constructs using a combination of cell-laden hydrogels and a 3D printed sacrificial polymer. Osteoblast-like Saos-2 cells were encapsulated within a gelatin methacrylate (GelMA) hydrogel and 3D printed polyvinyl alcohol (PVA) pipes were used to create perfusable channels. A custom-built bioreactor was used to perfuse osteogenic media directly through the channels in order to induce mineral deposition which was subsequently quantified via microCT. Histological staining was used to verify mineral deposition around the perfused channels, while COMSOL modeling was used to simulate oxygen diffusion between adjacent channels. This information was used to design a scaled-up construct containing a 3D array of perfusable channels within cell-laden GelMA. Progressive matrix mineralization was observed by cells surrounding perfused channels as opposed to random mineral deposition in static constructs. MicroCT confirmed that there was a direct relationship between channel mineralization within perfused constructs and time within the bioreactor. Furthermore, the scalable method presented in this work serves as a model on how large-scale bone tissue replacement constructs could be made using commonly available 3D printers, sacrificial materials, and hydrogels. © 2018 IOP Publishing Ltd.

Analysis of antigen-specific B-cell memory directly ex vivo.

Science.gov (United States)

McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

2004-01-01

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

Local differentiation of cell wall matrix polysaccharides in sinuous pavement cells: its possible involvement in the flexibility of cell shape.

Science.gov (United States)

Sotiriou, P; Giannoutsou, E; Panteris, E; Galatis, B; Apostolakos, P

2018-03-01

The distribution of homogalacturonans (HGAs) displaying different degrees of esterification as well as of callose was examined in cell walls of mature pavement cells in two angiosperm and two fern species. We investigated whether local cell wall matrix differentiation may enable pavement cells to respond to mechanical tension forces by transiently altering their shape. HGA epitopes, identified with 2F4, JIM5 and JIM7 antibodies, and callose were immunolocalised in hand-made or semithin leaf sections. Callose was also stained with aniline blue. The structure of pavement cells was studied with light and transmission electron microscopy (TEM). In all species examined, pavement cells displayed wavy anticlinal cell walls, but the waviness pattern differed between angiosperms and ferns. The angiosperm pavement cells were tightly interconnected throughout their whole depth, while in ferns they were interconnected only close to the external periclinal cell wall and intercellular spaces were developed between them close to the mesophyll. Although the HGA epitopes examined were located along the whole cell wall surface, the 2F4- and JIM5- epitopes were especially localised at cell lobe tips. In fern pavement cells, the contact sites were impregnated with callose and JIM5-HGA epitopes. When tension forces were applied on leaf regions, the pavement cells elongated along the stretching axis, due to a decrease in waviness of anticlinal cell walls. After removal of tension forces, the original cell shape was resumed. The presented data support that HGA epitopes make the anticlinal pavement cell walls flexible, in order to reversibly alter their shape. Furthermore, callose seems to offer stability to cell contacts between pavement cells, as already suggested in photosynthetic mesophyll cells. © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.

Regulation of extracellular matrix vesicles via rapid responses to steroid hormones during endochondral bone formation.

Science.gov (United States)

Asmussen, Niels; Lin, Zhao; McClure, Michael J; Schwartz, Zvi; Boyan, Barbara D

2017-12-09

Endochondral bone formation is a precise and highly ordered process whose exact regulatory framework is still being elucidated. Multiple regulatory pathways are known to be involved. In some cases, regulation impacts gene expression, resulting in changes in chondrocyte phenotypic expression and extracellular matrix synthesis. Rapid regulatory mechanisms are also involved, resulting in release of enzymes, factors and micro RNAs stored in extracellular matrisomes called matrix vesicles. Vitamin D metabolites modulate endochondral development via both genomic and rapid membrane-associated signaling pathways. 1α,25-dihydroxyvitamin D3 [1α,25(OH) 2 D 3 ] acts through the vitamin D receptor (VDR) and a membrane associated receptor, protein disulfide isomerase A3 (PDIA3). 24R,25-dihydroxyvitamin D3 [24R,25(OH) 2 D 3 ] affects primarily chondrocytes in the resting zone (RC) of the growth plate, whereas 1α,25(OH) 2 D 3 affects cells in the prehypertrophic and upper hypertrophic cell zones (GC). This includes genomically directing the cells to produce matrix vesicles with zone specific characteristics. In addition, vitamin D metabolites produced by the cells interact directly with the matrix vesicle membrane via rapid signal transduction pathways, modulating their activity in the matrix. The matrix vesicle payload is able to rapidly impact the extracellular matrix via matrix processing enzymes as well as providing a feedback mechanism to the cells themselves via the contained micro RNAs. Copyright © 2017. Published by Elsevier Inc.

Insulin-like growth factor I has independent effects on bone matrix formation and cell replication

International Nuclear Information System (INIS)

Hock, J.M.; Centrella, M.; Canalis, E.

1988-01-01

The effects of insulin-like growth factor-I (IGF-I) and insulin on bone matrix synthesis and bone cell replication were studied in cultured 21-day-old fetal rat calvariae. Histomorphometry techniques were developed to measure the incorporation of [2,3- 3 H]proline and [methyl- 3 H]thymidine into bone matrix and bone cell nuclei, respectively, using autoradiographs of sagittal sections of calvariae cultured with IGF-I, insulin, or vehicle for up to 96 h. To confirm an effect on bone formation, IGF-I was also studied for its effects on [ 3 H]proline incorporation into collagenase-digestible protein (CDP) and noncollagen protein and on [ 3 H]thymidine incorporation into acid-precipitable material (DNA). IGF-I at 10(-9)-10(-7) M significantly increased the rate of bone matrix apposition and CDP after 24 h by 45-50% and increased cell labeling by 8-fold in the osteoprogenitor cell zone, by 4-fold in the osteoblast cell zone, and by 2-fold in the periosteal fibroblast zone. Insulin at 10(-9)-10(-6) M also increased matrix apposition rate and CDP by 40-50%, but increased cell labeling by 2-fold only at a concentration of 10(-7) M or higher and then only in the osteoprogenitor cell zone. When hydroxyurea was added to IGF-I-treated bones, the effects of IGF-I on DNA synthesis were abolished, but the increase in bone matrix apposition induced by IGF-I was only partly diminished. In conclusion, IGF-I stimulates matrix synthesis in calvariae, an effect that is partly, although not completely, dependent on its stimulatory effect on DNA synthesis

Protein-scaffold Directed Nanoscale Assembly of T Cell Ligands: Artificial Antigen Presentation with Defined Valency, Density and Ratio.

Science.gov (United States)

Smith, Mason R; Tolbert, Stephanie V; Wen, Fei

2018-05-07

Tuning antigen presentation to T cells is a critical step in investigating key aspects of T cell activation. However, existing technologies have limited ability to control the spatial and stoichiometric organization of T cell ligands on 3D surfaces. Here, we developed an artificial antigen presentation platform based on protein-scaffold directed assembly that allows fine control over the spatial and stoichiometric organization of T cell ligands on a 3D yeast-cell surface. Using this system, we observed that the T cell activation threshold on a 3D surface is independent of peptide-major histocompatibility complex (pMHC) valency, but instead determined by the overall pMHC surface density. When intercellular adhesion molecule 1 (ICAM-1) was co-assembled with pMHC, it enhanced antigen recognition sensitivity by 6-fold. Further, T cells responded with different magnitudes to varying ratios of pMHC and ICAM-1 and exhibited a maximum response at a ratio of 15% pMHC and 85% ICAM-1, introducing an additional parameter for tuning T cell activation. This protein-scaffold directed assembly technology is readily transferrable to acellular surfaces for translational research as well as large-scale T-cell manufacturing.

Application of Bladder Acellular Matrix in Urinary Bladder Regeneration: The State of the Art and Future Directions

Directory of Open Access Journals (Sweden)

Marta Pokrywczynska

2015-01-01

Full Text Available Construction of the urinary bladder de novo using tissue engineering technologies is the “holy grail” of reconstructive urology. The search for the ideal biomaterial for urinary bladder reconstruction has been ongoing for decades. One of the most promising biomaterials for this purpose seems to be bladder acellular matrix (BAM. In this review we determine the most important factors, which may affect biological and physical properties of BAM and its regeneration potential in tissue engineered urinary bladder. We also point out the directions in modification of BAM, which include incorporation of exogenous growth factors into the BAM structure. Finally, we discuss the results of the urinary bladder regeneration with cell seeded BAM.

Collagen matrix as a tool in studying fibroblastic cell behavior.

Science.gov (United States)

Kanta, Jiří

2015-01-01

Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes.

The interaction between LYVE-1 with hyaluronan on the cell surface may play a role in the diversity of adhesion to cancer cells.

Science.gov (United States)

Du, Yan; Liu, Hua; He, Yiqing; Liu, Yiwen; Yang, Cuixia; Zhou, Muqing; Wang, Wenjuan; Cui, Lian; Hu, Jiajie; Gao, Feng

2013-01-01

Hyaluronan (HA), a simple disaccharide unit, can polymerize and is considered a primary component of the extracellular matrix, which has a wide range of biological functions. In recent years, HA was found on the surface of tumor cells. According to previous reports, differing HA content on the cell surface of tumor cells is closely related to lymph node metastases, but the mechanisms mediating this process remained unclear. This research intended to study the surface content of HA on tumor cells and analyze cell adhesive changes caused by the interaction between HA and its lymphatic endothelial receptor (LYVE-1). We screened and observed high HA content on HS-578T breast cells and low HA content on MCF-7 breast cells through particle exclusion, immunofluorescence and flow cytometry experiments. The expression of LYVE-1, the lymph-vessel specific HA receptor, was consistent with our previous report and enhanced the adhesion of HA(high)-HS-578T cells to COS-7(LYVE-1(+)) through HA in cell static adhesion and dynamic parallel plate flow chamber experiments. MCF-7 breast cells contain little HA on the surface; however, our results showed little adhesion difference between MCF-7 cells and COS-7(LYVE-1(+)) and COS-7(LYVE-1(-)) cells. Similar results were observed concerning the adhesion of HS-578T cells or MCF-7 cells to SVEC4-10 cells. Furthermore, we observed for the first time that the cell surface HA content of high transfer tumor cells was rich, and we visualized the cross-linking of HA cable structures, which may activate LYVE-1 on lymphatic endothelial cells, promoting tumor adhesion. In summary, high-low cell surface HA content of tumor cells through the interaction with LYVE-1 leads to adhesion differences.

Induction of mesenchymal stem cell chondrogenic differentiation and functional cartilage microtissue formation for in vivo cartilage regeneration by cartilage extracellular matrix-derived particles.

Science.gov (United States)

Yin, Heyong; Wang, Yu; Sun, Zhen; Sun, Xun; Xu, Yichi; Li, Pan; Meng, Haoye; Yu, Xiaoming; Xiao, Bo; Fan, Tian; Wang, Yiguo; Xu, Wenjing; Wang, Aiyuan; Guo, Quanyi; Peng, Jiang; Lu, Shibi

2016-03-01

We propose a method of preparing a novel cell carrier derived from natural cartilage extracellular matrix (ECM), designated cartilage ECM-derived particles (CEDPs). Through a series of processes involving pulverization, sieving, and decellularization, fresh cartilage was made into CEDPs with a median diameter of 263 ± 48 μm. Under microgravity culture conditions in a rotary cell culture system (RCCS), bone marrow stromal cells (BMSCs) can proliferate rapidly on the surface of CEDPs with high viability. Histological evaluation and gene expression analysis indicated that BMSCs were differentiated into mature chondrocytes after 21 days of culture without the use of exogenous growth factors. Functional cartilage microtissue aggregates of BMSC-laden CEDPs formed as time in culture increased. Further, the microtissue aggregates were directly implanted into trochlear cartilage defects in a rat model (CEDP+MSC group). Gait analysis and histological results indicated that the CEDP+MSC group obtained better and more rapid joint function recovery and superior cartilage repair compared to the control groups, in which defects were treated with CEDPs alone or only fibrin glue, at both 6 and 12 weeks after surgery. In conclusion, the innovative cell carrier derived from cartilage ECM could promote chondrogenic differentiation of BMSCs, and the direct use of functional cartilage microtissue facilitated cartilage regeneration. This strategy for cell culture, stem cell differentiation and one-step surgery using cartilage microtissue for cartilage repair provides novel prospects for cartilage tissue engineering and may have further broad clinical applications. We proposed a method to prepare a novel cell carrier derived from natural cartilage ECM, termed cartilage ECM-derived particles (CEDPs), which can support proliferation of MSCs and facilitate their chondrogenic differentiation. Further, the direct use of functional cartilage microtissue of MSC-laden CEDP aggregates for

Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies.

Science.gov (United States)

Laperle, Alex; Masters, Kristyn S; Palecek, Sean P

2015-01-01

Stem cells reside in specialized niches in vivo. Specific factors, including the extracellular matrix (ECM), in these niches are directly responsible for maintaining the stem cell population. During development, components of the stem cell microenvironment also control differentiation with precise spatial and temporal organization. The stem cell microenvironment is dynamically regulated by the cellular component, including stem cells themselves. Thus, a mechanism exists whereby stem cells modify the ECM, which in turn affects the fate of the stem cell. In this study, we investigated whether the type of ECM initially adsorbed to the culture substrate can influence the composition of the ECM deposited by human embryonic stem cells (hESCs) differentiating in embryoid bodies, and whether different ECM composition and deposition profiles elicit distinct differentiation fates. We have shown that the initial ECM environment hESCs are exposed to affects the fate decisions of those cells and that this initial ECM environment is constantly modified during the differentiation process. © 2014 American Institute of Chemical Engineers.

Analysis of the interaction of extracellular matrix and phenotype of bladder cancer cells

International Nuclear Information System (INIS)

Dozmorov, Mikhail G; Kyker, Kimberly D; Saban, Ricardo; Knowlton, Nicholas; Dozmorov, Igor; Centola, Michael B; Hurst, Robert E

2006-01-01

The extracellular matrix has a major effect upon the malignant properties of bladder cancer cells both in vitro in 3-dimensional culture and in vivo. Comparing gene expression of several bladder cancer cells lines grown under permissive and suppressive conditions in 3-dimensional growth on cancer-derived and normal-derived basement membrane gels respectively and on plastic in conventional tissue culture provides a model system for investigating the interaction of malignancy and extracellular matrix. Understanding how the extracellular matrix affects the phenotype of bladder cancer cells may provide important clues to identify new markers or targets for therapy. Five bladder cancer cell lines and one immortalized, but non-tumorigenic, urothelial line were grown on Matrigel, a cancer-derived ECM, on SISgel, a normal-derived ECM, and on plastic, where the only ECM is derived from the cells themselves. The transcriptomes were analyzed on an array of 1186 well-annotated cancer derived cDNAs containing most of the major pathways for malignancy. Hypervariable genes expressing more variability across cell lines than a set expressing technical variability were analyzed further. Expression values were clustered, and to identify genes most likely to represent biological factors, statistically over-represented ontologies and transcriptional regulatory elements were identified. Approximately 400 of the 1186 total genes were expressed 2 SD above background. Approximately 100 genes were hypervariable in cells grown on each ECM, but the pattern was different in each case. A core of 20 were identified as hypervariable under all 3 growth conditions, and 33 were hypervariable on both SISgel and Matrigel, but not on plastic. Clustering of the hypervariable genes showed very different patterns for the same 6 cell types on the different ECM. Even when loss of cell cycle regulation was identified, different genes were involved, depending on the ECM. Under the most permissive conditions

Fibronectin-cell interactions

DEFF Research Database (Denmark)

Couchman, J R; Austria, M R; Woods, A

1990-01-01

Fibronectins are widespread extracellular matrix and body fluid glycoproteins, capable of multiple interactions with cell surfaces and other matrix components. Their structure at a molecular level has been resolved, yet there are still many unanswered questions regarding their biologic activity...... in vivo. Much data suggests that fibronectins may promote extracellular matrix assembly, and cell adhesion to those matrices. However, one outstanding enigma is that fibronectins may, under different circumstances, promote both cell migration and anchorage. An analysis of the interaction of fibroblasts...... with proteolytically derived and purified domains of plasma fibronectin revealed that the type of adhesion and the correlated cytoskeletal organization depended on multiple interactions of fibronectin domains with the cell surface. Human dermal fibroblasts were capable of interacting with the integrin-binding domain...