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Sample records for actin-binding scaffold protein

  1. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    OpenAIRE

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesi...

  2. The actin binding protein adseverin regulates osteoclastogenesis.

    Science.gov (United States)

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W P; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  3. The actin binding protein adseverin regulates osteoclastogenesis.

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    Siavash Hassanpour

    Full Text Available Adseverin (Ads, a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG. Ads is induced during OCG downstream of RANK-ligand (RANKL stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion.

  4. Characterization of engineered actin binding proteins that control filament assembly and structure.

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    Crista M Brawley

    Full Text Available BACKGROUND: Eukaryotic cells strictly regulate the structure and assembly of their actin filament networks in response to various stimuli. The actin binding proteins that control filament assembly are therefore attractive targets for those who wish to reorganize actin filaments and reengineer the cytoskeleton. Unfortunately, the naturally occurring actin binding proteins include only a limited set of pointed-end cappers, or proteins that will block polymerization from the slow-growing end of actin filaments. Of the few that are known, most are part of large multimeric complexes that are challenging to manipulate. METHODOLOGY/PRINCIPAL FINDINGS: We describe here the use of phage display mutagenesis to generate of a new class of binding protein that can be targeted to the pointed-end of actin. These proteins, called synthetic antigen binders (sABs, are based on an antibody-like scaffold where sequence diversity is introduced into the binding loops using a novel "reduced genetic code" phage display library. We describe effective strategies to select and screen for sABs that ensure the generated sABs bind to the pointed-end surface of actin exclusively. CONCLUSIONS/SIGNIFICANCE: From our set of pointed-end binders, we identify three sABs with particularly useful properties to systematically probe actin dynamics: one protein that caps the pointed end, a second that crosslinks actin filaments, and a third that severs actin filaments and promotes disassembly.

  5. Identification of Actin-Binding Proteins from Maize Pollen

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    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  6. Harmonin-b, an actin-binding scaffold protein, is involved in the adaptation of mechanoelectrical transduction by sensory hair cells

    OpenAIRE

    Michalski, Nicolas; Michel, Vincent; Caberlotto, Elisa; Lefèvre, Gaelle M.; Van Aken, Alexander F J; Tinevez, Jean-Yves; Bizard, Emilie; Houbron, Christophe; Weil, Dominique; Hardelin, Jean-Pierre; Richardson, Guy P.; Kros, Corné J.; Martin, Pascal; Petit, Christine

    2009-01-01

    We assessed the involvement of harmonin-b, a submembranous protein containing PDZ domains, in the mechanoelectrical transduction machinery of inner ear hair cells. Harmonin-b is located in the region of the upper insertion point of the tip link that joins adjacent stereocilia from different rows and that is believed to gate transducer channel(s) located in the region of the tip link's lower insertion point. In Ush1c dfcr-2J/dfcr-2J mutant mice defective for harmonin-b, step deflections of the...

  7. Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring

    OpenAIRE

    1990-01-01

    Actin-binding protein (ABP-280, nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is...

  8. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

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    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  9. Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening.

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    Gentry, Brian S; van der Meulen, Stef; Noguera, Philippe; Alonso-Latorre, Baldomero; Plastino, Julie; Koenderink, Gijsje H

    2012-11-01

    Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP's main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP's flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP's effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP's ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP's network stiffening activity may be tuned by the local concentration of monomeric actin.

  10. Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8.

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    Eleanna Stamatakou

    Full Text Available Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1 and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.

  11. Isolation and identification of actin-binding proteins in Plasmodium falciparum by affinity chromatography

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    Claudia Forero

    2000-06-01

    Full Text Available The invasion of the erythrocyte by Plasmodium falciparum depends on the ability of the merozoite to move through the membrane invagination. This ability is probably mediated by actin dependent motors. Using affinity columns with G-actin and F-actin we isolated actin binding proteins from the parasite. By immunoblotting and immunoprecipitation with specific antibodies we identified the presence of tropomyosin, myosin, a-actinin, and two different actins in the eluate corresponding to F-actin binding proteins. In addition to these, a 240-260 kDa doublet, different in size from the erythrocyte spectrin, reacted with an antibody against human spectrin. All the above mentioned proteins were metabolically radiolabeled when the parasite was cultured with 35S-methionine. The presence of these proteins in P. falciparum is indicative of a complex cytoskeleton and supports the proposed role for an actin-myosin motor during invasion.

  12. Regulation of water flow by actin-binding protein-induced actin gelatin.

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    Ito, T.; Suzuki, A.; Stossel, T. P.

    1992-01-01

    Actin filaments inhibit osmotically driven water flow (Ito, T., K.S. Zaner, and T.P. Stossel. 1987. Biophys. J. 51: 745-753). Here we show that the actin gelation protein, actin-binding protein (ABP), impedes both osmotic shrinkage and swelling of an actin filament solution and reduces markedly the concentration of actin filaments required for this inhibition. These effects depend on actin filament immobilization, because the ABP concentration that causes initial impairment of water flow by a...

  13. Prediction and dissection of widely-varying association rate constants of actin-binding proteins.

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    Xiaodong Pang

    Full Text Available Actin is an abundant protein that constitutes a main component of the eukaryotic cytoskeleton. Its polymerization and depolymerization are regulated by a variety of actin-binding proteins. Their functions range from nucleation of actin polymerization to sequestering G-actin in 1∶1 complexes. The kinetics of forming these complexes, with rate constants varying at least three orders of magnitude, is critical to the distinct regulatory functions. Previously we have developed a transient-complex theory for computing protein association mechanisms and association rate constants. The transient complex refers to an intermediate in which the two associating proteins have near-native separation and relative orientation but have yet to form short-range specific interactions of the native complex. The association rate constant is predicted as k(a = k(a0 e(-ΔG(el*/k(BT, where k(a0 is the basal rate constant for reaching the transient complex by free diffusion, and the Boltzmann factor captures the bias of long-range electrostatic interactions. Here we applied the transient-complex theory to study the association kinetics of seven actin-binding proteins with G-actin. These proteins exhibit three classes of association mechanisms, due to their different molecular shapes and flexibility. The 1000-fold k(a variations among them can mostly be attributed to disparate electrostatic contributions. The basal rate constants also showed variations, resulting from the different shapes and sizes of the interfaces formed by the seven actin-binding proteins with G-actin. This study demonstrates the various ways that actin-binding proteins use physical properties to tune their association mechanisms and rate constants to suit distinct regulatory functions.

  14. Functional Analysis of Actin-Binding Proteins in the Central Nervous System of Drosophila.

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    He, Qi; Roblodowski, Christopher

    2016-01-01

    Using Drosophila actin-binding protein Dunc-115 as model system, this chapter describes a MARCM (mosaic analysis with a repressible cell marker)-based method for analyzing cytoskeletal components for their functions in the nervous system. Following a concise description about the principle, a step-by-step protocol is provided for generating the needed stocks and for histological analysis. Additional details and explanations have been given in the accompanying notes. Together, this should form a practical and sufficient recipe for performing at the single-cell-level loss-of-function and gain-of-function analyses of proteins associated with the cytoskeleton.

  15. Resemblance of actin-binding protein/actin gels to covalently crosslinked networks

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    Janmey, Paul A.; Hvidt, Søren; Lamb, Jennifer; Stossel, Thomas P.

    1990-05-01

    THE maintainance of the shape of cells is often due to their surface elasticity, which arises mainly from an actin-rich cytoplasmic cortex1,2. On locomotion, phagocytosis or fission, however, these cells become partially fluid-like. The finding of proteins that can bind to actin and control the assembly of, or crosslink, actin filaments, and of intracellular messages that regulate the activities of some of these actin-binding proteins, indicates that such 'gel sol' transformations result from the rearrangement of cortical actin-rich networks3. Alternatively, on the basis of a study of the mechanical properties of mixtures of actin filaments and an Acanthamoeba actin-binding protein, α-actinin, it has been proposed that these transformations can be accounted for by rapid exchange of crosslinks between actin filaments4: the cortical network would be solid when the deformation rate is greater than the rate of crosslink exchange, but would deform or 'creep' when deformation is slow enough to permit crosslinker molecules to rearrange. Here we report, however, that mixtures of actin filaments and actin-binding protein (ABP), an actin crosslinking protein of many higher eukaryotes, form gels Theologically equivalent to covalently crosslinked networks. These gels do not creep in response to applied stress on a time scale compatible with most cell-surface movements. These findings support a more complex and controlled mechanism underlying the dynamic mechanical properties of cortical cytoplasm, and can explain why cells do not collapse under the constant shear forces that often exist in tissues.

  16. Study of the influence of actin-binding proteins using linear analyses of cell deformability.

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    Plaza, Gustavo R; Uyeda, Taro Q P; Mirzaei, Zahra; Simmons, Craig A

    2015-07-21

    The actin cytoskeleton plays a key role in the deformability of the cell and in mechanosensing. Here we analyze the contributions of three major actin cross-linking proteins, myosin II, α-actinin and filamin, to cell deformability, by using micropipette aspiration of Dictyostelium cells. We examine the applicability of three simple mechanical models: for small deformation, linear viscoelasticity and drop of liquid with a tense cortex; and for large deformation, a Newtonian viscous fluid. For these models, we have derived linearized equations and we provide a novel, straightforward methodology to analyze the experiments. This methodology allowed us to differentiate the effects of the cross-linking proteins in the different regimes of deformation. Our results confirm some previous observations and suggest important relations between the molecular characteristics of the actin-binding proteins and the cell behavior: the effect of myosin is explained in terms of the relation between the lifetime of the bond to actin and the resistive force; the presence of α-actinin obstructs the deformation of the cytoskeleton, presumably mainly due to the higher molecular stiffness and to the lower dissociation rate constants; and filamin contributes critically to the global connectivity of the network, possibly by rapidly turning over cross-links during the remodeling of the cytoskeletal network, thanks to the higher rate constants, flexibility and larger size. The results suggest a sophisticated relationship between the expression levels of actin-binding proteins, deformability and mechanosensing. PMID:26059185

  17. Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring.

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    Gorlin, J B; Yamin, R; Egan, S; Stewart, M; Stossel, T P; Kwiatkowski, D J; Hartwig, J H

    1990-09-01

    Actin-binding protein (ABP-280, nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is near the amino-terminus of the subunit where the amino acid sequence is similar to other actin filament binding proteins, including alpha-actinin, beta-spectrin, dystrophin, and Dictyostelium abp-120. The remaining 90% of the sequence comprises 24 repeats, each approximately 96 residues long, predicted to have stretches of beta-sheet secondary structure interspersed with turns. The first 15 repeats may have substantial intrachain hydrophobic interactions and overlap in a staggered fashion to yield a backbone with mechanical resilience. Sequence insertions immediately before repeats 16 and 24 predict two hinges in the molecule near points where rotary-shadowed molecules appear to swivel in electron micrographs. Both putative hinge regions are susceptible to cleavage by proteases and the second also contains the site that binds the platelet glycoprotein Ib/IX complex. Phosphorylation consensus sequences are also located in the hinges or near them. Degeneracy within every even-numbered repeat between 16 and 24 and the insertion before repeat 24 may convert interactions within chains to interactions between chains to account for dimer formation within a domain of 7 kD at the carboxy-terminus. The structure of ABP dimers resembles a leaf spring. Interchain interactions hold the leaves firmly together at one end, whereas intrachain hydrophobic bonds reinforce the arms of the spring where the leaves diverge, making it sufficiently stiff to promote high-angle branching of actin

  18. Comparative mapping of the actin-binding protein 280 genes in human and mouse

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    Gariboldi, M.; Canzian, F.; Manenti, G.; De Gregorio, L. (Istituto Nazionale Tumori, Milan (Italy)); Maestrini, E.; Rivella, S. (Istituto di Genetica Biochimica ed Evoluzionistica, Pavia (Italy)); Chatterjee, A.; Herman, G.E. (Universita di Bari (Italy)); Archidiacono, N.; Antonacci, R. (Institute for Molecular Genetics, Houston, TX (United States)) (and others)

    1994-05-15

    Two genes encode actin-binding protein 280 isoforms. ABP-280 or filamin (FLN1) is present in the cytoskeleton of many cell types, whereas expression of FLN2 is limited to skeletal muscle and heart. FLN1 maps to human chromosome Xq28, and, by physical mapping in YAC clones, the authors have mapped the homologous murine locus (Fln1) to mouse chromosome X, in a region of syntenic homology with human chromosome X. They mapped FLN2 to human chromosome 7q32-q35 by analysis of somatic cell hybrids containing portions of chromosome 7, and, by using a mapping panel from an interspecific murine cross, they mapped the corresponding murine locus (Fln2) to murine chromosome 6 in a region homologous to human chromosome 7. 21 refs., 1 fig., 1 tab.

  19. The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter

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    Sérgio Carvalho Leite

    2016-04-01

    Full Text Available The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here, we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord and optic and sciatic nerves, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew, suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity but is necessary to control the diameter of both actin rings and axons and actin filament growth within rings.

  20. EhNCABP166: a nucleocytoplasmic actin-binding protein from Entamoeba histolytica.

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    Campos-Parra, A D; Hernández-Cuevas, N A; Hernandez-Rivas, R; Vargas, M

    2010-07-01

    The actin cytoskeleton consists of multiple actin binding proteins (ABPs) that participate cooperatively in different cellular functions such as the maintenance of polarity and cell motility as well as the invasion of target cells and regulation of gene expression, among others. Due to the important role of ABPs in the pathogenesis of Entamoeba histolytica, the role of a new nucleocytoplasmic ABP from E. histolytica named EhNCABP166 was investigated. The EhNCABP166 gene encodes a protein with an estimated molecular weight of 166kDa. Structurally, this peptide is composed of two CH domains arranged in tandem at the N-terminus of the protein, followed by an alpha-helical region containing a number of different domains with a low level of homology. Two (Bin1/Amphiphysin/Rvs167) (BAR) domains, one GTPase-binding/formin 3 homology (GBD/FH3) domain, three Bcl2-associated athanogene (BAG) domains, one basic-leucine zipper (bZIP) domain and one poly(A)-binding protein C-terminal (PABC) domain were also present. Molecular and biochemical studies showed that the EhNCABP166 protein is transcribed and translated in trophozoites of E. histolytica. It was also shown that the CH domains are functional and bind to F-actin, whereas the BAR and GBD/FH3 domains interact in vitro and in vivo with different families of GTPases such as Rho and Ras, and with different phosphoinositides. These findings suggest that these domains have the conserved functional properties described in other eukaryotic systems. These domains also interacted with additional GTPase and lipid targets that have not been previously described. Finally, cellular studies showed that EhNCABP166 is localized to the cytoplasm and nucleus of E. histolytica and that it has an important role in phagocytosis, proliferation, and motility of E. histolytica.

  1. MARCKS is a natively unfolded protein with an inaccessible actin-binding site: evidence for long-range intramolecular interactions.

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    Tapp, Hazel; Al-Naggar, Iman M; Yarmola, Elena G; Harrison, Alexis; Shaw, Gerry; Edison, Arthur S; Bubb, Michael R

    2005-03-18

    Myristoylated alanine-rich C kinase substrate (MARCKS) is an unfolded protein that contains well characterized actin-binding sites within the phosphorylation site domain (PSD), yet paradoxically, we now find that intact MARCKS does not bind to actin. Intact MARCKS also does not bind as well to calmodulin as does the PSD alone. Myristoylation at the N terminus alters how calmodulin binds to MARCKS, implying that, despite its unfolded state, the distant N terminus influences binding events at the PSD. We show that the free PSD binds with site specificity to MARCKS, suggesting that long-range intramolecular interactions within MARCKS are also possible. Because of the unusual primary sequence of MARCKS with an overall isoelectric point of 4.2 yet a very basic PSD (overall charge of +13), we speculated that ionic interactions between oppositely charged domains of MARCKS were responsible for long-range interactions within MARCKS that sterically influence binding events at the PSD and that explain the observed differences between properties of the PSD and MARCKS. Consistent with this hypothesis, chemical modifications of MARCKS that neutralize negatively charged residues outside of the PSD allow the PSD to bind to actin and increase the affinity of MARCKS for calmodulin. Similarly, both myristoylation of MARCKS and cleavage of MARCKS by calpain are shown to increase the availability of the PSD so as to activate its actin-binding activity. Because abundant evidence supports the conclusion that MARCKS is an important protein in regulating actin dynamics, our data imply that post-translational modifications of MARCKS are necessary and sufficient to regulate actin-binding activity. PMID:15640140

  2. NAC1 is an actin-binding protein that is essential for effective cytokinesis in cancer cells.

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    Yap, Kai Lee; Fraley, Stephanie I; Thiaville, Michelle M; Jinawath, Natini; Nakayama, Kentaro; Wang, Jianlong; Wang, Tian-Li; Wirtz, Denis; Shih, Ie-Ming

    2012-08-15

    NAC1 is a transcriptional corepressor protein that is essential to sustain cancer cell proliferation and migration. However, the underlying molecular mechanisms of NAC1 function in cancer cells remain unknown. In this study, we show that NAC1 functions as an actin monomer-binding protein. The conserved BTB protein interaction domain in NAC1 is the minimal region for actin binding. Disrupting NAC1 complex function by dominant-negative or siRNA strategies reduced cell retraction and abscission during late-stage cytokinesis, causing multinucleation in cancer cells. In Nac1-deficient murine fibroblasts, restoring NAC1 expression was sufficient to partially avert multinucleation. We found that siRNA-mediated silencing of the actin-binding protein profilin-1 in cancer cells caused a similar multinucleation phenotype and that NAC1 modulated the binding of actin to profillin-1. Taken together, our results indicate that the NAC1/actin/profilin-1 complex is crucial for cancer cell cytokinesis, with a variety of important biologic and clinical implications.

  3. The actin-binding protein capulet genetically interacts with the microtubule motor kinesin to maintain neuronal dendrite homeostasis.

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    Paul M B Medina

    Full Text Available BACKGROUND: Neurons require precise cytoskeletal regulation within neurites, containing microtubule tracks for cargo transport in axons and dendrites or within synapses containing organized actin. Due to the unique architecture and specialized function of neurons, neurons are particularly susceptible to perturbation of the cytoskeleton. Numerous actin-binding proteins help maintain proper cytoskeletal regulation. METHODOLOGY/PRINCIPAL FINDINGS: From a Drosophila forward genetic screen, we identified a mutation in capulet--encoding a conserved actin-binding protein--that causes abnormal aggregates of actin within dendrites. Through interaction studies, we demonstrate that simultaneous genetic inactivation of capulet and kinesin heavy chain, a microtubule motor protein, produces elongate cofilin-actin rods within dendrites but not axons. These rods resemble actin-rich structures induced in both mammalian neurodegenerative and Drosophila Alzheimer's models, but have not previously been identified by loss of function mutations in vivo. We further demonstrate that mitochondria, which are transported by Kinesin, have impaired distribution along dendrites in a capulet mutant. While Capulet and Cofilin may biochemically cooperate in certain circumstances, in neuronal dendrites they genetically antagonize each other. CONCLUSIONS/SIGNIFICANCE: The present study is the first molecularly defined loss of function demonstration of actin-cofilin rods in vivo. This study suggests that simultaneous, seemingly minor perturbations in neuronal dendrites can synergize producing severe abnormalities affecting actin, microtubules and mitochondria/energy availability in dendrites. Additionally, as >90% of Alzheimer's and Parkinson's cases are sporadic this study suggests mechanisms by which multiple mutations together may contribute to neurodegeneration instead of reliance on single mutations to produce disease.

  4. The parafibromin tumor suppressor protein interacts with actin-binding proteins actinin-2 and actinin-3

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    Marx Stephen J

    2008-08-01

    Full Text Available Abstract Background Germline and somatic inactivating mutations in the HRPT2 gene occur in the inherited hyperparathyroidism-jaw tumor syndrome, in some cases of parathyroid cancer and in some cases of familial hyperparathyroidism. HRPT2 encodes parafibromin. To identify parafibromin interacting proteins we used the yeast two-hybrid system for screening a heart cDNA library with parafibromin as the bait. Results Fourteen parafibromin interaction positive preys representing 10 independent clones encoding actinin-2 were isolated. Parafibromin interacted with muscle alpha-actinins (actinin-2 and actinin-3, but not with non-muscle alpha-actinins (actinin-1 and actinin-4. The parafibromin-actinin interaction was verified by yeast two-hybrid, GST pull-down, and co-immunoprecipitation. Yeast two-hybrid analysis revealed that the N-terminal region of parafibromin interacted with actinins. In actin sedimentation assays parafibromin did not dissociate skeletal muscle actinins from actin filaments, but interestingly, parafibromin could also bundle/cross-link actin filaments. Parafibromin was predominantly nuclear in undifferentiated proliferating myoblasts (C2C12 cells, but in differentiated C2C12 myotubes parafibromin co-localized with actinins in the cytoplasmic compartment. Conclusion These data support a possible contribution of parafibromin outside the nucleus through its interaction with actinins and actin bundling/cross-linking. These data also suggest that actinins (and actin participate in sequestering parafibromin in the cytoplasmic compartment.

  5. The Dynamic Pollen Tube Cytoskeleton: Live Cell Studies Using Actin-Binding and Microtubule-Binding Reporter Proteins

    Institute of Scientific and Technical Information of China (English)

    Alice Y. Cheung; Qiao-hong Duan; Silvia Santos Costa; Barend H.J.de Graaf; Veronica S.Di Stilio; Jose Feijo; Hen-Ming Wu

    2008-01-01

    Pollen tubes elongate within the pistil to transport sperm cells to the embryo sac for fertilization.Growth occurs exclusively at the tube apex,rendering pollen tube elongation a most dramatic polar cell growth process.A hall-mark pollen tube feature is its cytoskeleton,which comprises elaborately organized and dynamic actin microfilaments and microtubules.Pollen tube growth is dependent on the actin cytoskeleton;its organization and regulation have been exalined extensively by various approaches.including fluorescent protein labeled actin-binding proteins in live cell studies.Using the previously described GFP-NtADF1 and GFP-LIADF1, and a new actin reporter protein NtPLIM2b-GFP,we re-affirm that the predominant actin structures in elongating tobacco and lily pollen tubes are long,streaming actin cables along the pollen tube shank,and a subapical structure comprising shorter actin cables.The subapical collection of actin microfilaments undergoes dynamic changes,giving rise to the appearance of structures that range from basket-or funnel-shaped,mesh-like to a subtle ring.NtPLIM2b-GFP is used in combination with a guanine nucleotide exchange factor for the Rho GTPases,AtROP-GEF1,to illustrate the use of these actin reporter proteins to explore the linkage between the polar cell growth process and its actin cytoskeleton.Contrary to the actin cytoskeleton,microtubules appear not to play a direct role in supporting the polar cell growth process in angiosperm pollen tubes.Using a microtubule reporter protein based on the microtubule end-binding protein from Arabidopsis AtEB1,GFP-AtEB1,we show that the extensive microtubule network in elongating pollen tubes displays varying degrees of dynamics.These reporter proteins provide versatile tools to explore the functional connection between major structural and signaling components of the polar pollen tube growth process.

  6. Actin-binding Rho activating protein is expressed in the central nervous system of normal adult rats

    Institute of Scientific and Technical Information of China (English)

    Lihua Liu; Jutta Schaper; Mingying Luo; Baolin Yang; Xiaoqiong Wu; Wu Zhu; Yinglu Guan; Weijun Cai; Kerstin Troidl; Wolfgang Schaper

    2012-01-01

    Previous studies show that actin-binding Rho activating protein (Abra) is expressed in cardiomyocytes and vascular smooth muscle cells. In this study, we investigated the expression profile of Abra in the central nervous system of normal adult rats by confocal immunofluorescence.Results show ed that Abra immunostaining was located in neuronal nuclei, cytoplasm and processes in the central nervous system, with the strongest staining in the nuclei; in the cerebral cortex, Abra positive neuronal bodies and processes were distributed in six cortical layers including molecular layer, external granular layer, external pyramidal layer, internal granular layer, internal pyramidal layer and polymorphic layer; in the hippocampus, the cell bodies of Abra positive neurons were distributed evenly in pyramidal layer and granular layer, with pos itive processes in molecular layer and orien layer; in the cerebellar cortex, Abra staining showed the positive neuronal cell bodies in Purkinje cell layer and granular layer and positive processes in molecular layer; in the spinal cord, Abra-immunopositive products covered the whole gray matter and w hite matter; co-localization studies showed that Abra was co-stained with F-actin in neuronal cytoplasm and processes, but weakly in the nuclei. In addition, in the hippocampus, Abra was co-stained with F-actin only in neuronal processes, but not in the cell body. This study for the first time presents a comprehensive overview of Abra expression in the central nervous system, providing insights for further investigating the role of Abra in the mature central nervous system.

  7. Progressive hearing loss and gradual deterioration of sensory hair bundles in the ears of mice lacking the actin-binding protein Eps8L2

    OpenAIRE

    Furness, David N.; Johnson, Stuart L.; Manor, Uri; Rüttiger, Lukas; Tocchetti, Arianna; Offenhauser, Nina; Olt, Jennifer; Goodyear, Richard J.; Vijayakumar, Sarath; Dai, Yuhai; Hackney, Carole M.; Franz, Christoph; Di Fiore, Pier Paolo; Masetto, Sergio; Jones, Sherri M.

    2013-01-01

    Mechanotransduction in the mammalian auditory system depends on mechanosensitive channels in the hair bundles that project from the apical surface of the sensory hair cells. Individual stereocilia within each bundle contain a core of tightly packed actin filaments, whose length is dynamically regulated during development and in the adult. We show that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly i...

  8. XIRP2, an Actin-Binding Protein Essential for Inner Ear Hair-Cell Stereocilia

    Directory of Open Access Journals (Sweden)

    Déborah I. Scheffer

    2015-03-01

    Full Text Available Hair cells of the inner ear are mechanoreceptors for hearing and balance, and proteins highly enriched in hair cells may have specific roles in the development and maintenance of the mechanotransduction apparatus. We identified XIRP2/mXinβ as an enriched protein likely to be essential for hair cells. We found that different isoforms of this protein are expressed and differentially located: short splice forms (also called XEPLIN are targeted more to stereocilia, whereas two long isoforms containing a XIN-repeat domain are in both stereocilia and cuticular plates. Mice lacking the Xirp2 gene developed normal stereocilia bundles, but these degenerated with time: stereocilia were lost and long membranous protrusions emanated from the nearby apical surfaces. At an ultrastructural level, the paracrystalline actin filaments became disorganized. XIRP2 is apparently involved in the maintenance of actin structures in stereocilia and cuticular plates of hair cells, and perhaps in other organs where it is expressed.

  9. On the association of glycoprotein Ib and actin-binding protein in human platelets

    OpenAIRE

    1985-01-01

    Glycoprotein (GP) Ib was purified from lysates of human platelets prepared in the presence or absence of inhibitors of the endogenous calcium-activated neutral protease (CANP) by immunoaffinity chromatography, employing the GPIb-specific murine monoclonal antibody, AP1, coupled to Sepharose CL4B. When derived from lysates prepared in the presence of EDTA or leupeptin, the eluate from the AP1-affinity column contained a 240,000-260,000-mol-wt protein in addition to GPIb. In SDS PAGE, this prot...

  10. Cooperation between actin-binding proteins of invasive Salmonella: SipA potentiates SipC nucleation and bundling of actin

    OpenAIRE

    Emma J McGhie; Hayward, Richard D.; Koronakis, Vassilis

    2001-01-01

    Pathogen-induced remodelling of the host cell actin cytoskeleton drives internalization of invasive Salmon ella by non-phagocytic intestinal epithelial cells. Two Salmonella actin-binding proteins are involved in internalization: SipC is essential for the process, while SipA enhances its efficiency. Using purified SipC and SipA proteins in in vitro assays of actin dynamics and F-actin bundling, we demonstrate that SipA stimulates substantially SipC-mediated nucleation of actin polymerization....

  11. Drosophila myosin-XX functions as an actin-binding protein to facilitate the interaction between Zyx102 and actin.

    Science.gov (United States)

    Cao, Yang; White, Howard D; Li, Xiang-Dong

    2014-01-21

    The class XX myosin is a member of the diverse myosin superfamily and exists in insects and several lower invertebrates. DmMyo20, the class XX myosin in Drosophila, is encoded by dachs, which functions as a crucial downstream component of the Fat signaling pathway, influencing growth, affinity, and gene expression during development. Sequence analysis shows that DmMyo20 contains a unique N-terminal extension, the motor domain, followed by one IQ motif, and a C-terminal tail. To investigate the biochemical properties of DmMyo20, we expressed several DmMyo20 truncated constructs containing the motor domain in the baculovirus/Sf9 system. We found that the motor domain of DmMyo20 had neither ATPase activity nor the ability to bind to ATP, suggesting that DmMyo20 does not function as a molecular motor. We found that the motor domain of DmMyo20 could specifically bind to actin filaments in an ATP-independent manner and enhance the interaction between actin filaments and Zyx102, a downstream component of DmMyo20 in the Fat signaling pathway. These results suggest that DmMyo20 functions as a scaffold protein, but not as a molecular motor, in a signaling pathway controlling cell differentiation.

  12. Identification of regions within the Legionella pneumophila VipA effector protein involved in actin binding and polymerization and in interference with eukaryotic organelle trafficking.

    Science.gov (United States)

    Bugalhão, Joana N; Mota, Luís Jaime; Franco, Irina S

    2016-02-01

    The Legionella pneumophila effector protein VipA is an actin nucleator that co-localizes with actin filaments and early endosomes in infected macrophages and which interferes with organelle trafficking when expressed in yeast. To identify the regions of VipA involved in its subcellular localization and functions, we ectopically expressed specific VipA mutant proteins in eukaryotic cells. This indicated that the characteristic punctate distribution of VipA depends on its NH2 -terminal (amino acid residues 1-133) and central coiled-coil (amino acid residues 133-206) regions, and suggested a role for the COOH-terminal (amino acid residues 206-339) region in association with actin filaments and for the NH2 -terminal in co-localization with early endosomes. Co-immunoprecipitation and in vitro assays showed that the COOH-terminal region of VipA is necessary and sufficient to mediate actin binding, and is essential but insufficient to induce microfilament formation. Assays in yeast revealed that the NH2 and the COOH-terminal regions, and possibly an NPY motif within the NH2 region of VipA, are necessary for interference with organelle trafficking. Overall, this suggests that subversion of eukaryotic vesicular trafficking by VipA involves both its ability to associate with early endosomes via its NH2 -terminal region and its capacity to bind and polymerize actin through its COOH-terminal region. PMID:26626407

  13. LATS1 tumor suppressor is a novel actin-binding protein and negative regulator of actin polymerization

    Institute of Scientific and Technical Information of China (English)

    Stacy Visser-Grieve; Zhonghua Zhou; Yi-Min She; He Huang; Terry D Cyr; Tian Xu; Xiaolong Yang

    2011-01-01

    Dear Editor,The LATS tumor suppressor,conserved from Drosophila (dlats) to humans (LATS1,LATS2),plays a vital role in maintaining cellular homeostasis in humans since loss of either LATS1 or LATS2 leads to the development of numerous cancer types such as breast cancer and leukemia [1].Apart from its roles as a Ser/Thr kinase within the emerging Hippo pathway regulating cell proliferation and apoptosis,ultimately leading to the control of organ size and tumorigenesis [2],LATS is also implicated in a broad range of functions including regulation of genetic stability,transcription,and protein stability [1 ].Recently,tumor suppressors have also been shown to affect the later stages of tumorigenesis,including metastasis.Among this group of metastasis regulators are genes that can directly affect actin dynamics by binding to F-actin,such as the tumor suppressors p53 [3],NF2 [4] and APC [5].

  14. Arabidopsis AtADF1 is Functionally Affected by Mutations on Actin Binding Sites

    Institute of Scientific and Technical Information of China (English)

    Chun-Hai Dong; Wei-Ping Tang; Jia-Yao Liu

    2013-01-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin,and is directly involved in the depolymerization of actin filaments.To better understand the actin binding sites of the Arabidopsis thaliana L.AtADF1,we generated mutants of AtADF1 and investigated their functions in vitro and in vivo.Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G-and F-actin binding.The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A,R137/A) form another actin binding site that is important for F-actin binding.Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L.plants overexpressing these mutants,we analyzed how these mutant proteins regulate actin organization and affect seedling growth.Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional,unless the affinity foractin monomers is also affected.The G-actin binding activity of the ADF plays an essential role in actin binding,depolymerization of actin polymers,and therefore in the control of actin organization.

  15. Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion

    DEFF Research Database (Denmark)

    Galliano, M F; Huet, C; Frygelius, J;

    2000-01-01

    differentiation. Using the yeast two-hybrid screen, we found that the muscle-specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific interaction. The major binding site for alpha-actinin-2 was mapped to a short sequence in...

  16. Actin-binding protein regulation by microRNAs as a novel microbial strategy to modulate phagocytosis by host cells: the case of N-Wasp and miR-142-3p

    Directory of Open Access Journals (Sweden)

    Paulo eBettencourt

    2013-06-01

    Full Text Available Mycobacterium tuberculosis (Mtb is a successful intracellular pathogen that thrives in macrophages (Mφs. There is a need to better understand how Mtb hijacks cellular processes like phagolysosome biogenesis, a classical pathogenesis determinant. A central feature to this microbial strategy is the manipulation of actin in Mφs. Here, we examine the role of microRNAs (miRNAs as a potential mechanism in the regulation of actin-mediated events leading to phagocytosis in the context of mycobacteria infection. Given that non-virulent Mycobacterium smegmatis also controls actin filamentation to prolong its instracellular survival inside host cells, our approach was to perform a global transcriptomic approach to assess the modulation of miRNAs upon M. smegmatis infection of the murine Mφ cell line, J774A.1. This approach yielded miR-142-3p as a top candidate to be involved in the regulation of actin dynamics required in phagocytosis. We unequivocally demonstrate miR-142-3p targets N-Wasp, an actin-binding protein required during microbial challenge. A gain-of-function approach for miR-142-3p revealed a down-regulation of N-Wasp expression accompanied by a decrease of mycobacteria intake, while a loss-of-function approach yielded the reciprocal increase of the phagocytosis process. Equally important, we show Mtb induces the early expression of miR-142-3p and down-regulates N-Wasp protein levels in both the murine J774A.1 cell line and primary human Mφs. As proof of principle, the partial siRNA-mediated knock down of N-Wasp resulted in a decrease of Mtb intake by human Mφs, reflected in lower levels of colony-forming units counts over time. We therefore propose the modulation of miRNAs as a novel strategy in mycobacterial infection to control factors involved in actin filamentation and other early events of phagolysosome biogenesis, such as the case presented here for miR-142-3p influencing N-Wasp activity during the phagocytosis process.

  17. Filament assembly by Spire: key residues and concerted actin binding.

    Science.gov (United States)

    Rasson, Amy S; Bois, Justin S; Pham, Duy Stephen L; Yoo, Haneul; Quinlan, Margot E

    2015-02-27

    The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers.

  18. Comparative genome analysis of cortactin and HSI : the significance of the F-actin binding repeat domain

    NARCIS (Netherlands)

    van Rossum, AGSH; Schuuring-Scholtes, E; Seggelen, VV; Kluin, PM; Schuuring, E

    2005-01-01

    Background: In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp)2/3 mediated actin polymerization. It shares a high amino acid seque

  19. Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro.

    Directory of Open Access Journals (Sweden)

    Rohini Shrivastava

    Full Text Available Ezrin, a member of the ERM (Ezrin/Radixin/Moesin protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2 or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.

  20. 25 Years of Tension over Actin Binding to the Cadherin Cell Adhesion Complex: The Devil is in the Details.

    Science.gov (United States)

    Nelson, W James; Weis, William I

    2016-07-01

    Over the past 25 years, there has been a conceptual (re)evolution in understanding how the cadherin cell adhesion complex, which contains F-actin-binding proteins, binds to the actin cytoskeleton. There is now good synergy between structural, biochemical, and cell biological results that the cadherin-catenin complex binds to F-actin under force. PMID:27166091

  1. Systematic Prediction of Scaffold Proteins Reveals New Design Principles in Scaffold-Mediated Signal Transduction.

    Directory of Open Access Journals (Sweden)

    Jianfei Hu

    Full Text Available Scaffold proteins play a crucial role in facilitating signal transduction in eukaryotes by bringing together multiple signaling components. In this study, we performed a systematic analysis of scaffold proteins in signal transduction by integrating protein-protein interaction and kinase-substrate relationship networks. We predicted 212 scaffold proteins that are involved in 605 distinct signaling pathways. The computational prediction was validated using a protein microarray-based approach. The predicted scaffold proteins showed several interesting characteristics, as we expected from the functionality of scaffold proteins. We found that the scaffold proteins are likely to interact with each other, which is consistent with previous finding that scaffold proteins tend to form homodimers and heterodimers. Interestingly, a single scaffold protein can be involved in multiple signaling pathways by interacting with other scaffold protein partners. Furthermore, we propose two possible regulatory mechanisms by which the activity of scaffold proteins is coordinated with their associated pathways through phosphorylation process.

  2. Comparative genome analysis of cortactin and HS1: the significance of the F-actin binding repeat domain

    Directory of Open Access Journals (Sweden)

    Seggelen Vera

    2005-02-01

    Full Text Available Abstract Background In human carcinomas, overexpression of cortactin correlates with poor prognosis. Cortactin is an F-actin-binding protein involved in cytoskeletal rearrangements and cell migration by promoting actin-related protein (Arp2/3 mediated actin polymerization. It shares a high amino acid sequence and structural similarity to hematopoietic lineage cell-specific protein 1 (HS1 although their functions differ considerable. In this manuscript we describe the genomic organization of these two genes in a variety of species by a combination of cloning and database searches. Based on our analysis, we predict the genesis of the actin-binding repeat domain during evolution. Results Cortactin homologues exist in sponges, worms, shrimps, insects, urochordates, fishes, amphibians, birds and mammalians, whereas HS1 exists in vertebrates only, suggesting that both genes have been derived from an ancestor cortactin gene by duplication. In agreement with this, comparative genome analysis revealed very similar exon-intron structures and sequence homologies, especially over the regions that encode the characteristic highly conserved F-actin-binding repeat domain. Cortactin splice variants affecting this F-actin-binding domain were identified not only in mammalians, but also in amphibians, fishes and birds. In mammalians, cortactin is ubiquitously expressed except in hematopoietic cells, whereas HS1 is mainly expressed in hematopoietic cells. In accordance with their distinct tissue specificity, the putative promoter region of cortactin is different from HS1. Conclusions Comparative analysis of the genomic organization and amino acid sequences of cortactin and HS1 provides inside into their origin and evolution. Our analysis shows that both genes originated from a gene duplication event and subsequently HS1 lost two repeats, whereas cortactin gained one repeat. Our analysis genetically underscores the significance of the F-actin binding domain in

  3. Concentration profiles of actin-binding molecules in lamellipodia

    Science.gov (United States)

    Falcke, Martin

    2016-04-01

    Motile cells form lamellipodia in the direction of motion, which are flat membrane protrusions containing an actin filament network. The network flows rearward relative to the leading edge of the lamellipodium due to actin polymerization at the front. Thus, actin binding molecules are subject to transport towards the rear of the cell in the bound state and diffuse freely in the unbound state. We analyze this reaction-diffusion-advection process with respect to the concentration profiles of these species and provide an analytic approximation for them. Network flow may cause a depletion zone of actin binding molecules close to the leading edge. The existence of such zone depends on the free molecule concentration in the cell body, on the ratio of the diffusion length to the distance bound molecules travel rearward with the flow before dissociating, and the ratio of the diffusion length to the width of the region with network flow and actin binding. Our calculations suggest the existence of depletion zones for the F-actin cross-linkers filamin and α-actinin in fish keratocytes (and other cell types), which is in line with the small elastic moduli of the F-actin network close to the leading edge found in measurements of the force motile cells are able to exert.

  4. A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding.

    Science.gov (United States)

    Avery, Adam W; Crain, Jonathan; Thomas, David D; Hays, Thomas S

    2016-01-01

    Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration. PMID:26883385

  5. Human RNASET2 derivatives as potential anti-angiogenic agents: actin binding sequence identification and characterization

    Science.gov (United States)

    Nesiel-Nuttman, Liron; Doron, Shani; Schwartz, Betty; Shoseyov, Oded

    2015-01-01

    Human RNASET2 (hRNASET2) has been demonstrated to exert antiangiogenic and antitumorigenic effects independent of its ribonuclease capacity. We suggested that RNASET2 exerts its antiangiogenic and antitumorigenic activities via binding to actin and consequently inhibits cell motility. We focused herein on the identification of the actin binding site of hRNASET2 using defined sequences encountered within the whole hRNASET2 protein. For that purpose we designed 29 different hRNASET2-derived peptides. The 29 peptides were examined for their ability to bind immobilized actin. Two selected peptides-A103-Q159 consisting of 57 amino acids and peptide K108-K133 consisting of 26 amino acids were demonstrated to have the highest actin binding ability and concomitantly the most potent anti-angiogenic activity. Further analyses on the putative mechanisms associated with angiogenesis inhibition exerted by peptide K108-K133 involved its location during treatment within the HUVE cells. Peptide K108-K133 readily penetrates the cell membrane within 10 min of incubation. In addition, supplementation with angiogenin delays the entrance of peptide K108-K133 to the cell suggesting competition on the same cell internalization route. The peptide was demonstrated to co-localize with angiogenin, suggesting that both molecules bind analogous cellular epitopes, similar to our previously reported data for ACTIBIND and trT2-50. PMID:25815360

  6. Protein Scaffolding for Small Molecule Catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Baker, David [Univ. of Washington, Seattle, WA (United States)

    2014-09-14

    We aim to design hybrid catalysts for energy production and storage that combine the high specificity, affinity, and tunability of proteins with the potent chemical reactivities of small organometallic molecules. The widely used Rosetta and RosettaDesign methodologies will be extended to model novel protein / small molecule catalysts in which one or many small molecule active centers are supported and coordinated by protein scaffolding. The promise of such hybrid molecular systems will be demonstrated with the nickel-phosphine hydrogenase of DuBois et. al.We will enhance the hydrogenase activity of the catalyst by designing protein scaffolds that incorporate proton relays and systematically modulate the local environment of the catalyticcenter. In collaboration with DuBois and Shaw, the designs will be experimentally synthesized and characterized.

  7. Control of actin-based motility through localized actin binding

    International Nuclear Information System (INIS)

    A wide variety of cell biological and biomimetic systems use actin polymerization to drive motility. It has been suggested that an object such as a bacterium can propel itself by self-assembling a high concentration of actin behind it, if it is repelled by actin. However, it is also known that it is essential for the moving object to bind actin. Therefore, a key question is how the actin tail can propel an object when it both binds and repels the object. We present a physically consistent Brownian dynamics model for actin-based motility that includes the minimal components of the dendritic nucleation model and allows for both attractive and repulsive interactions between actin and a moveable disc. We find that the concentration gradient of filamentous actin generated by polymerization is sufficient to propel the object, even with moderately strong binding interactions. Additionally, actin binding can act as a biophysical cap, and may directly control motility through modulation of network growth. Overall, this mechanism is robust in that it can drive motility against a load up to a stall pressure that depends on the Young’s modulus of the actin network and can explain several aspects of actin-based motility. (paper)

  8. A Short Splice Form of Xin-Actin Binding Repeat Containing 2 (XIRP2) Lacking the Xin Repeats Is Required for Maintenance of Stereocilia Morphology and Hearing Function

    OpenAIRE

    Francis, Shimon P.; Krey, Jocelyn F.; Krystofiak, Evan S.; Cui, Runjia; Nanda, Sonali; Xu, Wenhao; Kachar, Bechara; Barr-Gillespie, Peter G.; Shin, Jung-Bum

    2015-01-01

    Approximately one-third of known deafness genes encode proteins located in the hair bundle, the sensory hair cell's mechanoreceptive organelle. In previous studies, we used mass spectrometry to characterize the hair bundle's proteome, resulting in the discovery of novel bundle proteins. One such protein is Xin-actin binding repeat containing 2 (XIRP2), an actin-cross-linking protein previously reported to be specifically expressed in striated muscle. Because mutations in other actin-cross-lin...

  9. Identification of sucrose synthase as an actin-binding protein

    Science.gov (United States)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  10. Scaffold diversification enhances effectiveness of a superlibrary of hyperthermophilic proteins.

    Science.gov (United States)

    Hussain, Mahmud; Gera, Nimish; Hill, Andrew B; Rao, Balaji M

    2013-01-18

    The use of binding proteins from non-immunoglobulin scaffolds has become increasingly common in biotechnology and medicine. Typically, binders are isolated from a combinatorial library generated by mutating a single scaffold protein. In contrast, here we generated a "superlibrary" or "library-of-libraries" of 4 × 10(8) protein variants by mutagenesis of seven different hyperthermophilic proteins; six of the seven proteins have not been used as scaffolds prior to this study. Binding proteins for five different model targets were successfully isolated from this library. Binders obtained were derived from five out of the seven scaffolds. Strikingly, binders from this modestly sized superlibrary have affinities comparable or higher than those obtained from a library with 1000-fold higher sequence diversity but derived from a single stable scaffold. Thus scaffold diversification, i.e., randomization of multiple different scaffolds, is a powerful alternate strategy for combinatorial library construction.

  11. Membrane-mediated interaction between strongly anisotropic protein scaffolds.

    Directory of Open Access Journals (Sweden)

    Yonatan Schweitzer

    2015-02-01

    Full Text Available Specialized proteins serve as scaffolds sculpting strongly curved membranes of intracellular organelles. Effective membrane shaping requires segregation of these proteins into domains and is, therefore, critically dependent on the protein-protein interaction. Interactions mediated by membrane elastic deformations have been extensively analyzed within approximations of large inter-protein distances, small extents of the protein-mediated membrane bending and small deviations of the protein shapes from isotropic spherical segments. At the same time, important classes of the realistic membrane-shaping proteins have strongly elongated shapes with large and highly anisotropic curvature. Here we investigated, computationally, the membrane mediated interaction between proteins or protein oligomers representing membrane scaffolds with strongly anisotropic curvature, and addressed, quantitatively, a specific case of the scaffold geometrical parameters characterizing BAR domains, which are crucial for membrane shaping in endocytosis. In addition to the previously analyzed contributions to the interaction, we considered a repulsive force stemming from the entropy of the scaffold orientation. We computed this interaction to be of the same order of magnitude as the well-known attractive force related to the entropy of membrane undulations. We demonstrated the scaffold shape anisotropy to cause a mutual aligning of the scaffolds and to generate a strong attractive interaction bringing the scaffolds close to each other to equilibrium distances much smaller than the scaffold size. We computed the energy of interaction between scaffolds of a realistic geometry to constitute tens of kBT, which guarantees a robust segregation of the scaffolds into domains.

  12. The conserved Tarp actin binding domain is important for chlamydial invasion.

    Directory of Open Access Journals (Sweden)

    Travis J Jewett

    Full Text Available The translocated actin recruiting phosphoprotein (Tarp is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.

  13. Soy Protein Scaffold Biomaterials for Tissue Engineering and Regenerative Medicine

    Science.gov (United States)

    Chien, Karen B.

    Developing functional biomaterials using highly processable materials with tailorable physical and bioactive properties is an ongoing challenge in tissue engineering. Soy protein is an abundant, natural resource with potential use for regenerative medicine applications. Preliminary studies show that soy protein can be physically modified and fabricated into various biocompatible constructs. However, optimized soy protein structures for tissue regeneration (i.e. 3D porous scaffolds) have not yet been designed. Furthermore, little work has established the in vivo biocompatibility of implanted soy protein and the benefit of using soy over other proteins including FDA-approved bovine collagen. In this work, freeze-drying and 3D printing fabrication processes were developed using commercially available soy protein to create porous scaffolds that improve cell growth and infiltration compared to other soy biomaterials previously reported. Characterization of scaffold structure, porosity, and mechanical/degradation properties was performed. In addition, the behavior of human mesenchymal stem cells seeded on various designed soy scaffolds was analyzed. Biological characterization of the cell-seeded scaffolds was performed to assess feasibility for use in liver tissue regeneration. The acute and humoral response of soy scaffolds implanted in an in vivo mouse subcutaneous model was also investigated. All fabricated soy scaffolds were modified using thermal, chemical, and enzymatic crosslinking to change properties and cell growth behavior. 3D printing allowed for control of scaffold pore size and geometry. Scaffold structure, porosity, and degradation rate significantly altered the in vivo response. Freeze-dried soy scaffolds had similar biocompatibility as freeze-dried collagen scaffolds of the same protein content. However, the soy scaffolds degraded at a much faster rate, minimizing immunogenicity. Interestingly, subcutaneously implanted soy scaffolds affected blood

  14. Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

    Science.gov (United States)

    Liu, Fang

    Globally, there is growing appreciation for developing a sustainable economy that uses eco-efficient bio-processes. Biotechnology provides an increasing range of tools for industry to help reduce cost and improve environmental performance. Inspired by the naturally evolved machineries of protein scaffolds and their binding ligands, synthetic protein scaffolds were engineered based on cohesin-dockerin interactions and metal chelating peptides to tackle the challenges and make improvements in three specific areas: (1) protein purification, (2) biofuel cells, and (3) nanomaterial synthesis. The first objective was to develop efficient and cost-effective non-chromatographic purification processes to purify recombinant proteins in an effort to meet the dramatically growing market of protein drugs. In our design, the target protein was genetically fused with a dockerin domain from Clostridium thermocellum and direct purification and recovery was achieved using thermo-responsive elastin-like polypeptide (ELP) scaffold containing the cohesin domain from the same species. By exploiting the highly specific interaction between the dockerin and cohesin domain and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as endoglucanase CelA, chloramphenicol acetyl transferase (CAT) and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single purification step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins by another cycle of thermal precipitation. The purification cost can be further reduced by regenerating and recycling the ELP-cohesin capturing scaffolds. However, due to the high binding affinity between cohesin and dockerin domains, the bound dockerin-intein tag cannot be completely disassociated from ELP-cohesin scaffold after binding. Therefore, a truncated dockerin with the calcium

  15. Disease-associated mutations in the actin-binding domain of filamin B cause cytoplasmic focal accumulations correlating with disease severity

    DEFF Research Database (Denmark)

    Daniel, Philip B; Morgan, Tim; Alanay, Yasemin;

    2012-01-01

    Dominant missense mutations in FLNB, encoding the actin-cross linking protein filamin B (FLNB), cause a broad range of skeletal dysplasias with varying severity by an unknown mechanism. Here these FLNB mutations are shown to cluster in exons encoding the actin-binding domain (ABD) and filamin rep...... actin-binding activity but suggest that substitutions affecting repeats near the flexible hinge region of FLNB precipitate the same phenotypes through a different mechanism....... repeats surrounding the flexible hinge 1 region of the FLNB rod domain. Despite being positioned in domains that bind actin, it is unknown if these mutations perturb cytoskeletal structure. Expression of several full-length FLNB constructs containing ABD mutations resulted in the appearance of actin...... exception disease-associated substitutions surrounding hinge 1 demonstrated no tendency to form actin-filamin foci. The exception, a substitution in filamin repeat 6, lies within a region previously implicated in filamin-actin binding. These data are consistent with mutations in the ABD conferring enhanced...

  16. The actin binding domain of βI-spectrin regulates the morphological and functional dynamics of dendritic spines.

    Directory of Open Access Journals (Sweden)

    Michael W Nestor

    Full Text Available Actin microfilaments regulate the size, shape and mobility of dendritic spines and are in turn regulated by actin binding proteins and small GTPases. The βI isoform of spectrin, a protein that links the actin cytoskeleton to membrane proteins, is present in spines. To understand its function, we expressed its actin-binding domain (ABD in CA1 pyramidal neurons in hippocampal slice cultures. The ABD of βI-spectrin bundled actin in principal dendrites and was concentrated in dendritic spines, where it significantly increased the size of the spine head. These effects were not observed after expression of homologous ABDs of utrophin, dystrophin, and α-actinin. Treatment of slice cultures with latrunculin-B significantly decreased spine head size and decreased actin-GFP fluorescence in cells expressing the ABD of α-actinin, but not the ABD of βI-spectrin, suggesting that its presence inhibits actin depolymerization. We also observed an increase in the area of GFP-tagged PSD-95 in the spine head and an increase in the amplitude of mEPSCs at spines expressing the ABD of βI-spectrin. The effects of the βI-spectrin ABD on spine size and mEPSC amplitude were mimicked by expressing wild-type Rac3, a small GTPase that co-immunoprecipitates specifically with βI-spectrin in extracts of cultured cortical neurons. Spine size was normal in cells co-expressing a dominant negative Rac3 construct with the βI-spectrin ABD. We suggest that βI-spectrin is a synaptic protein that can modulate both the morphological and functional dynamics of dendritic spines, perhaps via interaction with actin and Rac3.

  17. Protein-engineered biomaterials: highly tunable tissue engineering scaffolds.

    Science.gov (United States)

    Sengupta, Debanti; Heilshorn, Sarah C

    2010-06-01

    A common goal in tissue engineering is to attain the ability to tailor specific cell-scaffold interactions and thereby gain control over cell behavior. The tunable nature of protein-engineered biomaterials enables independent tailoring of a range of biomaterial properties, creating an attractive alternative to synthetic polymeric scaffolds or harvested natural scaffolds. Protein-engineered biomaterials are comprised of modular peptide domains with various functionalities that are encoded into a DNA plasmid, transfected into an organism of choice, and expressed and purified to yield a biopolymer with exact molecular-level sequence specification. Because of the modular design strategy of protein-engineered biomaterials, these scaffolds can be easily modified to enable optimization for specific tissue engineering applications. By including multiple peptide domains with different functionalities in a single, modular biomaterial, the scaffolds can be designed to mimic the diverse properties of the natural extracellular matrix, including cell adhesion, cell signaling, elasticity, and biodegradability. Recently, the field of protein-engineered biomaterials has expanded to include functional modules that are not normally present in the extracellular matrix, thus expanding the scope and functionality of these materials. For example, these modules can include noncanonical amino acids, inorganic-binding domains, and DNA-binding sequences. The modularity, tunability, and sequence specificity of protein-engineered biomaterials make them attractive candidates for use as substrates for a variety of tissue engineering applications. PMID:20141386

  18. Scaffolding proteins in membrane trafficking : the role of ELKS

    NARCIS (Netherlands)

    Yu, K.L.

    2015-01-01

    Intracellular membrane trafficking is an essential cellular process that involves cooperation of many factors such as scaffolding proteins, GTPases and SNAREs. These proteins work together to ensure proper delivery of different membrane-enclosed cargoes to specific cellular destinations. In this the

  19. The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

    Science.gov (United States)

    Ly, Thu; Moroz, Natalia; Pappas, Christopher T; Novak, Stefanie M; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M; Helms, Gregory; Gregorio, Carol C; Kostyukova, Alla S

    2016-08-15

    Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends.

  20. Gold nanoparticle capture within protein crystal scaffolds.

    Science.gov (United States)

    Kowalski, Ann E; Huber, Thaddaus R; Ni, Thomas W; Hartje, Luke F; Appel, Karina L; Yost, Jarad W; Ackerson, Christopher J; Snow, Christopher D

    2016-07-01

    DNA assemblies have been used to organize inorganic nanoparticles into 3D arrays, with emergent properties arising as a result of nanoparticle spacing and geometry. We report here the use of engineered protein crystals as an alternative approach to biologically mediated assembly of inorganic nanoparticles. The protein crystal's 13 nm diameter pores result in an 80% solvent content and display hexahistidine sequences on their interior. The hexahistidine sequence captures Au25(glutathione)∼17 (nitrilotriacetic acid)∼1 nanoclusters throughout a chemically crosslinked crystal via the coordination of Ni(ii) to both the cluster and the protein. Nanoparticle loading was validated by confocal microscopy and elemental analysis. The nanoparticles may be released from the crystal by exposure to EDTA, which chelates the Ni(ii) and breaks the specific protein/nanoparticle interaction. The integrity of the protein crystals after crosslinking and nanoparticle capture was confirmed by single crystal X-ray crystallography. PMID:27264210

  1. Gold nanoparticle capture within protein crystal scaffolds

    Science.gov (United States)

    Kowalski, Ann E.; Huber, Thaddaus R.; Ni, Thomas W.; Hartje, Luke F.; Appel, Karina L.; Yost, Jarad W.; Ackerson, Christopher J.; Snow, Christopher D.

    2016-06-01

    DNA assemblies have been used to organize inorganic nanoparticles into 3D arrays, with emergent properties arising as a result of nanoparticle spacing and geometry. We report here the use of engineered protein crystals as an alternative approach to biologically mediated assembly of inorganic nanoparticles. The protein crystal's 13 nm diameter pores result in an 80% solvent content and display hexahistidine sequences on their interior. The hexahistidine sequence captures Au25(glutathione)~17 (nitrilotriacetic acid)~1 nanoclusters throughout a chemically crosslinked crystal via the coordination of Ni(ii) to both the cluster and the protein. Nanoparticle loading was validated by confocal microscopy and elemental analysis. The nanoparticles may be released from the crystal by exposure to EDTA, which chelates the Ni(ii) and breaks the specific protein/nanoparticle interaction. The integrity of the protein crystals after crosslinking and nanoparticle capture was confirmed by single crystal X-ray crystallography.DNA assemblies have been used to organize inorganic nanoparticles into 3D arrays, with emergent properties arising as a result of nanoparticle spacing and geometry. We report here the use of engineered protein crystals as an alternative approach to biologically mediated assembly of inorganic nanoparticles. The protein crystal's 13 nm diameter pores result in an 80% solvent content and display hexahistidine sequences on their interior. The hexahistidine sequence captures Au25(glutathione)~17 (nitrilotriacetic acid)~1 nanoclusters throughout a chemically crosslinked crystal via the coordination of Ni(ii) to both the cluster and the protein. Nanoparticle loading was validated by confocal microscopy and elemental analysis. The nanoparticles may be released from the crystal by exposure to EDTA, which chelates the Ni(ii) and breaks the specific protein/nanoparticle interaction. The integrity of the protein crystals after crosslinking and nanoparticle capture was

  2. A Pareto-optimal refinement method for protein design scaffolds.

    Science.gov (United States)

    Nivón, Lucas Gregorio; Moretti, Rocco; Baker, David

    2013-01-01

    Computational design of protein function involves a search for amino acids with the lowest energy subject to a set of constraints specifying function. In many cases a set of natural protein backbone structures, or "scaffolds", are searched to find regions where functional sites (an enzyme active site, ligand binding pocket, protein-protein interaction region, etc.) can be placed, and the identities of the surrounding amino acids are optimized to satisfy functional constraints. Input native protein structures almost invariably have regions that score very poorly with the design force field, and any design based on these unmodified structures may result in mutations away from the native sequence solely as a result of the energetic strain. Because the input structure is already a stable protein, it is desirable to keep the total number of mutations to a minimum and to avoid mutations resulting from poorly-scoring input structures. Here we describe a protocol using cycles of minimization with combined backbone/sidechain restraints that is Pareto-optimal with respect to RMSD to the native structure and energetic strain reduction. The protocol should be broadly useful in the preparation of scaffold libraries for functional site design.

  3. Design, synthesis, and biological evaluation of simplified side chain hybrids of the potent actin binding polyketides rhizopodin and bistramide.

    Science.gov (United States)

    Herkommer, Daniel; Dreisigacker, Sandra; Sergeev, Galina; Sasse, Florenz; Gohlke, Holger; Menche, Dirk

    2015-03-01

    The natural products rhizopodin and bistramide belong to an elite class of highly potent actin binding agents. They show powerful antiproliferative activities against a range of tumor cell lines, with IC50 values in the low-nanomolar range. At the molecular level they disrupt the actin cytoskeleton by binding specifically to a few critical sites of G-actin, resulting in actin filament stabilization. The important biological properties of rhizopodin and bistramide, coupled with their unique and intriguing molecular architectures, render them attractive compounds for further development. However, this is severely hampered by the structural complexity of these metabolites. We initiated an interdisciplinary approach at the interface between molecular modeling, organic synthesis, and chemical biology to support further biological applications. We also wanted to expand structure-activity relationship studies with the goal of accessing simplified analogues with potent biological properties. We report computational analyses of actin-inhibitor interactions involving molecular docking, validated on known actin binding ligands, that show a close match between the crystal and modeled structures. Based on these results, the ligand shape was simplified, and more readily accessible rhizopodin-bistramide mimetics were designed. A flexible and modular strategy was applied for the synthesis of these compounds, enabling diverse access to dramatically simplified rhizopodin-bistramide hybrids. This novel analogue class was analyzed for its antiproliferative and actin binding properties. PMID:25641798

  4. Early events of fertilization in sea urchin eggs are sensitive to actin-binding organic molecules.

    Science.gov (United States)

    Chun, Jong T; Limatola, Nunzia; Vasilev, Filip; Santella, Luigia

    2014-08-01

    We previously demonstrated that many aspects of the intracellular Ca(2+) increase in fertilized eggs of starfish are significantly influenced by the state of the actin cytoskeleton. In addition, the actin cytoskeleton appeared to play comprehensive roles in modulating cortical granules exocytosis and sperm entry during the early phase of fertilization. In the present communication, we have extended our work to sea urchin which is believed to have bifurcated from the common ancestor in the phylogenetic tree some 500 million years ago. To corroborate our earlier findings in starfish, we have tested how the early events of fertilization in sea urchin eggs are influenced by four different actin-binding drugs that promote either depolymerization or stabilization of actin filaments. We found that all the actin drugs commonly blocked sperm entry in high doses and significantly reduced the speed of the Ca(2+) wave. At low doses, however, cytochalasin B and phalloidin increased the rate of polyspermy. Overall, certain aspects of Ca(2+) signaling in these eggs were in line with the morphological changes induced by the actin drugs. That is, the time interval between the cortical flash and the first Ca(2+) spot at the sperm interaction site (the latent period) was significantly prolonged in the eggs pretreated with cytochalasin B or latrunculin A, whereas the Ca(2+) decay kinetics after the peak was specifically attenuated in the eggs pretreated with jasplakinolide or phalloidin. In addition, the sperm interacting with the eggs pretreated with actin drugs often generated multiple Ca(2+) waves, but tended to fail to enter the egg. Thus, our results indicated that generation of massive Ca(2+) waves is neither indicative of sperm entry nor sufficient for cortical granules exocytosis in the inseminated sea urchin eggs, whereas the structure and functionality of the actin cytoskeleton are the major determining factors in the two processes.

  5. Requirement of JIP scaffold proteins for NMDA-mediated signal transduction

    OpenAIRE

    Kennedy, Norman J.; Martin, Gilles; Ehrhardt, Anka G.; Cavanagh-Kyros, Julie; Kuan, Chia-Yi; Rakic, Pasko; Richard A Flavell; Treistman, Steven N.; Davis, Roger J

    2007-01-01

    JIP scaffold proteins are implicated in the regulation of protein kinase signal transduction pathways. To test the physiological role of these scaffold proteins, we examined the phenotype of compound mutant mice that lack expression of JIP proteins. These mice were found to exhibit severe defects in N-methyl-D-aspartic acid (NMDA) receptor function, including decreased NMDA-evoked current amplitude, cytoplasmic Ca++, and gene expression. The decreased NMDA receptor activity in JIP-deficient n...

  6. Computation-Guided Backbone Grafting of a Discontinuous Motif onto a Protein Scaffold

    Energy Technology Data Exchange (ETDEWEB)

    Azoitei, Mihai L.; Correia, Bruno E.; Ban, Yih-En Andrew; Carrico, Chris; Kalyuzhniy, Oleksandr; Chen, Lei; Schroeter, Alexandria; Huang, Po-Ssu; McLellan, Jason S.; Kwong, Peter D.; Baker, David; Strong, Roland K.; Schief, William R. (UWASH); (FHCRC); (NIAID)

    2012-02-07

    The manipulation of protein backbone structure to control interaction and function is a challenge for protein engineering. We integrated computational design with experimental selection for grafting the backbone and side chains of a two-segment HIV gp120 epitope, targeted by the cross-neutralizing antibody b12, onto an unrelated scaffold protein. The final scaffolds bound b12 with high specificity and with affinity similar to that of gp120, and crystallographic analysis of a scaffold bound to b12 revealed high structural mimicry of the gp120-b12 complex structure. The method can be generalized to design other functional proteins through backbone grafting.

  7. Actin-binding proteins differentially regulate endothelial cell stiffness, ICAM-1 function and neutrophil transmigration

    NARCIS (Netherlands)

    Schaefer, A.; Riet, J. te; Ritz, K.; Hoogenboezem, M.; Anthony, E.C.; Mul, F.P.; Vries, C.J. de; Daemen, M.J.; Figdor, C.G.; Buul, J.D. van; Hordijk, P.L.

    2014-01-01

    Chronic vascular inflammation is driven by interactions between activated leukocytes and the endothelium. Leukocyte beta2-integrins bind to endothelial intercellular adhesion molecule 1 (ICAM-1), which allows leukocyte spreading, crawling and transendothelial migration. Leukocytes scan the vascular

  8. Arabidopsis scaffold protein RACK1A interacts with diverse environmental stress and photosynthesis related proteins.

    Science.gov (United States)

    Kundu, Nabanita; Dozier, Uvetta; Deslandes, Laurent; Somssich, Imre E; Ullah, Hemayet

    2013-05-01

    Scaffold proteins are known to regulate important cellular processes by interacting with multiple proteins to modulate molecular responses. RACK1 (Receptor for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in eukaryotes, from Chlamydymonas to plants and humans, expresses ubiquitously and plays regulatory roles in diverse signal transduction and stress response pathways. Here we present the use of Arabidopsis RACK1A, the predominant isoform of a 3-member family, as a bait to screen a split-ubiquitin based cDNA library. In total 97 proteins from dehydration, salt stress, ribosomal and photosynthesis pathways are found to potentially interact with RACK1A. False positive interactions were eliminated following extensive selection based growth potentials. Confirmation of a sub-set of selected interactions is demonstrated through the co-transformation with individual plasmid containing cDNA and the respective bait. Interaction of diverse proteins points to a regulatory role of RACK1A in the cross-talk between signaling pathways. Promoter analysis of the stress and photosynthetic pathway genes revealed conserved transcription factor binding sites. RACK1A is known to be a multifunctional protein and the current identification of potential interacting proteins and future in vivo elucidations of the physiological basis of such interactions will shed light on the possible molecular mechanisms that RACK1A uses to regulate diverse signaling pathways.

  9. Scaffold State Switching Amplifies, Accelerates, and Insulates Protein Kinase C Signaling*

    Science.gov (United States)

    Greenwald, Eric C.; Redden, John M.; Dodge-Kafka, Kimberly L.; Saucerman, Jeffrey J.

    2014-01-01

    Scaffold proteins localize two or more signaling enzymes in close proximity to their downstream effectors. A-kinase-anchoring proteins (AKAPs) are a canonical family of scaffold proteins known to bind protein kinase A (PKA) and other enzymes. Several AKAPs have been shown to accelerate, amplify, and specify signal transduction to dynamically regulate numerous cellular processes. However, there is little theory available to mechanistically explain how signaling on protein scaffolds differs from solution biochemistry. In our present study, we propose a novel kinetic mechanism for enzymatic reactions on protein scaffolds to explain these phenomena, wherein the enzyme-substrate-scaffold complex undergoes stochastic state switching to reach an active state. This model predicted anchored enzymatic reactions to be accelerated, amplified, and insulated from inhibition compared with those occurring in solution. We exploited a direct interaction between protein kinase C (PKC) and AKAP7α as a model to validate these predictions experimentally. Using a genetically encoded PKC activity reporter, we found that both the strength and speed of substrate phosphorylation were enhanced by AKAP7α. PKC tethered to AKAP7α was less susceptible to inhibition from the ATP-competitive inhibitor Gö6976 and the substrate-competitive inhibitor PKC 20-28, but not the activation-competitive inhibitor calphostin C. Model predictions and experimental validation demonstrated that insulation is a general property of scaffold tethering. Sensitivity analysis indicated that these findings may be applicable to many other scaffolds as well. Collectively, our findings provide theoretical and experimental evidence that scaffold proteins can amplify, accelerate, and insulate signal transduction. PMID:24302730

  10. Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds

    Directory of Open Access Journals (Sweden)

    Michaela Mühlberg

    2015-05-01

    Full Text Available To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at the N-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide–alkyne cycloaddition (CuAAC and oxime ligation. This method was applied to the conjugation of biotin and β-linked galactose residues to yield an enzymatically active thermophilic lipase, which revealed specific binding to Erythrina cristagalli lectin by SPR binding studies.

  11. Traffic of p24 Proteins and COPII Coat Composition Mutually Influence Membrane Scaffolding.

    Science.gov (United States)

    D'Arcangelo, Jennifer G; Crissman, Jonathan; Pagant, Silvere; Čopič, Alenka; Latham, Catherine F; Snapp, Erik L; Miller, Elizabeth A

    2015-05-18

    Eukaryotic protein secretion requires efficient and accurate delivery of diverse secretory and membrane proteins. This process initiates in the ER, where vesicles are sculpted by the essential COPII coat. The Sec13p subunit of the COPII coat contributes to membrane scaffolding, which enforces curvature on the nascent vesicle. A requirement for Sec13p can be bypassed when traffic of lumenally oriented membrane proteins is abrogated. Here we sought to further explore the impact of cargo proteins on vesicle formation. We show that efficient ER export of the p24 family of proteins is a major driver of the requirement for Sec13p. The scaffolding burden presented by the p24 complex is met in part by the cargo adaptor Lst1p, which binds to a subset of cargo, including the p24 proteins. We propose that the scaffolding function of Lst1p is required to generate vesicles that can accommodate difficult cargo proteins that include large oligomeric assemblies and asymmetrically distributed membrane proteins. Vesicles that contain such cargoes are also more dependent on scaffolding by Sec13p, and may serve as a model for large carrier formation in other systems.

  12. The Cytoskeletal Regulatory Scaffold Protein GIT2 Modulates Mesenchymal Stem Cell Differentiation and Osteoblastogenesis

    OpenAIRE

    Wang, Xiaojuan; Liao, Shaoxi; Nelson, Erik R.; Schmalzigaug, Robert; Spurney, Robert F.; Guilak, Farshid; Premont, Richard T.; Gesty-Palmer, Diane

    2012-01-01

    G protein-coupled receptor kinase interacting protein 2 (GIT2) is a signaling scaffold protein involved in the regulation of cytoskeletal structure, membrane trafficking, and G protein-coupled receptor internalization. Since dynamic cytoskeletal reorganization plays key roles both in osteoblast differentiation and in the maintenance of osteoclast polarity during bone resorption, we hypothesized that skeletal physiology would be altered in GIT2−/− mice. We found that adult GIT2−/− mice have de...

  13. Green fluorescent protein-mtalin causes defects in actin organization and cell expansion in Arabidopsis and inhibits actin depolymerizing factor's actin depolymerizing activity in vitro

    NARCIS (Netherlands)

    Ketelaar, T.; Anthony, R.G.; Hussey, P.J.

    2004-01-01

    Expression of green fluorescent protein (GFP) linked to an actin binding domain is a commonly used method for live cell imaging of the actin cytoskeleton. One of these chimeric proteins is GFP-mTalin (GFP fused to the actin binding domain of mouse talin). Although it has been demonstrated that GFP-m

  14. Design and modular parallel synthesis of a MCR derived α-helix mimetic protein-protein interaction inhibitor scaffold

    NARCIS (Netherlands)

    Antuch, Walfrido; Menon, Sanjay; Chen, Quin-Zene; Lu, Yingchun; Sakamuri, Sukumar; Beck, Barbara; Schauer-Vukašinović, Vesna; Agarwal, Seema; Hess, Sibylle; Dömling, Alexander

    2006-01-01

    A terphenyl α-helix mimetic scaffold recognized to be capable of disrupting protein-protein interactions was structurally morphed into an easily amenable and versatile multicomponent reaction (MCR) backbone. The design, modular in-parallel library synthesis, initial cell based biological data, and p

  15. A new biocompatible delivery scaffold containing heparin and bone morphogenetic protein 2.

    Science.gov (United States)

    Thanyaphoo, Suphannee; Kaewsrichan, Jasadee

    2016-09-01

    Silicon-substituted calcium phosphate (Si-CaP) was developed in our laboratory as a biomaterial for delivery in bone tissue engineering. It was fabricated as a 3D-construct of scaffolds using chitosan-trisodium polyphosphate (TPP) cross-linked networks. In this study, heparin was covalently bonded to the residual -NH2 groups of chitosan on the scaffold applying carbodiimide chemistry. Bonded heparin was not leached away from scaffold surfaces upon vigorous washing or extended storage. Recombinant human bone morphogenetic protein 2 (rhBMP-2) was bound to conjugated scaffolds by ionic interactions between the negatively charged SO42- clusters of heparin and positively charged amino acids of rhBMP-2. The resulting scaffolds were inspected for bone regenerative capacity by subcutaneous implanting in rats. Histological observation and mineralization assay were performed after 4 weeks of implantation. Results from both in vitro and in vivo experiments suggest the potential of the developed scaffolds for bone tissue engineering applications in the future. PMID:27383886

  16. A new biocompatible delivery scaffold containing heparin and bone morphogenetic protein 2

    Directory of Open Access Journals (Sweden)

    Thanyaphoo Suphannee

    2016-09-01

    Full Text Available Silicon-substituted calcium phosphate (Si-CaP was developed in our laboratory as a biomaterial for delivery in bone tissue engineering. It was fabricated as a 3D-construct of scaffolds using chitosan-trisodium polyphosphate (TPP cross-linked networks. In this study, heparin was covalently bonded to the residual -NH2 groups of chitosan on the scaffold applying carbodiimide chemistry. Bonded heparin was not leached away from scaffold surfaces upon vigorous washing or extended storage. Recombinant human bone morphogenetic protein 2 (rhBMP-2 was bound to conjugated scaffolds by ionic interactions between the negatively charged SO42- clusters of heparin and positively charged amino acids of rhBMP-2. The resulting scaffolds were inspected for bone regenerative capacity by subcutaneous implanting in rats. Histological observation and mineralization assay were performed after 4 weeks of implantation. Results from both in vitro and in vivo experiments suggest the potential of the developed scaffolds for bone tissue engineering applications in the future.

  17. Functional Modification of Fibrous PCL Scaffolds with Fusion Protein VEGF-HGFI Enhanced Cellularization and Vascularization.

    Science.gov (United States)

    Zhao, Liqiang; Ma, Shaoyang; Pan, Yiwa; Zhang, Qiuying; Wang, Kai; Song, Dongmin; Wang, Xiangxiang; Feng, Guowei; Liu, Ruming; Xu, Haijin; Zhang, Jun; Qiao, Mingqiang; Kong, Deling

    2016-09-01

    The lack of efficient vascularization within frequently used poly(ε-caprolactone) (PCL) scaffolds has hindered their application in tissue engineering. Hydrophobin HGFI, an amphiphilic protein, can form a self-assembly layer on the surface of PCL scaffolds and convert their wettability. In this study, a fusion protein consisting of HGFI and vascular endothelial growth factor (VEGF) is prepared by Pichia pastoris expression system. Sodium dodecyl sulface-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting confirm that the VEGF-HGFI is successfully isolated and purified. Transmission electron microscope and water contact angle measurement demonstrate that VEGF-HGFI can form a self-assembly layer with about 25 nm in thickness on electrospun PCL fibers and increase their hydrophilicity. VEGF-HGFI modification can effectively enhance the adhesion, migration, and proliferation of human umbilical vein endothelial cells. Near-infrared fluorescence imaging shows that the VEGF-HGFI modification on PCL scaffolds can exist at least 21 d in vitro and at least 14 d in vivo. Bioluminescence imaging shows that VEGF-HGFI can effectively activate vascular endothelial growth factor receptor 2 receptors. Subcutaneous implantation in mice and rats reveal that cellularization and vascularization are significantly improved in VEGF-HGFI modified PCL scaffolds. These results suggest that VEGF-HGFI is a useful molecule for functional modification of scaffolds to enhance cellularization and vascularization in tissue engineering. PMID:27391702

  18. Artificial Metalloenzymes for Asymmetric Catalysis by Creation of Novel Active Sites in Protein and DNA Scaffolds

    NARCIS (Netherlands)

    Drienovska, Ivana; Roelfes, Gerard

    2015-01-01

    Artificial metalloenzymes have emerged as a promising new approach to asymmetric catalysis. In our group, we are exploring novel artificial metalloenzyme designs involving creation of a new active site in a protein or DNA scaffold that does not have an existing binding pocket. In this review, we giv

  19. Controlled release of recombinant human cementum protein 1 from electrospun multiphasic scaffold for cementum regeneration.

    Science.gov (United States)

    Chen, Xiaofeng; Liu, Yu; Miao, Leiying; Wang, Yangyang; Ren, Shuangshuang; Yang, Xuebin; Hu, Yong; Sun, Weibin

    2016-01-01

    Periodontitis is a major cause for tooth loss, which affects about 15% of the adult population. Cementum regeneration has been the crux of constructing the periodontal complex. Cementum protein 1 (CEMP1) is a cementum-specific protein that can induce cementogenic differentiation. In this study, poly(ethylene glycol) (PEG)-stabilized amorphous calcium phosphate (ACP) nanoparticles were prepared by wet-chemical method and then loaded with recombinant human CEMP1 (rhCEMP1) for controlled release. An electrospun multiphasic scaffold constituted of poly(ε-caprolactone) (PCL), type I collagen (COL), and rhCEMP1/ACP was fabricated. The effects of rhCEMP1/ACP/PCL/COL scaffold on the attachment proliferation, osteogenic, and cementogenic differentiations of human periodontal ligament cells, (PDLCs) were systematically investigated. A critical size defect rat model was introduced to evaluate the effect of tissue regeneration of the scaffolds in vivo. The results showed that PEG-stabilized ACP nanoparticles formed a core-shell structure with sustained release of rhCEMP1 for up to 4 weeks. rhCEMP1/ACP/PCL/COL scaffold could suppress PDLCs proliferation behavior and upregulate the expression of cementoblastic markers including CEMP1 and cementum attachment protein while downregulating osteoblastic markers including osteocalcin and osteopontin when it was cocultured with PDLCs in vitro for 7 days. Histology analysis of cementum after being implanted with the scaffold in rats for 8 weeks showed that there was cementum-like tissue formation but little bone formation. These results indicated the potential of using electrospun multiphasic scaffolds for controlled release of rhCEMP1 for promoting cementum regeneration in reconstruction of the periodontal complex. PMID:27471382

  20. Controlled release of recombinant human cementum protein 1 from electrospun multiphasic scaffold for cementum regeneration

    Science.gov (United States)

    Chen, Xiaofeng; Liu, Yu; Miao, Leiying; Wang, Yangyang; Ren, Shuangshuang; Yang, Xuebin; Hu, Yong; Sun, Weibin

    2016-01-01

    Periodontitis is a major cause for tooth loss, which affects about 15% of the adult population. Cementum regeneration has been the crux of constructing the periodontal complex. Cementum protein 1 (CEMP1) is a cementum-specific protein that can induce cementogenic differentiation. In this study, poly(ethylene glycol) (PEG)-stabilized amorphous calcium phosphate (ACP) nanoparticles were prepared by wet-chemical method and then loaded with recombinant human CEMP1 (rhCEMP1) for controlled release. An electrospun multiphasic scaffold constituted of poly(ε-caprolactone) (PCL), type I collagen (COL), and rhCEMP1/ACP was fabricated. The effects of rhCEMP1/ACP/PCL/COL scaffold on the attachment proliferation, osteogenic, and cementogenic differentiations of human periodontal ligament cells, (PDLCs) were systematically investigated. A critical size defect rat model was introduced to evaluate the effect of tissue regeneration of the scaffolds in vivo. The results showed that PEG-stabilized ACP nanoparticles formed a core-shell structure with sustained release of rhCEMP1 for up to 4 weeks. rhCEMP1/ACP/PCL/COL scaffold could suppress PDLCs proliferation behavior and upregulate the expression of cementoblastic markers including CEMP1 and cementum attachment protein while downregulating osteoblastic markers including osteocalcin and osteopontin when it was cocultured with PDLCs in vitro for 7 days. Histology analysis of cementum after being implanted with the scaffold in rats for 8 weeks showed that there was cementum-like tissue formation but little bone formation. These results indicated the potential of using electrospun multiphasic scaffolds for controlled release of rhCEMP1 for promoting cementum regeneration in reconstruction of the periodontal complex. PMID:27471382

  1. Protein mimics by attachment of cyclic peptides to molecular scaffolds

    NARCIS (Netherlands)

    van de Langemheen, W.

    2014-01-01

    The interaction between proteins is important in all biological functions. In practically every cellular process protein complexes have been identified as essential components. Defects or disturbance in the regulation of protein-protein interactions are responsible for many diseases. Therefore, the

  2. A molecular switch in the scaffold NHERF1 enables misfolded CFTR to evade the peripheral quality control checkpoint.

    Science.gov (United States)

    Loureiro, Cláudia A; Matos, Ana Margarida; Dias-Alves, Ângela; Pereira, Joana F; Uliyakina, Inna; Barros, Patrícia; Amaral, Margarida D; Matos, Paulo

    2015-05-19

    The peripheral protein quality control (PPQC) checkpoint removes improperly folded proteins from the plasma membrane through a mechanism involving the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70 interacting protein). PPQC limits the efficacy of some cystic fibrosis (CF) drugs, such as VX-809, that improve trafficking to the plasma membrane of misfolded mutants of the CF transmembrane conductance regulator (CFTR), including F508del-CFTR, which retains partial functionality. We investigated the PPQC checkpoint in lung epithelial cells with F508del-CFTR that were exposed to VX-809. The conformation of the scaffold protein NHERF1 (Na(+)/H(+) exchange regulatory factor 1) determined whether the PPQC recognized "rescued" F508del-CFTR (the portion that reached the cell surface in VX-809-treated cells). Activation of the cytoskeletal regulator Rac1 promoted an interaction between the actin-binding adaptor protein ezrin and NHERF1, triggering exposure of the second PDZ domain of NHERF1, which interacted with rescued F508del-CFTR. Because binding of F508del-CFTR to the second PDZ of NHERF1 precluded the recruitment of CHIP, the coexposure of airway cells to Rac1 activator nearly tripled the efficacy of VX-809. Interference with the NHERF1-ezrin interaction prevented the increase of efficacy of VX-809 by Rac1 activation, but the actin-binding domain of ezrin was not required for the increase in efficacy. Thus, rather than mainly directing anchoring of F508del-CFTR to the actin cytoskeleton, induction of ezrin activation by Rac1 signaling triggered a conformational change in NHERF1, which was then able to bind and stabilize misfolded CFTR at the plasma membrane. These insights into the cell surface stabilization of CFTR provide new targets to improve treatment of CF.

  3. NF-κB signaling pathways regulated by CARMA family of scaffold proteins

    Institute of Scientific and Technical Information of China (English)

    Marzenna Blonska; Xin Lin

    2011-01-01

    The NF-κB family of transcription factors plays a crucial role in cell activation,survival and proliferation.Its aberrant activity results in cancer,immunodeficiency or autoimmune disorders.Over the past two decades,tremendous progress has been made in our understanding of the signals that regulate NF-κB activation,especially how scaffold proteins link different receptors to the NF-κB-activating complex,the IκB kinase complex.The growing number of these scaffolds underscores the complexity of the signaling networks in different cell types.In this review,we discuss the role of scaffold molecules in signaling cascades induced by stimulation of antigen receptors,G-protein-coupled receptors and C-type Lectin receptors,resulting in NF-κB activation.Especially,we focus on the family of Caspase recruitment domain(CARD)-containing proteins known as CARMA and their function in activation of NF-κB,as well as the link of these scaffolds to the development of various neoplastic diseases through regulation of NF-κB.

  4. Self-Assembled Proteins and Peptides as Scaffolds for Tissue Regeneration.

    Science.gov (United States)

    Loo, Yihua; Goktas, Melis; Tekinay, Ayse B; Guler, Mustafa O; Hauser, Charlotte A E; Mitraki, Anna

    2015-11-18

    Self-assembling proteins and peptides are increasingly gaining interest for potential use as scaffolds in tissue engineering applications. They self-organize from basic building blocks under mild conditions into supramolecular structures, mimicking the native extracellular matrix. Their properties can be easily tuned through changes at the sequence level. Moreover, they can be produced in sufficient quantities with chemical synthesis or recombinant technologies to allow them to address homogeneity and standardization issues required for applications. Here. recent advances in self-assembling proteins, peptides, and peptide amphiphiles that form scaffolds suitable for tissue engineering are reviewed. The focus is on a variety of motifs, ranging from minimalistic dipeptides, simplistic ultrashort aliphatic peptides, and peptide amphiphiles to large "recombinamer" proteins. Special emphasis is placed on the rational design of self-assembling motifs and biofunctionalization strategies to influence cell behavior and modulate scaffold stability. Perspectives for combination of these "bottom-up" designer strategies with traditional "top-down" biofabrication techniques for new generations of tissue engineering scaffolds are highlighted. PMID:26461979

  5. Surface studies of coated polymer microspheres and protein release from tissue-engineered scaffolds.

    Science.gov (United States)

    Meese, Thomas M; Hu, Yunhua; Nowak, Richard W; Marra, Kacey G

    2002-01-01

    The controlled release of growth factors from porous, polymer scaffolds is being studied for potential use as tissue-engineered scaffolds. Biodegradable polymer microspheres were coated with a biocompatible polymer membrane to permit the incorporation of the microspheres into tissue-engineered scaffolds. Surface studies with poly(D,L-lactic-co-glycolic acid) [PLGA], and poly(vinyl alcohol) [PVA] were conducted. Polymer films were dip-coated onto glass slides and water contact angles were measured. The contact angles revealed an initially hydrophobic PLGA film, which became hydrophilic after PVA coating. After immersion in water, the PVA coating was removed and a hydrophobic PLGA film remained. Following optimization using these 2D contact angle studies, biodegradable PLGA microspheres were prepared, characterized, and coated with PVA. X-ray photoelectron spectroscopy was used to further characterize coated slides and microspheres. The release of the model protein bovine serum albumin from PVA-coated PLGA microspheres was studied over 8 days. The release of BSA from PVA-coated PLGA microspheres embedded in porous PLGA scaffolds over 24 days was also examined. Coating of the PLGA microspheres with PVA permitted their incorporation into tissue-engineered scaffolds and resulted in a controlled release of BSA. PMID:12022746

  6. Plasma Surface Modification for Immobilization of Bone Morphogenic Protein-2 on Polycaprolactone Scaffolds

    Science.gov (United States)

    Kim, Byung Hoon; Myung, Sung Woon; Jung, Sang Chul; Ko, Yeong Mu

    2013-11-01

    The immobilization of recombinant human bone formation protein-2 (rhBMP-2) on polycaprolactone (PCL) scaffolds was performed by plasma polymerization. RhBMP-2, which induces osteoblast differentiation in various cell types, is a growth factor that plays an important role in bone formation and repair. The surface of the PCL scaffold was functionalized with the carboxyl groups of plasma-polymerized acrylic acid (PPAA) thin films. Plasma polymerization was carried out at a discharge power of 60 W at an acrylic acid flow rate of 7 sccm for 5 min. The PPAA thin film exhibited moderate hydrophilic properties and possessed a high density of carboxyl groups. Carboxyl groups and rhBMP-2 on the PCL scaffolds surface were identified by attenuated total reflection Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The alkaline phosphatase activity assay showed that the rhBMP-2 immobilized PCL scaffold increased the level of MG-63 cell differentiation. Plasma surface modification for the preparation of biomaterials, such as biofunctionalized polymer scaffolds, can be used for the binding of bioactive molecules in tissue engineering.

  7. Scaffold-Assisted Catalysis: A Novel Domain In the Ste5 Scaffold Protein is Required to Unlock the MAPK Fus3 for Phosphorylation by the MAPKK Ste7

    OpenAIRE

    Good, Matthew; Tang, Grace; Singleton, Julie; Remenyi, Attila; Lim, Wendell A.

    2009-01-01

    The scaffold protein Ste5 is required to properly direct signaling through the yeast mating pathway to the mitogen-activated protein kinase (MAPK), Fus3. Scaffolds are thought to function by tethering kinase and substrate in proximity. We find, however, that the previously identified Fus3-binding site on Ste5 is not required for signaling, suggesting an alternative mechanism controls Fus3’s activation by the MAPKK Ste7. Reconstituting MAPK signaling in vitro, we find that Fus3 is an intrinsic...

  8. Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila

    OpenAIRE

    1996-01-01

    Regulation of actin filament length and orientation is important in many actin-based cellular processes. This regulation is postulated to occur through the action of actin-binding proteins. Many actin-binding proteins that modify actin in vitro have been identified, but in many cases, it is not known if this activity is physiologically relevant. Capping protein (CP) is an actin-binding protein that has been demonstrated to control filament length in vitro by binding to the barbed ends and pre...

  9. Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein.

    Science.gov (United States)

    Coscia, Francesca; Estrozi, Leandro F; Hans, Fabienne; Malet, Hélène; Noirclerc-Savoye, Marjolaine; Schoehn, Guy; Petosa, Carlo

    2016-01-01

    Recent technical advances have revolutionized the field of cryo-electron microscopy (cryo-EM). However, most monomeric proteins remain too small (analysis. To overcome this limitation, we explored a strategy whereby a monomeric target protein is genetically fused to a homo-oligomeric scaffold protein and the junction optimized to allow the target to adopt the scaffold symmetry, thereby generating a chimeric particle suitable for cryo-EM. To demonstrate the concept, we fused maltose-binding protein (MBP), a 40 kDa monomer, to glutamine synthetase, a dodecamer formed by two hexameric rings. Chimeric constructs with different junction lengths were screened by biophysical analysis and negative-stain EM. The optimal construct yielded a cryo-EM reconstruction that revealed the MBP structure at sub-nanometre resolution. These findings illustrate the feasibility of using homo-oligomeric scaffolds to enable cryo-EM analysis of monomeric proteins, paving the way for applying this strategy to challenging structures resistant to crystallographic and NMR analysis. PMID:27485862

  10. Calcium Phosphate Scaffolds Combined with Bone Morphogenetic Proteins or Mesenchymal Stem Cells in Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Han Sun

    2015-01-01

    Full Text Available Objective: The purpose of this study was to review the current status of calcium phosphate (CaP scaffolds combined with bone morphogenetic proteins (BMPs or mesenchymal stem cells (MSCs in the field of bone tissue engineering (BTE. Date Sources: Data cited in this review were obtained primarily from PubMed and Medline in publications from 1979 to 2014, with highly regarded older publications also included. The terms BTE, CaP, BMPs, and MSC were used for the literature search. Study Selection: Reviews focused on relevant aspects and original articles reporting in vitro and/or in vivo results concerning the efficiency of CaP/BMPs or CaP/MSCs composites were retrieved, reviewed, analyzed, and summarized. Results: An ideal BTE product contains three elements: Scaffold, growth factors, and stem cells. CaP-based scaffolds are popular because of their outstanding biocompatibility, bioactivity, and osteoconductivity. However, they lack stiffness and osteoinductivity. To solve this problem, composite scaffolds of CaP with BMPs have been developed. New bone formation by CaP/BMP composites can reach levels similar to those of autografts. CaP scaffolds are compatible with MSCs and CaP/MSC composites exhibit excellent osteogenesis and stiffness. In addition, a CaP/MSC/BMP scaffold can repair bone defects more effectively than an autograft. Conclusions: Novel BTE products possess remarkable osteoconduction and osteoinduction capacities, and exhibit balanced degradation with osteogenesis. Further work should yield safe, viable, and efficient materials for the repair of bone lesions.

  11. Calcium Phosphate Scaffolds Combined with Bone Morphogenetic Proteins or Mesenchymal Stem Cells in Bone Tissue Engineering

    Institute of Scientific and Technical Information of China (English)

    Han Sun; Hui-Lin Yang

    2015-01-01

    Objective:The purpose of this study was to review the current status of calcium phosphate (CaP) scaffolds combined with bone morphogenetic proteins (BMPs) or mesenchymal stem cells (MSCs) in the field of bone tissue engineering (BTE).Date Sources:Data cited in this review were obtained primarily from PubMed and Medline in publications from 1979 to 2014,with highly regarded older publications also included.The terms BTE,CaP,BMPs,and MSC were used for the literature search.Study Selection:Reviews focused on relevant aspects and original articles reporting in vitro and/or in vivo results concerning the efficiency of CaP/BMPs or CaP/MSCs composites were retrieved,reviewed,analyzed,and summarized.Results:An ideal BTE product contains three elements:Scaffold,growth factors,and stem cells.CaP-based scaffolds are popular because of their outstanding biocompatibility,bioactivity,and osteoconductivity.However,they lack stiffness and osteoinductivity.To solve this problem,composite scaffolds of CaP with BMPs have been developed.New bone formation by CaP/BMP composites can reach levels similar to those of autografts.CaP scaffolds are compatible with MSCs and CaP/MSC composites exhibit excellent osteogenesis and stiffness.In addition,a CaP/MSC/BMP scaffold can repair bone defects more effectively than an autograft.Conclusions:Novel BTE products possess remarkable osteoconduction and osteoinduction capacities,and exhibit balanced degradation with osteogenesis.Further work should yield safe,viable,and efficient materials for the repair of bone lesions.

  12. Controlled release of recombinant human cementum protein 1 from electrospun multiphasic scaffold for cementum regeneration

    Directory of Open Access Journals (Sweden)

    Chen XF

    2016-07-01

    Full Text Available Xiaofeng Chen,1,* Yu Liu,1,* Leiying Miao,1 Yangyang Wang,2 Shuangshuang Ren,1 Xuebin Yang,3 Yong Hu,4 Weibin Sun1 1Department of Periodontology, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, Jiangsu, People’s Republic of China; 2Department of Materials Science and Engineering, College of Materials Science and Technology, Nanjing University of Aeronautics and Astronautics, Nanjing, Jiangsu, People’s Republic of China; 3Biomaterials and Tissue Engineering Group, Leeds Dental Institute, University of Leeds, Leeds, UK; 4Institute of Materials Engineering, National Laboratory of Solid State Micro Structure, College of Engineering and Applied Sciences, Nanjing University, Nanjing, Jiangsu, People’s Republic of China *These authors contributed equally to this work Abstract: Periodontitis is a major cause for tooth loss, which affects about 15% of the adult population. Cementum regeneration has been the crux of constructing the periodontal complex. Cementum protein 1 (CEMP1 is a cementum-specific protein that can induce cementogenic differentiation. In this study, poly(ethylene glycol (PEG-stabilized amorphous calcium phosphate (ACP nanoparticles were prepared by wet-chemical method and then loaded with recombinant human CEMP1 (rhCEMP1 for controlled release. An electrospun multiphasic scaffold constituted of poly(ε-caprolactone (PCL, type I collagen (COL, and rhCEMP1/ACP was fabricated. The effects of rhCEMP1/ACP/PCL/COL scaffold on the attachment proliferation, osteogenic, and cementogenic differentiations of human periodontal ligament cells, (PDLCs were systematically investigated. A critical size defect rat model was introduced to evaluate the effect of tissue regeneration of the scaffolds in vivo. The results showed that PEG-stabilized ACP nanoparticles formed a core-shell structure with sustained release of rhCEMP1 for up to 4 weeks. rhCEMP1/ACP/PCL/COL scaffold could suppress PDLCs proliferation behavior

  13. RACK1, A multifaceted scaffolding protein: Structure and function

    Directory of Open Access Journals (Sweden)

    Kiely Patrick A

    2011-10-01

    Full Text Available Abstract The Receptor for Activated C Kinase 1 (RACK1 is a member of the tryptophan-aspartate repeat (WD-repeat family of proteins and shares significant homology to the β subunit of G-proteins (Gβ. RACK1 adopts a seven-bladed β-propeller structure which facilitates protein binding. RACK1 has a significant role to play in shuttling proteins around the cell, anchoring proteins at particular locations and in stabilising protein activity. It interacts with the ribosomal machinery, with several cell surface receptors and with proteins in the nucleus. As a result, RACK1 is a key mediator of various pathways and contributes to numerous aspects of cellular function. Here, we discuss RACK1 gene and structure and its role in specific signaling pathways, and address how posttranslational modifications facilitate subcellular location and translocation of RACK1. This review condenses several recent studies suggesting a role for RACK1 in physiological processes such as development, cell migration, central nervous system (CN function and circadian rhythm as well as reviewing the role of RACK1 in disease.

  14. RACK1, A Multifaceted Scaffolding Protein: Structure and Function

    LENUS (Irish Health Repository)

    Adams, David R

    2011-10-06

    Abstract The Receptor for Activated C Kinase 1 (RACK1) is a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins and shares significant homology to the β subunit of G-proteins (Gβ). RACK1 adopts a seven-bladed β-propeller structure which facilitates protein binding. RACK1 has a significant role to play in shuttling proteins around the cell, anchoring proteins at particular locations and in stabilising protein activity. It interacts with the ribosomal machinery, with several cell surface receptors and with proteins in the nucleus. As a result, RACK1 is a key mediator of various pathways and contributes to numerous aspects of cellular function. Here, we discuss RACK1 gene and structure and its role in specific signaling pathways, and address how posttranslational modifications facilitate subcellular location and translocation of RACK1. This review condenses several recent studies suggesting a role for RACK1 in physiological processes such as development, cell migration, central nervous system (CN) function and circadian rhythm as well as reviewing the role of RACK1 in disease.

  15. Optimization of protein cross-linking in bicomponent electrospun scaffolds for therapeutic use

    Energy Technology Data Exchange (ETDEWEB)

    Papa, Antonio [Institute for Polymers, Composites and Biomaterials, National Research Council of Italy (IPCB-CNR), V.le Kennedy 54, Naples 80125 (Italy); IMAST SCaRL, Piazza Bovio 22, 80133 Naples (Italy); Guarino, Vincenzo, E-mail: vincenzo.guarino@cnr.it; Cirillo, Valentina; Oliviero, Olimpia; Ambrosio, Luigi [Institute for Polymers, Composites and Biomaterials, National Research Council of Italy (IPCB-CNR), V.le Kennedy 54, Naples 80125 (Italy)

    2015-12-17

    Bio-instructive electrospun scaffolds based on the combination of synthetic polymers, such as PCL or PLLA, and natural polymers (e.g., collagen) have been extensively investigated as temporary extracellular matrix (ECM) analogues able to support cell proliferation and stem cell differentiation for the regeneration of several tissues. The growing use of natural polymers as carrier of bioactive molecules is introducing new ideas for the design of polymeric drug delivery systems based on electrospun fibers with improved bioavailability, therapeutic efficacy and programmed drug release. In particular, the release mechanism is driven by the use of water soluble proteins (i.e., collagen, gelatin) which fully degrade in in vitro microenvironment, thus delivering the active principles. However, these protein are generally rapidly digested by enzymes (i.e., collagenase) produced by many different cell types, both in vivo and in vitro with significant drawbacks in tissue engineering and controlled drug delivery. Here, we aim at investigating different chemical strategies to improve the in vitro stability and mechanical strength of scaffolds against enzymatic degradation, by modifying the biodegradation rates of proteins embedded in bicomponent fibers. By comparing scaffolds treated by different cross-linking agents (i.e., GC, EDC, BDDGE), we have provided an extensive morphological/chemical/physical characterization via SEM and TGA to identify the best conditions to control drug release via protein degradation from bicomponent fibers without compromising in vitro cell response.

  16. Optimization of protein cross-linking in bicomponent electrospun scaffolds for therapeutic use

    Science.gov (United States)

    Papa, Antonio; Guarino, Vincenzo; Cirillo, Valentina; Oliviero, Olimpia; Ambrosio, Luigi

    2015-12-01

    Bio-instructive electrospun scaffolds based on the combination of synthetic polymers, such as PCL or PLLA, and natural polymers (e.g., collagen) have been extensively investigated as temporary extracellular matrix (ECM) analogues able to support cell proliferation and stem cell differentiation for the regeneration of several tissues. The growing use of natural polymers as carrier of bioactive molecules is introducing new ideas for the design of polymeric drug delivery systems based on electrospun fibers with improved bioavailability, therapeutic efficacy and programmed drug release. In particular, the release mechanism is driven by the use of water soluble proteins (i.e., collagen, gelatin) which fully degrade in in vitro microenvironment, thus delivering the active principles. However, these protein are generally rapidly digested by enzymes (i.e., collagenase) produced by many different cell types, both in vivo and in vitro with significant drawbacks in tissue engineering and controlled drug delivery. Here, we aim at investigating different chemical strategies to improve the in vitro stability and mechanical strength of scaffolds against enzymatic degradation, by modifying the biodegradation rates of proteins embedded in bicomponent fibers. By comparing scaffolds treated by different cross-linking agents (i.e., GC, EDC, BDDGE), we have provided an extensive morphological/chemical/physical characterization via SEM and TGA to identify the best conditions to control drug release via protein degradation from bicomponent fibers without compromising in vitro cell response.

  17. Optimization of protein cross-linking in bicomponent electrospun scaffolds for therapeutic use

    International Nuclear Information System (INIS)

    Bio-instructive electrospun scaffolds based on the combination of synthetic polymers, such as PCL or PLLA, and natural polymers (e.g., collagen) have been extensively investigated as temporary extracellular matrix (ECM) analogues able to support cell proliferation and stem cell differentiation for the regeneration of several tissues. The growing use of natural polymers as carrier of bioactive molecules is introducing new ideas for the design of polymeric drug delivery systems based on electrospun fibers with improved bioavailability, therapeutic efficacy and programmed drug release. In particular, the release mechanism is driven by the use of water soluble proteins (i.e., collagen, gelatin) which fully degrade in in vitro microenvironment, thus delivering the active principles. However, these protein are generally rapidly digested by enzymes (i.e., collagenase) produced by many different cell types, both in vivo and in vitro with significant drawbacks in tissue engineering and controlled drug delivery. Here, we aim at investigating different chemical strategies to improve the in vitro stability and mechanical strength of scaffolds against enzymatic degradation, by modifying the biodegradation rates of proteins embedded in bicomponent fibers. By comparing scaffolds treated by different cross-linking agents (i.e., GC, EDC, BDDGE), we have provided an extensive morphological/chemical/physical characterization via SEM and TGA to identify the best conditions to control drug release via protein degradation from bicomponent fibers without compromising in vitro cell response

  18. Protein scaffolds for selective enrichment of metal ions

    Energy Technology Data Exchange (ETDEWEB)

    He, Chuan; Zhou, Lu; Bosscher, Michael

    2016-02-09

    Polypeptides comprising high affinity for the uranyl ion are provided. Methods for binding uranyl using such proteins are likewise provided and can be used, for example, in methods for uranium purification or removal.

  19. The human Na(+)/H(+) exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2

    DEFF Research Database (Denmark)

    Hendus-Altenburger, Ruth; Pedraz-Cuesta, Elena; Olesen, Christina W;

    2016-01-01

    BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. METHODS AND RESULTS: Here, we identify...... the human Na(+)/H(+) exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D...... and ERK2, and provides a molecular mechanism for the important ERK2 scaffolding function of the membrane protein hNHE1, which regulates the phosphorylation of both hNHE1 and ERK2....

  20. Evolutionary implications of localization of the signaling scaffold protein parafusin to both cilia and the nucleus.

    Science.gov (United States)

    Satir, Birgit Hegner; Wyroba, Elzbieta; Liu, Li; Lethan, Mette; Satir, Peter; Christensen, Søren Tvorup

    2015-02-01

    Parafusin (PFUS), a 63 kDa protein first discovered in the eukaryote Paramecium and known for its role in apicomplexan exocytosis, provides a model for the common origin of cellular systems employing scaffold proteins for targeting and signaling. PFUS is closely related to eubacterial rather than archeal phosphoglucomutases (PGM) - as we proved by comparison of their 88 sequences - but has no PGM activity. Immunofluorescence microscopy analysis with a PFUS-specific peptide antibody showed presence of this protein around the base region of primary cilia in a variety of mammalian cell types, including mouse embryonic (MEFs) and human foreskin fibroblasts (hFFs), human carcinoma stem cells (NT-2 cells), and human retinal pigment epithelial (RPE) cells. Further, PFUS localized to the nucleus of fibroblasts, and prominently to nucleoli of MEFs. Localization studies were confirmed by Western blot analysis, showing that the PFUS antibody specifically recognizes a single protein of ca. 63 kDa in both cytoplasmic and nuclear fractions. Finally, immunofluorescence microscopy analysis showed that PFUS localized to nuclei and cilia in Paramecium. These results support the suggestion that PFUS plays a role in signaling between nucleus and cilia, and that the cilium and the nucleus both evolved around the time of eukaryotic emergence. We hypothesize that near the beginnings of eukaryotic cell evolution, scaffold proteins such as PFUS arose as peripheral membrane protein identifiers for cytoplasmic membrane trafficking and were employed similarly during the subsequent evolution of exocytic, nuclear transport, and ciliogenic mechanisms.

  1. pH induced protein-scaffold biosynthesis of tunable shape gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Xiaorong; He Xiaoxiao; Wang Kemin; Ren Fang; Qin Zhihe, E-mail: kmwang@hnu.cn [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha 410082 (China)

    2011-09-02

    In this paper, a pH-inductive protein-scaffold biosynthesis of shape-tunable crystalline gold nanoparticles at room temperature has been developed. By simple manipulation of the reaction solution's pH, anisotropic gold nanoparticles including spheres, triangles and cubes could be produced by incubating an aqueous solution of sodium tetrachloroaurate with Dolichomitriopsis diversiformis biomasses after immersion in ultrapure Millipore water overnight. A moss protein with molecular weight of about 71 kDa and pI of 4.9 was the primary biomolecule involved in the biosynthesis of gold nanoparticles. The secondary configuration of the proteins by CD spectrum implied that the moss protein could display different secondary configurations including random coil, {alpha}-helix and intermediate conformations between random coil and {alpha}-helix for the experimental pH solution. The growth process of gold nanoparticles further showed that the moss protein with different configurations provided the template scaffold for the shape-controlled biosynthesis of gold nanoparticles. The constrained shape of the gold nanoparticles, however, disappeared in boiled moss extract. The gold nanoparticles with designed morphology were successfully reconstructed using the moss protein purified from the gold nanoparticles. Structural characterizations by SEM, TEM and SAED showed that the triangular and cubic gold nanoparticles were single crystalline.

  2. Staphylococcal Bap Proteins Build Amyloid Scaffold Biofilm Matrices in Response to Environmental Signals.

    Science.gov (United States)

    Taglialegna, Agustina; Navarro, Susanna; Ventura, Salvador; Garnett, James A; Matthews, Steve; Penades, José R; Lasa, Iñigo; Valle, Jaione

    2016-06-01

    Biofilms are communities of bacteria that grow encased in an extracellular matrix that often contains proteins. The spatial organization and the molecular interactions between matrix scaffold proteins remain in most cases largely unknown. Here, we report that Bap protein of Staphylococcus aureus self-assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental conditions. Specifically, Bap is processed and fragments containing at least the N-terminus of the protein become aggregation-prone and self-assemble into amyloid-like structures under acidic pHs and low concentrations of calcium. The molten globule-like state of Bap fragments is stabilized upon binding of the cation, hindering its self-assembly into amyloid fibers. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein to promote biofilm development under specific environmental conditions. Since the pH-driven multicellular behavior mediated by Bap occurs in coagulase-negative staphylococci and many other bacteria exploit Bap-like proteins to build a biofilm matrix, the mechanism of amyloid-like aggregation described here may be widespread among pathogenic bacteria.

  3. Staphylococcal Bap Proteins Build Amyloid Scaffold Biofilm Matrices in Response to Environmental Signals

    Science.gov (United States)

    Taglialegna, Agustina; Navarro, Susanna; Ventura, Salvador; Garnett, James A.; Matthews, Steve; Penades, José R.; Lasa, Iñigo; Valle, Jaione

    2016-01-01

    Biofilms are communities of bacteria that grow encased in an extracellular matrix that often contains proteins. The spatial organization and the molecular interactions between matrix scaffold proteins remain in most cases largely unknown. Here, we report that Bap protein of Staphylococcus aureus self-assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental conditions. Specifically, Bap is processed and fragments containing at least the N-terminus of the protein become aggregation-prone and self-assemble into amyloid-like structures under acidic pHs and low concentrations of calcium. The molten globule-like state of Bap fragments is stabilized upon binding of the cation, hindering its self-assembly into amyloid fibers. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein to promote biofilm development under specific environmental conditions. Since the pH-driven multicellular behavior mediated by Bap occurs in coagulase-negative staphylococci and many other bacteria exploit Bap-like proteins to build a biofilm matrix, the mechanism of amyloid-like aggregation described here may be widespread among pathogenic bacteria. PMID:27327765

  4. Synthetic protein scaffolds based on peptide motifs and cognate adaptor domains for improving metabolic productivity

    Directory of Open Access Journals (Sweden)

    Anselm H.C. Horn

    2015-11-01

    Full Text Available The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity.

  5. Nanofibrous yet injectable polycaprolactone-collagen bone tissue scaffold with osteoprogenitor cells and controlled release of bone morphogenetic protein-2

    Energy Technology Data Exchange (ETDEWEB)

    Subramanian, Gayathri; Bialorucki, Callan [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Yildirim-Ayan, Eda, E-mail: eda.yildirimayan@utoledo.edu [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Department of Orthopaedic Surgery, University of Toledo Medical Center, Toledo, OH 43614 (United States)

    2015-06-01

    In this work, we developed a nanofibrous, yet injectable orthobiologic tissue scaffold that is capable of hosting osteoprogenitor cells and controlling kinetic release profile of the encapsulated pro-osteogenic factor without diminishing its bioactivity over 21 days. This innovative injectable scaffold was synthesized by incorporating electrospun and subsequently O{sub 2} plasma-functionalized polycaprolactone (PCL) nanofibers within the collagen type-I solution along with MC3T3-E1 cells (pre-osteoblasts) and bone morphogenetic protein-2 (BMP2). Through changing the PCL nanofiber concentration within the injectable scaffolds, we were able to tailor the mechanical strength, protein retention capacity, bioactivity preservation, and osteoinductive potential of the scaffolds. The nanofibrous internal structure of the scaffold allowed us to use a low dose of BMP2 (200 ng/ml) to achieve osteoblastic differentiation in in vitro culture. The osteogenesis capacity of the injectable scaffolds were evaluated though measuring MC3T3-E1 cell proliferation, ALP activity, matrix mineralization, and early- and late-osteoblast specific gene expression profiles over 21 days. The results demonstrated that the nanofibrous injectable scaffold provides not only an osteoinductive environment for osteoprogenitor cells to differentiate, but also a suitable biomechanical and biochemical environment to act as a reservoir for osteogenic factors with controlled release profile. - Highlights: • Injectable nanofibrous scaffold with osteoprogenitor cells and BMP2 was synthesized. • PCL nanofiber concentration within collagen scaffold affected the BMP2 retention and bioactivity. • Optimal PCL concentration was identified for mechanical stability, injectability, and osteogenic activity. • Scaffolds exhibited long-term osteoinductive capacity for bone repair and regeneration.

  6. PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena

    NARCIS (Netherlands)

    Hughes, Shannon K; Oudin, Madeleine J; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A; Gertler, Frank B

    2015-01-01

    During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. Howev

  7. Arabidopsis scaffold protein RACK1A modulates rare sugar D-allose regulated gibberellin signaling

    OpenAIRE

    Fennell, Herman; Olawin, Abdulquadri; Mizanur, Rahman M; Izumori, Ken; Chen, Jin-Gui; Ullah, Hemayet

    2012-01-01

    As energy sources and structural components, sugars are the central regulators of plant growth and development. In addition to the abundant natural sugars in plants, more than 50 different kinds of rare sugars exist in nature, several of which show distinct roles in plant growth and development. Recently, one of the rare sugars, D-allose, an epimer of D-glucose at C3, is found to suppress plant hormone gibberellin (GA) signaling in rice. Scaffold protein RACK1A in the model plant Arabidopsis ...

  8. Bone Tissue Engineering Using High Permeability Poly-epsilon-caprolactone Scaffolds Conjugated with Bone Morphogenetic Protein-2

    Science.gov (United States)

    Mitsak, Anna Guyer

    Bone is the second most commonly transplanted tissue in the United States. Limitations of current bone defect treatment options include morbidity at the autograft harvest site, mechanical failure, and poorly controlled growth factor delivery. Combining synthetic scaffolds with biologics may address these issues and reduce dependency on autografts. The ideal scaffolding system should promote tissue in-growth and nutrient diffusion, control delivery of biologics and maintain mechanical integrity during bone formation. This dissertation evaluates how scaffold permeability, conjugated bone morphogenetic protein-2 (BMP-2) and differentiation medium affect osteogenesis in vitro and bone growth in vivo.. "High" and "low" permeability polycaprolactone (PCL) scaffolds with regular architectures were manufactured using solid free form fabrication. Bone growth in vivo was evaluated in an ectopic mouse model. High permeability scaffolds promoted better 8 week bone growth, supported tissue penetration into the scaffold core, and demonstrated increased mechanical properties due to newly formed bone. Next, the effects of differentiation medium and conjugated BMP-2 on osteogenesis were compared. Conjugation may improve BMP-2 loading efficiency, help localize bone growth and control release. High permeability scaffolds were conjugated with BMP-2 using the crosslinker, sulfo-SMCC. When adipose-derived and bone marrow stromal cells were seeded onto constructs (with or without BMP-2), BMSC expressed more differentiation markers, and differentiation medium affected differentiation more than BMP-2. In vivo, scaffolds with ADSC pre-differentiated in osteogenic medium (with and without BMP-2) and scaffolds with only BMP-2 grew the most bone. Bone volume did not differ among these groups, but constructs with ADSC had evenly distributed, scaffold-guided bone growth. Analysis of two additional BMP-2 attachment methods (heparin and adsorption) showed highest conjugation efficiency for the

  9. Emerging role of the scaffolding protein Dlg1 in vesicle trafficking.

    Science.gov (United States)

    Walch, Laurence

    2013-09-01

    Discs large 1 (Dlg1) is a modular scaffolding protein implicated in the control of cell polarity through assembly of specific multiprotein complexes, including receptors, ion channels and signaling proteins, at specialized zones of the plasma membrane. Recent data have shown that in addition to these well-known interaction partners, Dlg1 may also recruit components of the vesicle trafficking machinery either to the plasma membrane or to transport vesicles. Here, we discuss Dlg1 function in vesicle formation, targeting, tethering and fusion, in both the exocytotic and endocytotic pathways. These pathways contribute to cell functions as major and diverse as glutamatergic activity in the neurons, membrane homeostasis in Schwann cell myelination, insulin stimulation of glucose transport in adipocytes, or endothelial secretion of the hemostatic protein, von Willebrand factor (VWF).

  10. Influence of Histidine-Containing Tags on the Biodistribution of ADAPT Scaffold Proteins.

    Science.gov (United States)

    Lindbo, Sarah; Garousi, Javad; Åstrand, Mikael; Honarvar, Hadis; Orlova, Anna; Hober, Sophia; Tolmachev, Vladimir

    2016-03-16

    Engineered scaffold proteins (ESP) are high-affinity binders that can be used as probes for radionuclide imaging. Histidine-containing tags enable both efficient purification of ESP and radiolabeling with (99m)Tc(CO)3. Earlier studies demonstrated that the use of a histidine-glutamate-histidine-glutamate-histidine-glutamate (HE)3-tag instead of the commonly used hexahistidine (H6)-tag reduces hepatic uptake of radiolabeled ESP and short peptides. Here, we investigated the influence of histidine-containing tags on the biodistribution of a novel type of ESP, ADAPTs. A series of anti-HER2 ADAPT probes having H6- or (HE)3-tags in the N-termini were prepared. The constructs, (HE)3-ADAPT6 and H6-ADAPT6, were labeled with two different nuclides, (99m)Tc or (111)In. The labeling with (99m)Tc(CO)3 utilized the histidine-containing tags, while (111)In was attached through a maleimido derivative of DOTA conjugated to the N-terminus. For (111)In-labeled ADAPTs, the use of (HE)3 provided a significantly (p < 0.05) lower hepatic uptake at 1 h after injection, but there was no significant difference in hepatic uptake of (111)In-(HE)3-ADAPT6 and H6-ADAPT6 at later time points. Interestingly, in the case of (99m)Tc, (99m)Tc(CO)3-H6-ADAPT6 provided significantly (p < 0.05) lower uptake in a number of normal tissues and was more suitable as an imaging probe. Thus, the influence of histidine-containing tags on the biodistribution of the novel ADAPT scaffold proteins was different compared to its influence on other ESPs studied so far. Apparently, the effect of a histidine-containing tag on the biodistribution is highly dependent on the scaffold composition of the ESP. PMID:26781756

  11. Phage display selection of tight specific binding variants from a hyperthermostable Sso7d scaffold protein library.

    Science.gov (United States)

    Zhao, Ning; Schmitt, Margaret A; Fisk, John D

    2016-04-01

    Antibodies, the quintessential biological recognition molecules, are not ideal for many applications because of their large size, complex modifications, and thermal and chemical instability. Identifying alternative scaffolds that may be evolved into tight, specific binding molecules with improved physical properties is of increasing interest, particularly for biomedical applications in resource-limited environments. Hyperthermophilic organisms, such as Sulfolobus solfataricus, are an attractive source of highly stable proteins that may serve as starting points for alternative molecular recognition scaffolds. We describe the first application of phage display to identify binding proteins based on the S. solfataricus protein Sso7d scaffold. Sso7d is a small cysteine-free DNA-binding protein (approximately 7 kDa, 63 amino acids), with a melting temperature of nearly 100 °C. Tight-binding Sso7d variants were selected for a diverse set of protein targets from a 10(10) member library, demonstrating the versatility of the scaffold. These Sso7d variants are able to discriminate among closely related human, bovine and rabbit serum albumins. Equilibrium dissociation constants in the nanomolar to low micromolar range were measured via competitive ELISA. Importantly, the Sso7d variants continue to bind their targets in the absence of the phage context. Furthermore, phage-displayed Sso7d variants retain their binding affinity after exposure to temperatures up to 70 °C. Taken together, our results suggest that the Sso7d scaffold will be a complementary addition to the range of non-antibody scaffold proteins that may be utilized in phage display. Variants of hyperthermostable binding proteins have potential applications in diagnostics and therapeutics for environments with extreme conditions of storage and deployment.

  12. Regulation of cell signaling dynamics by the protein kinase-scaffold Ste5.

    Science.gov (United States)

    Hao, Nan; Nayak, Sujata; Behar, Marcelo; Shanks, Ryan H; Nagiec, Michal J; Errede, Beverly; Hasty, Jeffrey; Elston, Timothy C; Dohlman, Henrik G

    2008-06-01

    Cell differentiation requires the ability to detect and respond appropriately to a variety of extracellular signals. Here we investigate a differentiation switch induced by changes in the concentration of a single stimulus. Yeast cells exposed to high doses of mating pheromone undergo cell division arrest. Cells at intermediate doses become elongated and divide in the direction of a pheromone gradient (chemotropic growth). Either of the pheromone-responsive MAP kinases, Fus3 and Kss1, promotes cell elongation, but only Fus3 promotes chemotropic growth. Whereas Kss1 is activated rapidly and with a graded dose-response profile, Fus3 is activated slowly and exhibits a steeper dose-response relationship (ultrasensitivity). Fus3 activity requires the scaffold protein Ste5; when binding to Ste5 is abrogated, Fus3 behaves like Kss1, and the cells no longer respond to a gradient or mate efficiently with distant partners. We propose that scaffold proteins serve to modulate the temporal and dose-response behavior of the MAP kinase. PMID:18538663

  13. Regulation of the actin cytoskeleton by an interaction of IQGAP related protein GAPA with filamin and cortexillin I.

    Directory of Open Access Journals (Sweden)

    Subhanjan Mondal

    Full Text Available Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin.

  14. The role of zinc in the stability of the marginally stable IscU scaffold protein.

    Science.gov (United States)

    Iannuzzi, Clara; Adrover, Miquel; Puglisi, Rita; Yan, Robert; Temussi, Piero Andrea; Pastore, Annalisa

    2014-09-01

    Understanding the factors that determine protein stability is interesting because it directly reflects the evolutionary pressure coming from function and environment. Here, we have combined experimental and computational methods to study the stability of IscU, a bacterial scaffold protein highly conserved in most organisms and an essential component of the iron-sulfur cluster biogenesis pathway. We demonstrate that the effect of zinc and its consequence strongly depend on the sample history. IscU is a marginally stable protein at low ionic strength to the point that undergoes cold denaturation at around -8°C with a corresponding dramatic decrease of enthalpy, which is consistent with the fluxional nature of the protein. Presence of constitutively bound zinc appreciably stabilizes the IscU fold, whereas it may cause protein aggregation when zinc is added back posthumously. We discuss how zinc coordination can be achieved by different side chains spatially available and all competent for tetrahedral coordination. The individual absence of some of these residues can be largely compensated by small local rearrangements of the others. We discuss the potential importance of our findings in vitro for the function in vivo of the protein.

  15. Targeting diverse protein-protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold.

    Science.gov (United States)

    Checco, James W; Kreitler, Dale F; Thomas, Nicole C; Belair, David G; Rettko, Nicholas J; Murphy, William L; Forest, Katrina T; Gellman, Samuel H

    2015-04-14

    Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and β-amino acid residues ("α/β-peptides") that mimic several peptides derived from the three-helix bundle "Z-domain" scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF165-induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain-mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.

  16. Biomimetic macroporous hydrogels: protein ligand distribution and cell response to the ligand architecture in the scaffold.

    Science.gov (United States)

    Savina, Irina N; Dainiak, Maria; Jungvid, Hans; Mikhalovsky, Sergey V; Galaev, Igor Yu

    2009-01-01

    Macroporous hydrogels (MHs), cryogels, are a new type of biomaterials for tissue engineering that can be produced from any natural or synthetic polymer that forms a gel. Synthetic MHs are rendered bioactive by surface or bulk modifications with extracellular matrix components. In this study, cell response to the architecture of protein ligands, bovine type-I collagen (CG) and human fibrinogen (Fg), immobilised using different methods on poly(2-hydroxyethyl methacrylate) (pHEMA) macroporous hydrogels (MHs) was analysed. Bulk modification was performed by cross-linking cryo-co-polymerisation of HEMA and poly(ethylene glycol)diacrylate (PEGA) in the presence of proteins (CG/pHEMA and Fg/pHEMA MHs). The polymer surface was modified by covalent immobilisation of the proteins to the active epoxy (ep) groups present on pHEMA after hydrogel fabrication (CG-epHEMA and Fg-epHEMA MHs). The concentration of proteins in protein/pHEMA and protein-epHEMA MHs was 80-85 and 130-140 mug/ml hydrogel, respectively. It was demonstrated by immunostaining and confocal laser scanning microscopy that bulk modification resulted in spreading of CG in the polymer matrix and spot-like distribution of Fg. On the contrary, surface modification resulted in spot-like distribution of CG and uniform spreading of Fg, which evenly coated the surface. Proliferation rate of fibroblasts was higher on MHs with even distribution of the ligands, i.e., on Fg-epHEMA and CG/pHEMA. After 30 days of growth, fibroblasts formed several monolayers and deposited extracellular matrix filling the pores of these MHs. The best result in terms of cell proliferation was obtained on Fg-epHEMA. The ligands displayed on surface of these scaffolds were in native conformation, while in bulk-modified CG/pHEMA MHs most of the proteins were buried inside the polymer matrix and were less accessible for interactions with specific antibodies and cells. The method used for MH modification with bioligands strongly affects spatial

  17. A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protien-mediated kinetic scaffolding.

    Science.gov (United States)

    Zhong, Huailing; Wade, Susan M; Woolf, Peter J; Linderman, Jennifer J; Traynor, John R; Neubig, Richard R

    2003-02-28

    Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to G(i) by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH(2)- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPgammaS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic "spatial focusing" of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Galpha-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Galpha-GDP is not depleted there. Thus, a novel RGS-mediated "kinetic scaffolding" mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of G(i) protein signals.

  18. Selective Inhibitors of Fibroblast Activation Protein (FAP) with a (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine Scaffold.

    Science.gov (United States)

    Jansen, Koen; Heirbaut, Leen; Cheng, Jonathan D; Joossens, Jurgen; Ryabtsova, Oxana; Cos, Paul; Maes, Louis; Lambeir, Anne-Marie; De Meester, Ingrid; Augustyns, Koen; Van der Veken, Pieter

    2013-05-01

    Fibroblast activation protein (FAP) is a serine protease that is generally accepted to play an important role in tumor growth and other diseases involving tissue remodeling. Currently there are no FAP inhibitors with reported selectivity toward both the closely related dipeptidyl peptidases (DPPs) and prolyl oligopeptidase (PREP). We present the discovery of a new class of FAP inhibitors with a N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) scaffold. We have explored the effects of substituting the quinoline ring and varying the position of its sp(2) hybridized nitrogen atom. The most promising inhibitors combined low nanomolar FAP inhibition and high selectivity indices (>10(3)) with respect to both the DPPs and PREP. Preliminary experiments on a representative inhibitor demonstrate that plasma stability, kinetic solubility, and log D of this class of compounds can be expected to be satisfactory. PMID:24900696

  19. Chemical shift assignment of the alternative scaffold protein IscA.

    Science.gov (United States)

    Popovic, Matija; Pastore, Annalisa

    2016-04-01

    The IscA protein (11.5 kDa) is an essential component of the iron sulphur cluster biogenesis machine. In bacteria, the machine components are clustered in operons, amongst which the most important is the isc operon. Bacterial IscA has direct homologues also in eukaryotes. Like the protein IscU, IscA is thought to assist cluster formation as an alternative scaffold protein which receives the cluster before transferring it further to the final acceptors. Several crystal structures have been published. They all report an IscA dimeric form, although the packing of the protomers in the dimers differs amongst structures. No solution studies have currently been reported. Here we report the (1)H, (13)C and (15)N backbone and side-chain chemical shift assignments of the cluster-free E. coli IscA as a starting point for further studies of the structure and functions of this still poorly characterized protein. We show that IscA exists in solution as an equilibrium between different species. Spectrum assignment was thus challenging given the heterogeneous nature of the sample but doable through judicious choice of selective labelling and concentration dependent studies.

  20. Biophysical characterization and modeling of human Ecdysoneless (ECD protein supports a scaffolding function

    Directory of Open Access Journals (Sweden)

    Nicholas P. Schafer

    2016-03-01

    Full Text Available The human homolog of Drosophila ecdysoneless protein (ECD is a p53 binding protein that stabilizes and enhances p53 functions. Homozygous deletion of mouse Ecd is early embryonic lethal and Ecd deletion delays G1-S cell cycle progression. Importantly, ECD directly interacts with the Rb tumor suppressor and competes with the E2F transcription factor for binding to Rb. Further studies demonstrated ECD is overexpressed in breast and pancreatic cancers and its overexpression correlates with poor patient survival. ECD overexpression together with Ras induces cellular transformation through upregulation of autophagy. Recently we demonstrated that CK2 mediated phosphorylation of ECD and interaction with R2TP complex are important for its cell cycle regulatory function. Considering that ECD is a component of multiprotein complexes and its crystal structure is unknown, we characterized ECD structure by circular dichroism measurements and sequence analysis software. These analyses suggest that the majority of ECD is composed of α-helices. Furthermore, small angle X-ray scattering (SAXS analysis showed that deletion fragments, ECD(1–432 and ECD(1–534, are both well-folded and reveals that the first 400 residues are globular and the next 100 residues are in an extended cylindrical structure. Taking all these results together, we speculate that ECD acts like a structural hub or scaffolding protein in its association with its protein partners. In the future, the hypothetical model presented here for ECD will need to be tested experimentally.

  1. A targeted library screen reveals a new inhibitor scaffold for protein kinase D.

    Directory of Open Access Journals (Sweden)

    Manuj Tandon

    Full Text Available Protein kinase D (PKD has emerged as a potential therapeutic target in multiple pathological conditions, including cancer and heart diseases. Potent and selective small molecule inhibitors of PKD are valuable for dissecting PKD-mediated cellular signaling pathways and for therapeutic application. In this study, we evaluated a targeted library of 235 small organic kinase inhibitors for PKD1 inhibitory activity at a single concentration. Twenty-eight PKD inhibitory chemotypes were identified and six exhibited excellent PKD1 selectivity. Five of the six lead structures share a common scaffold, with compound 139 being the most potent and selective for PKD vs PKC and CAMK. Compound 139 was an ATP-competitive PKD1 inhibitor with a low double-digit nanomolar potency and was also cell-active. Kinase profiling analysis identified this class of small molecules as pan-PKD inhibitors, confirmed their selectivity again PKC and CAMK, and demonstrated an overall favorable selectivity profile that could be further enhanced through structural modification. Furthermore, using a PKD homology model based on similar protein kinase structures, docking modes for compound 139 were explored and compared to literature examples of PKD inhibition. Modeling of these compounds at the ATP-binding site of PKD was used to rationalize its high potency and provide the foundation for future further optimization. Accordingly, using biochemical screening of a small number of privileged scaffolds and computational modeling, we have identified a new core structure for highly potent PKD inhibition with promising selectivity against closely related kinases. These lead structures represent an excellent starting point for the further optimization and the design of selective and therapeutically effective small molecule inhibitors of PKD.

  2. A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK 1/2 to promote fascin-1/actin binding and filopodia stability

    Directory of Open Access Journals (Sweden)

    Jayo Asier

    2012-08-01

    applicability for analyzing the activities of actin-binding proteins in intact cells.

  3. Enhanced healing of rabbit segmental radius defects with surface-coated calcium phosphate cement/bone morphogenetic protein-2 scaffolds

    International Nuclear Information System (INIS)

    Large osseous defects remain a difficult clinical problem in orthopedic surgery owing to the limited effective therapeutic options, and bone morphogenetic protein-2 (BMP-2) is useful for its potent osteoinductive properties in bone regeneration. Here we build a strategy to achieve prolonged duration time and help inducting new bone formation by using water-soluble polymers as a protective film. In this study, calcium phosphate cement (CPC) scaffolds were prepared as the matrix and combined with sodium carboxymethyl cellulose (CMC-Na), hydroxypropylmethyl cellulose (HPMC), and polyvinyl alcohol (PVA) respectively to protect from the digestion of rhBMP-2. After being implanted in the mouse thigh muscles, the surface-modified composite scaffolds evidently induced ectopic bone formation. In addition, we further evaluated the in vivo effects of surface-modified scaffolds in a rabbit radius critical defect by radiography, three dimensional micro-computed tomographic (μCT) imaging, synchrotron radiation-based micro-computed tomographic (SRμCT) imaging, histological analysis, and biomechanical measurement. The HPMC-modified CPC scaffold was regarded as the best combination for segmental bone regeneration in rabbit radius. - Highlights: • A simple surface-coating method was used to fabricate composite scaffolds. • Growth factor was protected from rapid depletion via superficial coating. • Significant promotion of bone regeneration was achieved. • HPMC-modification displayed optimal effect of bone regeneration

  4. Enhanced healing of rabbit segmental radius defects with surface-coated calcium phosphate cement/bone morphogenetic protein-2 scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yi; Hou, Juan; Yin, ManLi [Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Wang, Jing, E-mail: biomatwj@163.com [Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Liu, ChangSheng, E-mail: csliu@sh163.net [Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China); Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China)

    2014-11-01

    Large osseous defects remain a difficult clinical problem in orthopedic surgery owing to the limited effective therapeutic options, and bone morphogenetic protein-2 (BMP-2) is useful for its potent osteoinductive properties in bone regeneration. Here we build a strategy to achieve prolonged duration time and help inducting new bone formation by using water-soluble polymers as a protective film. In this study, calcium phosphate cement (CPC) scaffolds were prepared as the matrix and combined with sodium carboxymethyl cellulose (CMC-Na), hydroxypropylmethyl cellulose (HPMC), and polyvinyl alcohol (PVA) respectively to protect from the digestion of rhBMP-2. After being implanted in the mouse thigh muscles, the surface-modified composite scaffolds evidently induced ectopic bone formation. In addition, we further evaluated the in vivo effects of surface-modified scaffolds in a rabbit radius critical defect by radiography, three dimensional micro-computed tomographic (μCT) imaging, synchrotron radiation-based micro-computed tomographic (SRμCT) imaging, histological analysis, and biomechanical measurement. The HPMC-modified CPC scaffold was regarded as the best combination for segmental bone regeneration in rabbit radius. - Highlights: • A simple surface-coating method was used to fabricate composite scaffolds. • Growth factor was protected from rapid depletion via superficial coating. • Significant promotion of bone regeneration was achieved. • HPMC-modification displayed optimal effect of bone regeneration.

  5. Mechanically unfolding proteins: The effect of unfolding history and the supramolecular scaffold

    Science.gov (United States)

    Zinober, Rebecca C.; Brockwell, David J.; Beddard, Godfrey S.; Blake, Anthony W.; Olmsted, Peter D.; Radford, Sheena E.; Smith, D. Alastair

    2002-01-01

    The mechanical resistance of a folded domain in a polyprotein of five mutant I27 domains (C47S, C63S I27)5is shown to depend on the unfolding history of the protein. This observation can be understood on the basis of competition between two effects, that of the changing number of domains attempting to unfold, and the progressive increase in the compliance of the polyprotein as domains unfold. We present Monte Carlo simulations that show the effect and experimental data that verify these observations. The results are confirmed using an analytical model based on transition state theory. The model and simulations also predict that the mechanical resistance of a domain depends on the stiffness of the surrounding scaffold that holds the domain in vivo, and on the length of the unfolded domain. Together, these additional factors that influence the mechanical resistance of proteins have important consequences for our understanding of natural proteins that have evolved to withstand force. PMID:12441375

  6. Synthesis of a metal binding protein designed on the alpha/beta scaffold of charybdotoxin.

    Science.gov (United States)

    Pierret, B; Virelizier, H; Vita, C

    1995-12-01

    The alpha/beta scaffold of the scorpion toxin charybdotoxin has been used for the engineering of a metal binding site. Nine substitutions, including three histidines as metal ligands, have been introduced into the original toxin sequence. The newly designed sequence, 37 amino acids long, has been assembled by solid-phase synthesis and HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) coupling of Fmoc-protected amino acids. Formation of the three disulfide bonds occurred efficiently and rapidly in the presence of glutathione, and this post-synthesis modification has facilitated the purification task enormously. The process of synthesis and purification was performed in less than a week with an overall 10.2% yield. Circular dichroism analysis showed that the newly designed protein is folded in a alpha/beta structure, similarly to the parent toxin. Electronic absorption spectroscopy, circular dichroism and gel filtration experiments have been used to show that Cu2+ and Zn2+ ions bind with high affinity to the newly engineered protein. These results demonstrate that the alpha/beta fold, common to all scorpion toxins, is a very versatile basic structure, tolerant for substitutions and able to present new sequences in a predetermined conformation. The chemical approach is shown to be effective, rapid and practical for the production of novel designed small proteins.

  7. Distribution of the scaffolding proteins PSD-95, PSD-93, and SAP97 in isolated PSDs.

    Science.gov (United States)

    DeGiorgis, Joseph A; Galbraith, James A; Dosemeci, Ayse; Chen, Xiaobing; Reese, Thomas S

    2006-12-01

    We compared the distribution of three scaffolding proteins, all belonging to a family of membrane-associated guanylate kinases, thought to have key roles in the organization of the postsynaptic density (PSD). Isolated PSDs readily adhered to treated glass coverslips where they were labeled with immunogold and rotary shadowed for analysis by EM. The distribution of proteins within individual PSDs were measured by counting and mapping individual immunogold particles. PSD-95, as previously described, is distributed evenly throughout the PSD. We find here that PSD-93 has a nearly identical distribution suggesting that PSD-95 and PSD-93 could perform similar roles. SAP97, in contrast, is concentrated near edges of cleft sides of the PSDs, and in small clumps on their cytoplasmic sides. The homogenous distribution of PSD-95 and PSD-93 throughout the PSD is consistent with their being part of a backbone that stabilizes their various binding partners within the PSD. The distribution of SAP97 confirms that this protein is actually an integral component of the PSD, and suggests that it may have a role in inserting or stabilizing its main binding partner, Glu-R1, at the edge of the PSD. PMID:18392731

  8. Mitogen-activated protein kinases with distinct requirements for Ste5 scaffolding influence signaling specificity in Saccharomyces cerevisiae.

    Science.gov (United States)

    Flatauer, Laura J; Zadeh, Sheena F; Bardwell, Lee

    2005-03-01

    Scaffold proteins are believed to enhance specificity in cell signaling when different pathways share common components. The prototype scaffold Ste5 binds to multiple components of the Saccharomyces cerevisiae mating pheromone response pathway, thereby conducting the mating signal to the Fus3 mitogen-activated protein kinase (MAPK). Some of the kinases that Ste5 binds to, however, are also shared with other pathways. Thus, it has been presumed that Ste5 prevents its bound kinases from transgressing into other pathways and protects them from intrusions from those pathways. Here we found that Fus3MAPK required Ste5 scaffolding to receive legitimate signals from the mating pathway as well as misdirected signals leaking from other pathways. Furthermore, increasing the cellular concentration of active Ste5 enhanced the channeling of inappropriate stimuli to Fus3. This aberrant signal crossover resulted in the erroneous induction of cell cycle arrest and mating. In contrast to Fus3, the Kss1 MAPK did not require Ste5 scaffolding to receive either authentic or leaking signals. Furthermore, the Ste11 kinase, once activated via Ste5, was able to signal to Kss1 independently of Ste5 scaffolding. These results argue that Ste5 does not act as a barrier that actively prevents signal crossover to Fus3 and that Ste5 may not effectively sequester its activated kinases away from other pathways. Rather, we suggest that specificity in this network is promoted by the selective activation of Ste5 and the distinct requirements of the MAPKs for Ste5 scaffolding. PMID:15713635

  9. Synapse associated protein 102 (SAP102 binds the C-terminal part of the scaffolding protein neurobeachin.

    Directory of Open Access Journals (Sweden)

    Juliane Lauks

    Full Text Available Neurobeachin (Nbea is a multidomain scaffold protein abundant in the brain, where it is highly expressed during development. Nbea-null mice have severe defects in neuromuscular synaptic transmission resulting in lethal paralysis of the newborns. Recently, it became clear that Nbea is important also for the functioning of central synapses, where it is suggested to play a role in trafficking membrane proteins to both, the pre- and post-synaptic sites. So far, only few binding partners of Nbea have been found and the precise mechanism of their trafficking remains unclear. Here, we used mass spectrometry to identify SAP102, a MAGUK protein implicated in trafficking of the ionotropic glutamate AMPA- and NMDA-type receptors during synaptogenesis, as a novel Nbea interacting protein in mouse brain. Experiments in heterologous cells confirmed this interaction and revealed that SAP102 binds to the C-terminal part of Nbea that contains the DUF, PH, BEACH and WD40 domains. Furthermore, we discovered that introducing a mutation in Nbea's PH domain, which disrupts its interaction with the BEACH domain, abolishes this binding, thereby creating an excellent starting point to further investigate Nbea-SAP102 function in the central nervous system.

  10. Diurnal rhythms in neurexins transcripts and inhibitory/excitatory synapse scaffold proteins in the biological clock.

    Directory of Open Access Journals (Sweden)

    Mika Shapiro-Reznik

    Full Text Available The neurexin genes (NRXN1/2/3 encode two families (α and β of highly polymorphic presynaptic proteins that are involved in excitatory/inhibitory synaptic balance. Recent studies indicate that neuronal activation and memory formation affect NRXN1/2/3α expression and alternative splicing at splice sites 3 and 4 (SS#3/SS#4. Neurons in the biological clock residing in the suprachiasmatic nuclei of the hypothalamus (SCN act as self-sustained oscillators, generating rhythms in gene expression and electrical activity, to entrain circadian bodily rhythms to the 24 hours day/night cycles. Cell autonomous oscillations in NRXN1/2/3α expression and SS#3/SS#4 exons splicing and their links to rhythms in excitatory/inhibitory synaptic balance in the circadian clock were explored. NRXN1/2/3α expression and SS#3/SS#4 splicing, levels of neurexin-2α and the synaptic scaffolding proteins PSD-95 and gephyrin (representing excitatory and inhibitory synapses, respectively were studied in mRNA and protein extracts obtained from SCN of C3H/J mice at different times of the 24 hours day/night cycle. Further studies explored the circadian oscillations in these components and causality relationships in immortalized rat SCN2.2 cells. Diurnal rhythms in mNRXN1α and mNRXN2α transcription, SS#3/SS#4 exon-inclusion and PSD-95 gephyrin and neurexin-2α levels were found in the SCN in vivo. No such rhythms were found with mNRXN3α. SCN2.2 cells also exhibited autonomous circadian rhythms in rNRXN1/2 expression SS#3/SS#4 exon inclusion and PSD-95, gephyrin and neurexin-2α levels. rNRXN3α and rNRXN1/2β were not expressed. Causal relationships were demonstrated, by use of specific siRNAs, between rNRXN2α SS#3 exon included transcripts and gephyrin levels in the SCN2.2 cells. These results show for the first time dynamic, cell autonomous, diurnal rhythms in expression and splicing of NRXN1/2 and subsequent effects on the expression of neurexin-2α and postsynaptic

  11. Degradability, biocompatibility, and osteogenesis of biocomposite scaffolds containing nano magnesium phosphate and wheat protein both in vitro and in vivo for bone regeneration.

    Science.gov (United States)

    Xia, Yan; Zhou, Panyu; Wang, Fei; Qiu, Chao; Wang, Panfeng; Zhang, Yuntong; Zhao, Liming; Xu, Shuogui

    2016-01-01

    In this study, bioactive scaffold of nano magnesium phosphate (nMP)/wheat protein (WP) composite (MWC) was fabricated. The results revealed that the MWC scaffolds had interconnected not only macropores (sized 400-600 μm) but also micropores (sized 10-20 μm) on the walls of macropores. The MWC scaffolds containing 40 w% nMP had an appropriate degradability in phosphate-buffered saline and produced a weak alkaline microenvironment. In cell culture experiments, the results revealed that the MWC scaffolds significantly promoted the MC3T3-E1 cell proliferation, differentiation, and growth into the scaffolds. The results of synchrotron radiation microcomputed tomography and analysis of the histological sections of the in vivo implantation revealed that the MWC scaffolds evidently improved the new bone formation and bone defects repair as compared with WP scaffolds. Moreover, it was found that newly formed bone tissue continued to increase with the gradual reduction of materials residual in the MWC scaffolds. Furthermore, the immunohistochemical analysis further offered the evidence of the stimulatory effects of MWC scaffolds on osteogenic-related cell differentiation and new bone regeneration. The results indicated that MWC scaffolds with good biocompability and degradability could promote osteogenesis in vivo, which would have potential for bone tissue repair. PMID:27555766

  12. Degradability, biocompatibility, and osteogenesis of biocomposite scaffolds containing nano magnesium phosphate and wheat protein both in vitro and in vivo for bone regeneration

    Science.gov (United States)

    Xia, Yan; Zhou, Panyu; Wang, Fei; Qiu, Chao; Wang, Panfeng; Zhang, Yuntong; Zhao, Liming; Xu, Shuogui

    2016-01-01

    In this study, bioactive scaffold of nano magnesium phosphate (nMP)/wheat protein (WP) composite (MWC) was fabricated. The results revealed that the MWC scaffolds had interconnected not only macropores (sized 400–600 μm) but also micropores (sized 10–20 μm) on the walls of macropores. The MWC scaffolds containing 40 w% nMP had an appropriate degradability in phosphate-buffered saline and produced a weak alkaline microenvironment. In cell culture experiments, the results revealed that the MWC scaffolds significantly promoted the MC3T3-E1 cell proliferation, differentiation, and growth into the scaffolds. The results of synchrotron radiation microcomputed tomography and analysis of the histological sections of the in vivo implantation revealed that the MWC scaffolds evidently improved the new bone formation and bone defects repair as compared with WP scaffolds. Moreover, it was found that newly formed bone tissue continued to increase with the gradual reduction of materials residual in the MWC scaffolds. Furthermore, the immunohistochemical analysis further offered the evidence of the stimulatory effects of MWC scaffolds on osteogenic-related cell differentiation and new bone regeneration. The results indicated that MWC scaffolds with good biocompability and degradability could promote osteogenesis in vivo, which would have potential for bone tissue repair. PMID:27555766

  13. Clinical Application of a Silk Fibroin Protein Biologic Scaffold for Abdominal Wall Fascial Reinforcement

    Science.gov (United States)

    Downey, Susan; Agullo, Frank; Lehfeldt, Max R.; Kind, Gabriel M.; Palladino, Humberto; Marshall, Deirdre; Jewell, Mark L.; Mathur, Anshu B.; Bengtson, Bradley P.

    2014-01-01

    Background: Preclinical studies have demonstrated that macroporous silk fibroin protein scaffolds are capable of promoting physiologically durable supportive tissue, which favors application of these engineered tissues for clinical implantation. The safety and effectiveness of a long-lasting, transitory, 510(k)-cleared purified silk fibroin biologic scaffold (SBS) are investigated for soft-tissue support and repair of the abdominal wall. Methods: We conducted a multicenter retrospective review of all consecutive patients who underwent abdominal wall soft-tissue reinforcement with an SBS device between 2011 and 2013. Indications, comorbid conditions, surgical technique, complications, and outcomes were evaluated. Results: We reviewed the records of 172 consecutive patients who received an SBS for soft-tissue support. Of those, 77 patients underwent abdominal wall fascial repair, with a mean follow-up of 18.4 ± 7.5 months. Procedures using an SBS included reinforcement of an abdominal-based flap donor site (31.2%), ventral hernia repair (53.2%), and abdominoplasty (15.6%). The overall complication rate was 6.5%, consisting of 2 wound dehiscences, 1 with device exposure, 1 seroma, 1 infection with explantation, and a perioperative bulge requiring reoperation. There were no reports of hernia. Conclusions: Postoperative complication rates after 18 months were low, and most surgical complications were managed nonoperatively on an outpatient basis without mesh removal. To our knowledge, this is the only series to report on a long-lasting, transitory SBS for abdominal wall repair and reinforcement. Procedure-specific outcome studies are warranted to delineate optimal patient selection and define potential device characteristic advantages. PMID:25506529

  14. Conformational coupling between receptor and kinase binding sites through a conserved salt bridge in a signaling complex scaffold protein.

    Directory of Open Access Journals (Sweden)

    Davi R Ortega

    Full Text Available Bacterial chemotaxis is one of the best studied signal transduction pathways. CheW is a scaffold protein that mediates the association of the chemoreceptors and the CheA kinase in a ternary signaling complex. The effects of replacing conserved Arg62 of CheW with other residues suggested that the scaffold protein plays a more complex role than simply binding its partner proteins. Although R62A CheW had essentially the same affinity for chemoreceptors and CheA, cells expressing the mutant protein are impaired in chemotaxis. Using a combination of molecular dynamics simulations (MD, NMR spectroscopy, and circular dichroism (CD, we addressed the role of Arg62. Here we show that Arg62 forms a salt bridge with another highly conserved residue, Glu38. Although this interaction is unimportant for overall protein stability, it is essential to maintain the correct alignment of the chemoreceptor and kinase binding sites of CheW. Computational and experimental data suggest that the role of the salt bridge in maintaining the alignment of the two partner binding sites is fundamental to the function of the signaling complex but not to its assembly. We conclude that a key feature of CheW is to maintain the specific geometry between the two interaction sites required for its function as a scaffold.

  15. Quantification of extracellular matrix proteins from a rat lung scaffold to provide a molecular readout for tissue engineering.

    Science.gov (United States)

    Hill, Ryan C; Calle, Elizabeth A; Dzieciatkowska, Monika; Niklason, Laura E; Hansen, Kirk C

    2015-04-01

    The use of extracellular matrix (ECM) scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs.

  16. Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors

    Directory of Open Access Journals (Sweden)

    Nir Qvit

    2016-04-01

    Full Text Available Parasitic diseases cause ∼500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK (Leishmania's receptor for activated C-kinase and TRACK (Trypanosoma receptor for activated C-kinase. We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase, may be interaction sites for signaling proteins that are critical for the parasites' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs.

  17. Second-Site Suppressors of a Cold-Sensitive External Scaffolding Protein of Bacteriophage φX174

    OpenAIRE

    Fane, B. A; Shien, S.; Hayashi, M

    1993-01-01

    This report describes the isolation and characterization of second-site suppressors of a cold-sensitive (cs) external scaffolding protein, gpD, of bacteriophage φX174. Seven genetically distinct suppressors were isolated. Six of them are located in gene F which encodes the major coat protein of the virus. The seventh is located in gene J which encodes the DNA-binding protein. A subset of the suppressors are trans-acting. These second-site suppressors do not exhibit allele specificity; they ar...

  18. Self-assembly of silk-elastinlike protein polymers into three-dimensional scaffolds for biomedical applications

    Science.gov (United States)

    Zeng, Like

    Production of brand new protein-based materials with precise control over the amino acid sequences at single residue level has been made possible by genetic engineering, through which artificial genes can be developed that encode protein-based materials with desired features. As an example, silk-elastinlike protein polymers (SELPs), composed of tandem repeats of amino acid sequence motifs from Bombyx mori (silkworm) silk and mammalian elastin, have been produced in this approach. SELPs have been studied extensively in the past two decades, however, the fundamental mechanism governing the self-assembly process to date still remains largely unresolved. Further, regardless of the unprecedented success when exploited in areas including drug delivery, gene therapy, and tissue augmentation, SELPs scaffolds as a three-dimensional cell culture model system are complicated by the inability of SELPs to provide the embedded tissue cells with appropriate biochemical stimuli essential for cell survival and function. In this dissertation, it is reported that the self-assembly of silk-elastinlike protein polymers (SELPs) into nanofibers in aqueous solutions can be modulated by tuning the curing temperature, the size of the silk blocks, and the charge of the elastin blocks. A core-sheath model was proposed for nanofiber formation, with the silk blocks in the cores and the hydrated elastin blocks in the sheaths. The folding of the silk blocks into stable cores -- affected by the size of the silk blocks and the charge of the elastin blocks -- plays a critical role in the assembly of silk-elastin nanofibers. The assembled nanofibers further form nanofiber clusters on the microscale, and the nanofiber clusters then coalesce into nanofiber micro-assemblies, interconnection of which eventually leads to the formation of three-dimensional scaffolds with distinct nanoscale and microscale features. SELP-Collagen hybrid scaffolds were also fabricated to enable independent control over the

  19. Degradability, biocompatibility, and osteogenesis of biocomposite scaffolds containing nano magnesium phosphate and wheat protein both in vitro and in vivo for bone regeneration

    Directory of Open Access Journals (Sweden)

    Xia Y

    2016-07-01

    Full Text Available Yan Xia,1 Panyu Zhou,1,* Fei Wang,2 Chao Qiu,2 Panfeng Wang,1 Yuntong Zhang,1 Liming Zhao,3 Shuogui Xu1,2 1Department of Emergency, 2Department of Orthopedics, Changhai Hospital, Second Military Medical University, 3State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: In this study, bioactive scaffold of nano magnesium phosphate (nMP/wheat protein (WP composite (MWC was fabricated. The results revealed that the MWC scaffolds had interconnected not only macropores (sized 400–600 µm but also micropores (sized 10–20 µm on the walls of macropores. The MWC scaffolds containing 40 w% nMP had an appropriate degradability in phosphate-buffered saline and produced a weak alkaline microenvironment. In cell culture experiments, the results revealed that the MWC scaffolds significantly promoted the MC3T3-E1 cell proliferation, differentiation, and growth into the scaffolds. The results of synchrotron radiation microcomputed tomography and analysis of the histological sections of the in vivo implantation revealed that the MWC scaffolds evidently improved the new bone formation and bone defects repair as compared with WP scaffolds. Moreover, it was found that newly formed bone tissue continued to increase with the gradual reduction of materials residual in the MWC scaffolds. Furthermore, the immunohistochemical analysis further offered the evidence of the stimulatory effects of MWC scaffolds on osteogenic-related cell differentiation and new bone regeneration. The results indicated that MWC scaffolds with good biocompability and degradability could promote osteogenesis in vivo, which would have potential for bone tissue repair. Keywords: nano magnesium phosphate, wheat protein, composite scaffolds, degradation, bone regeneration

  20. Hypoxia and Amino Acid Supplementation Synergistically Promote the Osteogenesis of Human Mesenchymal Stem Cells on Silk Protein Scaffolds

    OpenAIRE

    Sengupta, Sejuti; Park, Sang-Hyug; Patel, Atur; Carn, Julia; Lee, Kyongbum; Kaplan, David L.

    2010-01-01

    Tailoring tissue engineering strategies to match patient- and tissue-specific bone regeneration needs offers to improve clinical outcomes. As a step toward this goal, osteogenic outcomes and metabolic parameters were assessed when varying inputs into the bone formation process. Silk protein scaffolds seeded with human mesenchymal stem cells in osteogenic differentiation media were used to study in vitro osteogenesis under varied conditions of amino acid (lysine and proline) concentration and ...

  1. Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold.

    Directory of Open Access Journals (Sweden)

    Anna V Lomonosova

    Full Text Available Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use. Here, the 50S ribosomal RNA-binding protein L35Ae from the extremophilic archaea Pyrococcus horikoshii has been probed for its potential to serve as a backbone in alternative scaffold engineering. The recombinant wild type L35Ae has a native-like secondary structure, extreme thermal stability (mid-transition temperature of 90°C and a moderate resistance to the denaturation by guanidine hydrochloride (half-transition at 2.6 M. Chemical crosslinking and dynamic light scattering data revealed that the wild type L35Ae protein has a propensity for multimerization and aggregation correlating with its non-specific binding to a model cell surface of HEK293 cells, as evidenced by flow cytometry. To suppress these negative features, a 10-amino acid mutant (called L35Ae 10X was designed, which lacks the interaction with HEK293 cells, is less susceptible to aggregation, and maintains native-like secondary structure and thermal stability. However, L35Ae 10X also shows lowered resistance to guanidine hydrochloride (half-transition at 2.0M and is more prone to oligomerization. This investigation of an extremophile protein's scaffolding potential demonstrates that lowered resistance to charged chemical denaturants and increased propensity to multimerization may limit the utility of extremophile proteins as alternative scaffolds.

  2. CARMA3: A novel scaffold protein in regulation of NF-κB activation and diseases

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    CARD recruited membrane associated protein 3 (CARMA3) is a novel scaffold protein. It belongs to the CARMA protein family, and is known to activate nuclear factor (NF)- κB. However, it is still unknown which receptor functions upstream of CARMA3 to trigger NF-κB activation. Recently, several studies have demonstrated that CARMA3 serves as an indispensable adaptor protein in NF-κB signaling under some G protein-coupled receptors (GP- CRs), such as lysophosphatidic acid (LPA) receptor and angiotensin (Ang) Ⅱ receptor. Mechanistically, CARMA3 recruits its essential downstream molecules Bcl10 and MALT1 to form the CBM (CARMA3-Bcl10-MALT1) signalosome whereby it triggers NF-κB activation. GPCRs and NF-κB play pivotal roles in the regulation of various cellular functions, therefore, aberrant regulation of the GPCR/NF-κB signaling axis leads to the development of many types of diseases, such as cancer and atherogenesis. Recently, the GPCR/CARMA3/NF-κB signaling axis has been confirmed in these specific diseases and it plays crucial roles in the pathogenesis of disease progression. In ovarian cancer cell lines, knockdown of CARMA3 abolishes LPA receptor-induced NF-κB activation, and reduces LPA-induced ovarian cancer invasion. In vascular smooth cells, downregulation of CARMA3 substantially impairs Ang-Ⅱ-receptor-induced NF-κB activation, and in vivo studies have confirmed that Bcl10- deficient mice are protected from developing Ang-Ⅱ-receptor-induced atherosclerosis and aortic aneurysms. In this review, we summarize the biology of CARMA3, describe the role of the GPCR/CARMA3/NF-κB signaling axis in ovarian cancer and atherogenesis, and speculate about the potential roles of this signaling axis in other types of cancer and diseases. With a significant increase in the identification of LPA- and Ang-Ⅱ-like ligands, such as endothelin-1, which also activates NF-κB via CARMA3 and contributes to the development of many diseases, CARMA3 is emerging as a novel

  3. HOMER2, a stereociliary scaffolding protein, is essential for normal hearing in humans and mice.

    Directory of Open Access Journals (Sweden)

    Hela Azaiez

    2015-03-01

    Full Text Available Hereditary hearing loss is a clinically and genetically heterogeneous disorder. More than 80 genes have been implicated to date, and with the advent of targeted genomic enrichment and massively parallel sequencing (TGE+MPS the rate of novel deafness-gene identification has accelerated. Here we report a family segregating post-lingual progressive autosomal dominant non-syndromic hearing loss (ADNSHL. After first excluding plausible variants in known deafness-causing genes using TGE+MPS, we completed whole exome sequencing in three hearing-impaired family members. Only a single variant, p.Arg185Pro in HOMER2, segregated with the hearing-loss phenotype in the extended family. This amino acid change alters a highly conserved residue in the coiled-coil domain of HOMER2 that is essential for protein multimerization and the HOMER2-CDC42 interaction. As a scaffolding protein, HOMER2 is involved in intracellular calcium homeostasis and cytoskeletal organization. Consistent with this function, we found robust expression in stereocilia of hair cells in the murine inner ear and observed that over-expression of mutant p.Pro185 HOMER2 mRNA causes anatomical changes of the inner ear and neuromasts in zebrafish embryos. Furthermore, mouse mutants homozygous for the targeted deletion of Homer2 present with early-onset rapidly progressive hearing loss. These data provide compelling evidence that HOMER2 is required for normal hearing and that its sequence alteration in humans leads to ADNSHL through a dominant-negative mode of action.

  4. Designed armadillo repeat proteins as general peptide-binding scaffolds: consensus design and computational optimization of the hydrophobic core

    DEFF Research Database (Denmark)

    Parmeggiani, Fabio; Pellarin, Riccardo; Larsen, Anders Peter;

    2007-01-01

    Armadillo repeat proteins are abundant eukaryotic proteins involved in several cellular processes, including signaling, transport, and cytoskeletal regulation. They are characterized by an armadillo domain, composed of tandem armadillo repeats of approximately 42 amino acids, which mediates...... interactions with peptides or parts of proteins in extended conformation. The conserved binding mode of the peptide in extended form, observed for different targets, makes armadillo repeat proteins attractive candidates for the generation of modular peptide-binding scaffolds. Taking advantage of the large...... number of repeat sequences available, a consensus-based approach combined with a force field-based optimization of the hydrophobic core was used to derive soluble, highly expressed, stable, monomeric designed proteins with improved characteristics compared to natural armadillo proteins. These sequences...

  5. Solution structure and peptide binding of the PTB domain from the AIDA1 postsynaptic signaling scaffolding protein.

    Directory of Open Access Journals (Sweden)

    Ekaterina Smirnova

    Full Text Available AIDA1 links persistent chemical signaling events occurring at the neuronal synapse with global changes in gene expression. Consistent with its role as a scaffolding protein, AIDA1 is composed of several protein-protein interaction domains. Here we report the NMR structure of the carboxy terminally located phosphotyrosine binding domain (PTB that is common to all AIDA1 splice variants. A comprehensive survey of peptides identified a consensus sequence around an NxxY motif that is shared by a number of related neuronal signaling proteins. Using peptide arrays and fluorescence based assays, we determined that the AIDA1 PTB domain binds amyloid protein precursor (APP in a similar manner to the X11/Mint PTB domain, albeit at reduced affinity (∼10 µM that may allow AIDA1 to effectively sample APP, as well as other protein partners in a variety of cellular contexts.

  6. Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Wieczorek Andrew S

    2012-12-01

    Full Text Available Abstract Background The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes crystalline cellulose. Enzymes interact with protein scaffolds via type 1 dockerin/cohesin interactions, while scaffolds in turn bind surface anchor proteins by means of type 2 dockerin/cohesin interactions, which demonstrate a different binding specificity than their type 1 counterparts. Recombinant chimeric scaffold proteins containing cohesins of different specificity allow binding of multiple enzymes to specific sites within an engineered complex. Results We report the successful display of engineered chimeric scaffold proteins containing both type 1 and type 2 cohesins on the surface of Lactococcus lactis cells. The chimeric scaffold proteins were able to form complexes with the Escherichia coli β-glucuronidase fused to either type 1 or type 2 dockerin, and differences in binding efficiencies were correlated with scaffold architecture. We used E. coli β-galactosidase, also fused to type 1 or type 2 dockerins, to demonstrate the targeted incorporation of two enzymes into the complexes. The simultaneous binding of enzyme pairs each containing a different dockerin resulted in bi-enzymatic complexes tethered to the cell surface. The sequential binding of the two enzymes yielded insights into parameters affecting assembly of the complex such as protein size and position within the scaffold. Conclusions The spatial organization of enzymes into complexes is an important strategy for increasing the efficiency of biochemical pathways. In this study

  7. Plasma treatment induces internal surface modifications of electrospun poly(L-lactic) acid scaffold to enhance protein coating

    Energy Technology Data Exchange (ETDEWEB)

    Jin Seo, Hyok; Hee Lee, Mi; Kwon, Byeong-Ju; Kim, Hye-Lee; Park, Jong-Chul [Cellbiocontrol Laboratory, Department of Medical Engineering, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Jin Lee, Seung [Department of Industrial Pharmacy, College of Pharmacy, Ewha Womans University, Seoul 120-750 (Korea, Republic of); Kim, Bong-Jin; Wang, Kang-Kyun; Kim, Yong-Rok [Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-Gu, Seoul 120-749 (Korea, Republic of)

    2013-08-21

    Advanced biomaterials should also be bioactive with regard to desirable cellular responses, such as selective protein adsorption and cell attachment, proliferation, and differentiation. To enhance cell-material interactions, surface modifications have commonly been performed. Among the various surface modification approaches, atmospheric pressure glow discharge plasma has been used to change a hydrophobic polymer surface to a hydrophilic surface. Poly(L-lactic acid) (PLLA)-derived scaffolds lack cell recognition signals and the hydrophobic nature of PLLA hinders cell seeding. To make PLLA surfaces more conducive to cell attachment and spreading, surface modifications may be used to create cell-biomaterial interfaces that elicit controlled cell adhesion and maintain differentiated phenotypes. In this study, (He) gaseous atmospheric plasma glow discharge was used to change the characteristics of a 3D-type polymeric scaffold from hydrophobic to hydrophilic on both the outer and inner surfaces of the scaffold and the penetration efficiency with fibronectin was investigated. Field-emission scanning electron microscope images showed that some grooves were formed on the PLLA fibers after plasma treatment. X-ray photoelectron spectroscopy data also showed chemical changes in the PLLA structure. After plasma treatment, -CN (285.76 eV) was increased in C1s and -NH{sub 2} (399.70 eV) was increased significantly and –N=CH (400.80 eV) and –NH{sub 3}{sup +} (402.05 eV) were newly appeared in N1s. These changes allowed fibronectin to penetrate into the PLLA scaffold; this could be observed by confocal microscopy. In conclusion, helium atmospheric pressure plasma treatment was effective in modifying the polymeric scaffold, making it hydrophilic, and this treatment can also be used in tissue engineering research as needed to make polymers hydrophilic.

  8. Plasma treatment induces internal surface modifications of electrospun poly(L-lactic) acid scaffold to enhance protein coating

    Science.gov (United States)

    Jin Seo, Hyok; Hee Lee, Mi; Kwon, Byeong-Ju; Kim, Hye-Lee; Jin Lee, Seung; Kim, Bong-Jin; Wang, Kang-Kyun; Kim, Yong-Rok; Park, Jong-Chul

    2013-08-01

    Advanced biomaterials should also be bioactive with regard to desirable cellular responses, such as selective protein adsorption and cell attachment, proliferation, and differentiation. To enhance cell-material interactions, surface modifications have commonly been performed. Among the various surface modification approaches, atmospheric pressure glow discharge plasma has been used to change a hydrophobic polymer surface to a hydrophilic surface. Poly(L-lactic acid) (PLLA)-derived scaffolds lack cell recognition signals and the hydrophobic nature of PLLA hinders cell seeding. To make PLLA surfaces more conducive to cell attachment and spreading, surface modifications may be used to create cell-biomaterial interfaces that elicit controlled cell adhesion and maintain differentiated phenotypes. In this study, (He) gaseous atmospheric plasma glow discharge was used to change the characteristics of a 3D-type polymeric scaffold from hydrophobic to hydrophilic on both the outer and inner surfaces of the scaffold and the penetration efficiency with fibronectin was investigated. Field-emission scanning electron microscope images showed that some grooves were formed on the PLLA fibers after plasma treatment. X-ray photoelectron spectroscopy data also showed chemical changes in the PLLA structure. After plasma treatment, -CN (285.76 eV) was increased in C1s and -NH2 (399.70 eV) was increased significantly and -N=CH (400.80 eV) and -NH3+ (402.05 eV) were newly appeared in N1s. These changes allowed fibronectin to penetrate into the PLLA scaffold; this could be observed by confocal microscopy. In conclusion, helium atmospheric pressure plasma treatment was effective in modifying the polymeric scaffold, making it hydrophilic, and this treatment can also be used in tissue engineering research as needed to make polymers hydrophilic.

  9. Optimization of the silk scaffold sericin removal process for retention of silk fibroin protein structure and mechanical properties

    Energy Technology Data Exchange (ETDEWEB)

    Teh, Thomas K H; Toh, Siew-Lok; Goh, James C H, E-mail: dosgohj@nus.edu.s, E-mail: dostkh@nus.edu.s, E-mail: bietohsl@nus.edu.s [Division of Bioengineering, National University of Singapore (Singapore)

    2010-06-01

    In the process of removing sericin (degumming) from a raw silk scaffold, the fibroin structural integrity is often challenged, leading to mechanical depreciation. This study aims to identify the factors and conditions contributing to fibroin degradation during alkaline degumming and to perform an optimization study of the parameters involved to achieve preservation of fibroin structure and properties. The methodology involves degumming knitted silk scaffolds for various durations (5-90 min) and temperatures (60-100 {sup 0}C). Mechanical agitation and use of the refreshed solution during degumming are included to investigate how these factors contribute to degumming efficiency and fibroin preservation. Characterizations of silk fibroin morphology, mechanical properties and protein components are determined by scanning electron microscopy (SEM), single fiber tensile tests and gel electrophoresis (SDS-PAGE), respectively. Sericin removal is ascertained via SEM imaging and a protein fractionation method involving SDS-PAGE. The results show that fibroin fibrillation, leading to reduced mechanical integrity, is mainly caused by prolonged degumming duration. Through a series of optimization, knitted scaffolds are observed to be optimally degummed and experience negligible mechanical and structural degradation when subjected to alkaline degumming with mechanical agitation for 30 min at 100 {sup 0}C.

  10. Protein growth factors loaded highly porous chitosan scaffold: A comparison of bone healing properties

    Energy Technology Data Exchange (ETDEWEB)

    Nandi, Samit K., E-mail: samitnandi1967@gmail.com [Department of Veterinary Surgery and Radiology, West Bengal University of Animal and Fishery Sciences, Kolkata (India); Kundu, Biswanath, E-mail: biswa_kundu@rediffmail.com [Bioceramics and Coating Division, CSIR—Central Glass and Ceramic Research Institute, Kolkata (India); Basu, Debabrata [Bioceramics and Coating Division, CSIR—Central Glass and Ceramic Research Institute, Kolkata (India)

    2013-04-01

    Present study aimed to investigate and compare effectiveness of porous chitosan alone and in combination with insulin like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) in bone healing. Highly porous (85 ± 2%) with wide distribution of macroporous (70–900 μm) chitosan scaffolds were fabricated as bone substitutes by employing a simple liquid hardening method using 2% (w/v) chitosan suspension. IGF-1 and BMP-2 were infiltrated using vacuum infiltration with freeze drying method. Adsorption efficiency was found to be 87 ± 2 and 90 ± 2% for BMP-2 and IGF-1 respectively. After thorough material characterization (pore details, FTIR and SEM), samples were used for subsequent in vivo animal trial. Eighteen rabbit models were used to evaluate and compare control (chitosan) (group A), chitosan with IGF-1 (group B) and chitosan with BMP-2 (group C) in the repair of critical size bone defect in tibia. Radiologically, there was evidence of radiodensity in defect area from 60th day (initiated on 30th day) in groups B and C as compared to group A and attaining nearly bony density in most of the part at day 90. Histological results depicted well developed osteoblastic proliferation around haversian canal along with proliferating fibroblast, vascularization and reticular network which was more pronounced in group B followed by groups C and A. Fluorochrome labeling and SEM studies in all groups showed similar outcome. Hence, porous chitosan alone and in combination with growth factors (GFs) can be successfully used for bone defect healing with slight advantage of IGF-1 in chitosan samples. - Highlights: ► Fabrication and characterization of porous chitosan with or without IGF-1 and BMP-2 ► Highly porous growth factor loaded chitosan studied in animal subjects for 3 months ► Parameters studied: histopathology, radiology and fluorochrome labeling ► IGF-1 loaded porous chitosan found to be very effective for bone defect healing.

  11. Protein growth factors loaded highly porous chitosan scaffold: A comparison of bone healing properties

    International Nuclear Information System (INIS)

    Present study aimed to investigate and compare effectiveness of porous chitosan alone and in combination with insulin like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) in bone healing. Highly porous (85 ± 2%) with wide distribution of macroporous (70–900 μm) chitosan scaffolds were fabricated as bone substitutes by employing a simple liquid hardening method using 2% (w/v) chitosan suspension. IGF-1 and BMP-2 were infiltrated using vacuum infiltration with freeze drying method. Adsorption efficiency was found to be 87 ± 2 and 90 ± 2% for BMP-2 and IGF-1 respectively. After thorough material characterization (pore details, FTIR and SEM), samples were used for subsequent in vivo animal trial. Eighteen rabbit models were used to evaluate and compare control (chitosan) (group A), chitosan with IGF-1 (group B) and chitosan with BMP-2 (group C) in the repair of critical size bone defect in tibia. Radiologically, there was evidence of radiodensity in defect area from 60th day (initiated on 30th day) in groups B and C as compared to group A and attaining nearly bony density in most of the part at day 90. Histological results depicted well developed osteoblastic proliferation around haversian canal along with proliferating fibroblast, vascularization and reticular network which was more pronounced in group B followed by groups C and A. Fluorochrome labeling and SEM studies in all groups showed similar outcome. Hence, porous chitosan alone and in combination with growth factors (GFs) can be successfully used for bone defect healing with slight advantage of IGF-1 in chitosan samples. - Highlights: ► Fabrication and characterization of porous chitosan with or without IGF-1 and BMP-2 ► Highly porous growth factor loaded chitosan studied in animal subjects for 3 months ► Parameters studied: histopathology, radiology and fluorochrome labeling ► IGF-1 loaded porous chitosan found to be very effective for bone defect healing

  12. Protein adsorption and cell adhesion on three-dimensional polycaprolactone scaffolds with respect to plasma modification by etching and deposition techniques

    Science.gov (United States)

    Myung, Sung Woon; Ko, Yeong Mu; Kim, Byung Hoon

    2014-11-01

    In this work, protein adsorption and cell adhesion on three-dimensional (3D) polycaprolactone (PCL) scaffolds treated by plasma etching and deposition were performed. The 3D PCL scaffold used as a substrate of a bone tissue was fabricated by recent rapid prototype techniques. To increase surface properties, such as hydrophilicity, roughness, and surface chemistry, through good protein adhesion on scaffolds, oxygen (O2) plasma etching and acrylic acid or allyamine plasma deposition were performed on the 3D PCL scaffolds. The O2 plasma etching induced the formation of random nanoporous structures on the roughened surfaces of the 3D PCL scaffolds. The plasma deposition with acrylic acid and allyamine induced the chemical modification for introducing a functional group. The protein adsorption increased on the O2 plasma-etched surface compared with an untreated 3D PCL scaffold. MC3T3-E1 cells adhered bioactively on the etched and deposited surface compared with the untreated surface. The present plasma modification might be sought as an effective technique for enhancing protein adsorption and cell adhesion.

  13. Crystal structure of Gib2, a signal-transducing protein scaffold associated with ribosomes in Cryptococcus neoformans

    Science.gov (United States)

    Ero, Rya; Dimitrova, Valya Tenusheva; Chen, Yun; Bu, Wenting; Feng, Shu; Liu, Tongbao; Wang, Ping; Xue, Chaoyang; Tan, Suet Mien; Gao, Yong-Gui

    2015-03-01

    The atypical Gβ-like/RACK1 Gib2 protein promotes cAMP signalling that plays a central role in regulating the virulence of Cryptococcus neoformans. Gib2 contains a seven-bladed β transducin structure and is emerging as a scaffold protein interconnecting signalling pathways through interactions with various protein partners. Here, we present the crystal structure of Gib2 at a 2.2-Å resolution. The structure allows us to analyse the association between Gib2 and the ribosome, as well as to identify the Gib2 amino acid residues involved in ribosome binding. Our studies not only suggest that Gib2 has a role in protein translation but also present Gib2 as a physical link at the crossroads of various regulatory pathways important for the growth and virulence of C. neoformans.

  14. Accelerated differentiation of osteoblast cells on polycaprolactone scaffolds driven by a combined effect of protein coating and plasma modification

    Energy Technology Data Exchange (ETDEWEB)

    Yildirim, Eda D; Gueceri, Selcuk; Sun, Wei [Department of Mechanical Engineering and Mechanics, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104 (United States); Besunder, Robyn; Allen, Fred [Drexel University, School of Biomedical Engineering Science and Health System, 3141 Chestnut Street, Philadelphia, PA 19104 (United States); Pappas, Daphne, E-mail: edy22@drexel.ed [Army Research Laboratory, Aberdeen Proving Ground, MD 21005 (United States)

    2010-03-15

    A combined effect of protein coating and plasma modification on the quality of the osteoblast-scaffold interaction was investigated. Three-dimensional polycaprolactone (PCL) scaffolds were manufactured by the precision extrusion deposition (PED) system. The structural, physical, chemical and biological cues were introduced to the surface through providing 3D structure, coating with adhesive protein fibronectin and modifying the surface with oxygen-based plasma. The changes in the surface properties of PCL after those modifications were examined by contact angle goniometry, surface energy calculation, surface chemistry analysis (XPS) and surface topography measurements (AFM). The effects of modification techniques on osteoblast short-term and long-term functions were examined by cell adhesion, proliferation assays and differentiation markers, namely alkaline phosphatase activity (ALP) and osteocalcin secretion. The results suggested that the physical and chemical cues introduced by plasma modification might be sufficient for improved cell adhesion, but for accelerated osteoblast differentiation the synergetic effects of structural, physical, chemical and biological cues should be introduced to the PCL surface.

  15. Targeting diverse protein–protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold

    Energy Technology Data Exchange (ETDEWEB)

    Checco, James W.; Kreitler, Dale F.; Thomas, Nicole C.; Belair, David G.; Rettko, Nicholas J.; Murphy, William L.; Forest, Katrina T.; Gellman, Samuel H. (UW)

    2015-04-14

    Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and β-amino acid residues ("α/β-peptides") that mimic several peptides derived from the three-helix bundle "Z-domain" scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF₁₆₅-induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain–mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.

  16. The Scaffold Protein Muscle A-Kinase Anchoring Protein β Orchestrates Cardiac Myocyte Hypertrophic Signaling Required for the Development of Heart Failure

    Science.gov (United States)

    Kritzer, Michael D.; Li, Jinliang; Passariello, Catherine L.; Gayanilo, Marjorie; Thakur, Hrishikesh; Dayan, Joseph; Dodge-Kafka, Kimberly; Kapiloff, Michael S.

    2014-01-01

    Background Cardiac myocyte hypertrophy is regulated by an extensive intracellular signal transduction network. In vitro evidence suggests that the scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) serves as a nodal organizer of hypertrophic signaling. However, the relevance of mAKAPβ signalosomes to pathological remodeling and heart failure in vivo remains unknown. Methods and Results Using conditional, cardiac myocyte–specific gene deletion, we now demonstrate that mAKAPβ expression in mice is important for the cardiac hypertrophy induced by pressure overload and catecholamine toxicity. mAKAPβ targeting prevented the development of heart failure associated with long-term transverse aortic constriction, conferring a survival benefit. In contrast to 29% of control mice (n=24), only 6% of mAKAPβ knockout mice (n=31) died in the 16 weeks of pressure overload (P=0.02). Accordingly, mAKAPβ knockout inhibited myocardial apoptosis and the development of interstitial fibrosis, left atrial hypertrophy, and pulmonary edema. This improvement in cardiac status correlated with the attenuated activation of signaling pathways coordinated by the mAKAPβ scaffold, including the decreased phosphorylation of protein kinase D1 and histone deacetylase 4 that we reveal to participate in a new mAKAP signaling module. Furthermore, mAKAPβ knockout inhibited pathological gene expression directed by myocyte-enhancer factor-2 and nuclear factor of activated T-cell transcription factors that associate with the scaffold. Conclusions mAKAPβ orchestrates signaling that regulates pathological cardiac remodeling in mice. Targeting of the underlying physical architecture of signaling networks, including mAKAPβ signalosome formation, may constitute an effective therapeutic strategy for the prevention and treatment of pathological remodeling and heart failure. PMID:24812305

  17. Selective Allosteric Antagonists for the G Protein-Coupled Receptor GPRC6A Based on the 2-Phenylindole Privileged Structure Scaffold

    DEFF Research Database (Denmark)

    Johansson, Henrik; Boesgaard, Michael Worch; Nørskov-Lauritsen, Lenea;

    2015-01-01

    G protein-coupled receptors (GPCRs) represent a biological target class of fundamental importance in drug therapy. The GPRC6A receptor is a newly deorphanized class C GPCR that we recently reported for the first allosteric antagonists based on the 2-arylindole privileged structure scaffold (e.g., 1...

  18. Design, synthesis and biological profile of new inhibitors of multidrug resistance associated proteins carrying a polycyclic scaffold.

    Science.gov (United States)

    Bisi, Alessandra; Gobbi, Silvia; Merolle, Lucia; Farruggia, Giovanna; Belluti, Federica; Rampa, Angela; Molnar, Joseph; Malucelli, Emil; Cappadone, Concettina

    2015-03-01

    Following the identification of a novel polycyclic scaffold, leading to the previously reported potent P-gp modulator 1, a small series of easily affordable derivatives bearing a properly selected nitrogen-containing but-2-ynyl side chain was now synthesized and tested to evaluate the MDR reverting activity on two different experimental models. All compounds proved not to be cytotoxic when tested alone and more potent chemosensitizers than the reference verapamil. Some of them showed remarkable effects in combination with doxorubicin, being able to induce apoptotic cell death due to their reverting activity. In particular, 2a and 2c could be regarded as non-toxic new potential chemosensitizers, being able to interfere with different ABC proteins. Moreover, the intrinsic cytotoxicity of compound 1 could broaden its employment as MDR modulator. These results also seem to confirm the polycyclic core of these compounds as a potential new pharmacophoric carrier in medicinal chemistry. PMID:25596478

  19. Actin-binding proteins from Burkholderia mallei and Burkholderia thailandensis can functionally compensate for the actin-based motility defect of a Burkholderia pseudomallei bimA mutant

    OpenAIRE

    Stevens, J. M.; Ulrich, R L; Taylor, L A; Wood, M W; DeShazer, D; M.P. Stevens; Galyov, E. E.

    2005-01-01

    Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind ...

  20. Actin-Binding Proteins from Burkholderia mallei and Burkholderia thailandensis Can Functionally Compensate for the Actin-Based Motility Defect of a Burkholderia pseudomallei bimA Mutant

    OpenAIRE

    Stevens, Joanne M; Ulrich, Ricky L.; Taylor, Lowrie A.; Wood, Michael W.; DeShazer, David; Stevens, Mark P.; Galyov, Edouard E.

    2005-01-01

    Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind ...

  1. Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2

    OpenAIRE

    Motawea, Hanaa K. B.; Selvi C. Jeyaraj; Eid, Ali H; Mitra, Srabani; Unger, Nicholas T.; Ahmed, Amany A. E.; Flavahan, Nicholas A.; Chotani, Maqsood A.

    2013-01-01

    The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expressio...

  2. The Chromatin Scaffold Protein SAFB1 Renders Chromatin Permissive for DNA Damage Signaling

    DEFF Research Database (Denmark)

    Altmeyer, Matthias; Toledo Lazaro, Luis Ignacio; Gudjonsson, Thorkell;

    2013-01-01

    Although the general relevance of chromatin modifications for genotoxic stress signaling, cell-cycle checkpoint activation, and DNA repair is well established, how these modifications reach initial thresholds in order to trigger robust responses remains largely unexplored. Here, we identify...... the chromatin-associated scaffold attachment factor SAFB1 as a component of the DNA damage response and show that SAFB1 cooperates with histone acetylation to allow for efficient γH2AX spreading and genotoxic stress signaling. SAFB1 undergoes a highly dynamic exchange at damaged chromatin in a poly......(ADP-ribose)-polymerase 1- and poly(ADP-ribose)-dependent manner and is required for unperturbed cell-cycle checkpoint activation and guarding cells against replicative stress. Altogether, our data reveal that transient recruitment of an architectural chromatin component is required in order to overcome physiological...

  3. Recombinant Bone Morphogenetic Protein 2 Stimulates the Remodeling Chitosan-Based Porous Scaffold Into Hyaline-like Cartilage: Study in Heterotopic Implantation

    Directory of Open Access Journals (Sweden)

    Nurshat M. Gaifullin

    2013-09-01

    Full Text Available To study the morphology of remodeling the chitosan-based three-dimensional porous scaffold, containing bone morphogenetic protein-2 (BMP-2 for chondroinduction, the experiments with heterotopic implantation using 28 Wistar rats were carried out. Scaffolds with growth factor (n=12 or without it (n=12, against intact control (n=4 were implanted subcutaneously. Classical methods of histology and morphometry as well as immune histochemical markers (CD-68, CD-31, MMP-9, TIMP-1, and osteonectin expression, one used to investigate zone of remodeling in euthanized animals at 4 and 8 weeks after implantation. The BMP-2 application provides more intensive and rapid new cartilage formation from the scaffold matter. The additional chondroinductive effect proved more intensive settlement and proliferation of chondral cells in the regenerate, expression of chondral phenotype with the building the hyaline-like matrix, and the supporting necessary balance between the matrix metalloproteinases and their tissue inhibitors.

  4. Control of MAPK signaling specificity by a conserved residue in the MEK-binding domain of the yeast scaffold protein Ste5.

    Science.gov (United States)

    Schwartz, Monica A; Madhani, Hiten D

    2006-06-01

    The yeast kinase scaffold Ste5 has been proposed to prevent unwanted cross-talk between the pheromone response pathway and other MAPK cascades. Protein fusion experiments have demonstrated that covalently tethering signaling components to each other or to Ste5 can determine the outcome of signaling. However, these do not fully test the role of scaffolds in signaling specificity, since fusing components precludes differential dissociation of subpopulations. We performed a targeted genetic screen on STE5 and repeatedly identified recessive mutations in a conserved residue, E756, in the Ste7/MEK-binding domain that caused erroneous activation of the filamentation MAPK pathway by pheromone signaling. Mutant cells exhibited a shift in the MAPK activation pattern such that the filamentation MAPK Kss1 was predominately activated in response to pheromone. Velocity sedimentation studies showed that the mutant scaffold was defective in binding to a phosphorylated subpopulation of Ste7. Our data suggest that increased dissociation of activated Ste7 kinase from the mutant scaffold may cause the observed shift in MAPK activation from Fus3 to Kss1 and the resulting loss of specificity. Cross-talk in ste5-E756G cells was due to both increased activation of Kss1 and reduced Fus3-dependent degradation of the filamentation pathway transcription factor Tec1. These studies demonstrate a role for an endogenous scaffold in signaling specificity. PMID:16463042

  5. Multiplexed multi-scale imaging: novel roles for the scaffold protein IQGAP1 in epithelial cell development (Conference Presentation)

    Science.gov (United States)

    Schweikhard, Volker

    2016-02-01

    The precise sub-cellular spatial localization of multi-protein complexes is increasingly recognized as a key mechanism governing the organization of mammalian cells. Consequently, there is a need for novel microscopy techniques capable of investigating such sub-cellular architectures in comprehensive detail. Here, we applied a novel multiplexed STORM super-resolution microscopy technique, in combination with high-throughput immunofluorescence microscopy and live-cell imaging, to investigate the roles of the scaffold protein IQGAP1 in epithelial cells. IQGAP1 is known to orchestrate a wide range of biological processes, including intracellular signaling, cytoskeletal regulation, cell-cell adhesion, and protein trafficking, by forming distinct complexes with a number of known interaction partners, and recruiting these complexes to specific subcellular locations. Our results demonstrate that, in addition to supporting epithelial adherens junctions by associating with specialized cortical actin structures, IQGAP1 plays a second role in which it controls the confinement of a unique, previously undocumented class of membranous compartments to the basal actin cortex. These largely immotile yet highly dynamic structures appear transiently as cells merge into clusters and establish of apical-basolateral (epithelial) polarity, and are identified as an intermediate compartment in the endocytic recycling pathways for cell junction complexes and cell surface receptors. Although these two functions of IQGAP1 occur in parallel and largely independently of each other, they both support the maturation and maintenance of polarized epithelial cell architectures.

  6. In vivo evaluation of whey protein-based biofilms as scaffolds for cutaneous cell cultures and biomedical applications

    International Nuclear Information System (INIS)

    This study evaluated the toxicity, biodegradability and immunogenicity of newly developed whey protein-based biofilms for possible use as biomaterials for medical applications. Biofilms were prepared using (A) a whey protein isolate plasticized with either diethylene glycol (DEG) or glycerol (GLY), and (B) β-lactoglobulin (βLGA) plasticized with DEG. The biofilms were implanted subcutaneously into Balb/c mice. Analyses were performed at various time points. At 15, 30 and 60 days post-implantation, no necrotic zones or exudates were present at the recipient sites. The biofilms began to degrade as early as 15 days post-implantation, as evidenced by erosion and crumbling. The macroscopic observations were supported by tissue analyses revealing no tissue necrosis or degradation and confirming that the biodegradation of the biofilms began as early as 15 days post-implantation and was almost complete after 60 days. The biodegradation was accompanied by significant leukocyte infiltration at 15 days which significantly decreased at 60 days. The absence of splenomagaly in the implanted mice confirms that these biofilms were not immunogenic. Whey protein-based biofilms are biocompatible and biodegradable and may be of interest for medical applications such as scaffolds for cutaneous cell cultures and skin recovery in burn patients

  7. Composites containing albumin protein or cyanoacrylate adhesives and biodegradable scaffolds: II. In vivo wound closure study in a rat model

    Science.gov (United States)

    McNally-Heintzelman, Karen M.; Heintzelman, Douglas L.; Duffy, Mark T.; Bloom, Jeffrey N.; Soller, Eric C.; Gilmour, Travis M.; Hoffman, Grant T.; Edward, Deepak

    2004-07-01

    Our Scaffold-Enhanced Biological Adhesive (SEBA) system was investigated as an alternative to sutures or adhesives alone for repair of wounds. Two scaffold materials were investigated: (i) a synthetic biodegradable material fabricated from poly(L-lactic-co-glycolic acid); and (ii) a biologic material, small intestinal submucosa, manufactured by Cook BioTech. Two adhesive materials were also investigated: (i) a biologic adhesive composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser; and (ii) Ethicon"s Dermabond, a 2-octyl-cyanoacrylate. The tensile strength and time-to-failure of skin incisions repaired in vivo in a rat model were measured at seven days postoperative. Incisions closed by protein solder alone, by Dermabond alone, or by suture, were also tested for comparison. The tensile strength of repairs formed using the SEBA system were 50% to 65% stronger than repairs formed by suture or either adhesive alone, with significantly less variations within each experimental group (average standard deviations of 15% for SEBA versus 38% for suture and 28% for adhesive alone). In addition, the time-to-failure curves showed a longevity not previously seen with the suture or adhesive alone techniques. The SEBA system acts to keep the dermis in tight apposition during the critical early phase of wound healing when tissue gaps are bridged by scar and granulation tissue. It has the property of being more flexible than either of the adhesives alone and may allow the apposed edges to move in conjunction with each other as a unit for a longer period of time and over a greater range of stresses than adhesives alone. This permits more rapid healing and establishment of integrity since the microgaps between the dermis edges are significantly reduced. By the time the scaffolds are sloughed from the wound site, there is greater strength and healing than that produced by adhesive alone or

  8. The therapeutic effect of monocyte chemoattractant protein-1 delivered by an electrospun scaffold for hyperglycemia and nephrotic disorders

    Directory of Open Access Journals (Sweden)

    Yong C

    2014-02-01

    Full Text Available Cai Yong,2,* Zhengxin Wang,1,* Xing Zhang,3 Xiaomin Shi,1 Zhijia Ni,1 Hong Fu,1 Guoshan Ding,1 Zhiren Fu,1 Hao Yin1,3 1Department of Surgery, Organ Transplant Center, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, People's Republic of China; 2Department of Transplantation, First Affiliated Hospital of Wenzhou Medical College, Wenzhou, People's Republic of China; 3Department of Surgery, University of Chicago, Chicago, IL, USA *These authors contributed equally to this article Abstract: Here, we investigated in diabetic mice the therapeutic effect of monocyte chemoattractant protein-1 (MCP-1, locally delivered by an electrospun scaffold, on transplanted islets. This therapeutic scheme is expected to exert a synergistic effect to ameliorate hyperglycemia and its associated nephrotic disorders. The cumulative amount of MCP-1 released from the scaffold in vitro within a 3-week window was 267.77±32.18 ng, without a compromise in bioactivity. After 8 weeks following the transplantation, the islet population stimulated by MCP-1 was 35.14%±7.23% larger than the non-stimulated islet population. Moreover, MCP-1 increased concentrations of blood insulin and C-peptide 2 by 49.83%±5.29% and 43.49%±9.21%, respectively. Consequently, the blood glucose concentration in the MCP-1 group was significantly lower than that in the control group at week 2 post-surgery. MCP-1 also enhanced the tolerance of sudden oral glucose challenge. The rapid decrease of blood creatinine, urine creatinine, and blood urea nitrogen suggested that the recovery of renal functions compromised by hyperglycemia could also be attributed to MCP-1. Our study shed new light on a synergistic strategy to alleviate hyperglycemia and nephrotic disorders in diabetic patients. Keywords: MCP-1, electrospinning, islet transplantation, diabetes

  9. A novel 18F-labeled two-helix scaffold protein for PET imaging of HER2-positive tumor

    International Nuclear Information System (INIS)

    Two-helix scaffold proteins (∝ 5 kDa) against human epidermal growth factor receptor type 2 (HER2) have been discovered in our previous work. In this research we aimed to develop an 18F-labeled two-helix scaffold protein for positron emission tomography (PET) imaging of HER2-positive tumors. An aminooxy-functionalized two-helix peptide (AO-MUT-DS) with high HER2 binding affinity was synthesized through conventional solid phase peptide synthesis. The purified linear peptide was cyclized by I2 oxidation to form a disulfide bridge. The cyclic peptide was then conjugated with a radiofluorination synthon, 4-18F-fluorobenzyl aldehyde (18F-FBA), through the aminooxy functional group at the peptide N terminus (30% yield, non-decay corrected). The binding affinities of the peptides were analyzed by Biacore analysis. Cell uptake assay of the resulting PET probe, 18F-FBO-MUT-DS, was performed at 37 C. 18F-FBO-MUT-DS with high specific activity (20-32 MBq/nmol, 88-140 μCi/μg, end of synthesis) was injected into mice xenograft model bearing SKOV3 tumor. MicroPET and biodistribution and metabolic stability studies were then conducted. Cell uptake assays showed high and specific cell uptake (∝12% applied activity at 1 h) by incubation of 18F-FBO-MUT-DS with HER2 high-expressing SKOV3 ovarian cancer cells. The affinities (KD) of AO-MUT-DS and FBO-MUT-DS as tested by Biacore analysis were 2 and 1 nM, respectively. In vivo small animal PET demonstrated fast tumor targeting, high tumor accumulation, and good tumor to normal tissue contrast of 18F-FBO-MUT-DS. Biodistribution studies further revealed that the probe had excellent tumor uptake (6.9%ID/g at 1 h post-injection) and was cleared through both liver and kidneys. Co-injection of the probe with 500 μg of HER2 Affibody protein reduced the tumor uptake (6.9 vs 1.8%ID/g, p 18F-based PET probes. (orig.)

  10. Fibrinogen and fibrin based micro and nano scaffolds incorporated with drugs, proteins, cells and genes for therapeutic biomedical applications

    Directory of Open Access Journals (Sweden)

    Rajangam T

    2013-09-01

    Full Text Available Thanavel Rajangam, Seong Soo A An Department of Bionanotechnology, Gachon University, Seongnam-Si, Republic of Korea Abstract: Over the past two decades, many types of natural and synthetic polymer-based micro- and nanocarriers, with exciting properties and applications, have been developed for application in various types of tissue regeneration, including bone, cartilage, nerve, blood vessels, and skin. The development of suitable polymers scaffold designs to aid the repair of specific cell types have created diverse and important potentials in tissue restoration. Fibrinogen (Fbg- and fibrin (Fbn-based micro- and nanostructures can provide suitable natural matrix environments. Since these primary materials are abundantly available in blood as the main coagulation proteins, they can easily interact with damaged tissues and cells through native biochemical interactions. Fbg- and Fbn-based micro and nanostructures can also be consecutively furnished/or encapsulated and specifically delivered, with multiple growth factors, proteins, and stem cells, in structures designed to aid in specific phases of the tissue regeneration process. The present review has been carried out to demonstrate the progress made with micro and nanoscaffold applications and features a number of applications of Fbg- and Fbn-based carriers in the field of biomaterials, including the delivery of drugs, active biomolecules, cells, and genes, that have been effectively used in tissue engineering and regenerative medicine. Keywords: biomaterial, polymer composite, cross-linking, growth factor, drug delivery, controlled release, tissue regeneration

  11. Urolithin as a converging scaffold linking ellagic acid and coumarin analogues: design of potent protein kinase CK2 inhibitors.

    Science.gov (United States)

    Cozza, Giorgio; Gianoncelli, Alessandra; Bonvini, Paolo; Zorzi, Elisa; Pasquale, Riccardo; Rosolen, Angelo; Pinna, Lorenzo A; Meggio, Flavio; Zagotto, Giuseppe; Moro, Stefano

    2011-12-01

    Casein kinase 2 (CK2) is a ubiquitous, essential, and highly pleiotropic protein kinase; its abnormally high constitutive activity is suspected to underlie its pathogenic potential in neoplasia and other relevant diseases. Previously, using different in silico screening approaches, two potent and selective CK2 inhibitors were identified by our group: ellagic acid, a naturally occurring tannic acid derivative (K(i)=20 nM) and 3,8-dibromo-7-hydroxy-4-methylchromen-2-one (DBC, K(i)=60 nM). Comparing the crystallographic binding modes of both ellagic acid and DBC, an X-ray structure-driven merging approach was taken to design novel CK2 inhibitors with improved target affinity. A urolithin moiety is proposed as a possible bridging scaffold between the two known CK2 inhibitors, ellagic acid and DBC. Optimization of urolithin A as the bridging moiety led to the identification of 4-bromo-3,8-dihydroxy-benzo[c]chromen-6-one as a novel, potent and selective CK2 inhibitor, which shows a K(i) value of 7 nM against the protein kinase, representing a significant improvement in affinity for the target compared with the two parent fragments. PMID:21972104

  12. Interplay between self-assembled structure of bone morphogenetic protein-2 (BMP-2) and osteoblast functions in three-dimensional titanium alloy scaffolds: Stimulation of osteogenic activity.

    Science.gov (United States)

    Nune, K C; Kumar, A; Murr, L E; Misra, R D K

    2016-02-01

    Three-dimensional cellular scaffolds are receiving significant attention in bone tissue engineering to treat segmental bone defects. However, there are indications of lack of significant osteoinductive ability of three-dimensional cellular scaffolds. In this regard, the objective of the study is to elucidate the interplay between bone morphogenetic protein (BMP-2) and osteoblast functions on 3D mesh structures with different porosities and pore size that were fabricated by electron beam melting. Self-assembled dendritic microstructure with interconnected cellular-type morphology of BMP-2 on 3D scaffolds stimulated osteoblast functions including adhesion, proliferation, and mineralization, with prominent effect on 2-mm mesh. Furthermore, immunofluorescence studies demonstrated higher density and viability of osteoblasts on lower porosity mesh structure (2 mm) as compared to 3- and 4-mm mesh structures. Enhanced filopodia cellular extensions with extensive cell spreading was observed on BMP-2 treated mesh structures, a behavior that is attributed to the unique self-assembled structure of BMP-2 that effectively communicates with the cells. The study underscores the potential of BMP-2 in imparting osteoinductive capability to the 3D printed scaffolds.

  13. The Use of Platelet Rich Plasma, Bone Morphogenetic Protein-2 and Different Scaffolds in Oral and Maxillofacial Surgery - Literature Review in Comparison with Own Clinical Experience

    OpenAIRE

    Karl-Heinz Schuckert; Stefan Jopp; Magdalena Osadnik

    2011-01-01

    ABSTRACT Objectives The purpose of this article was to review and critically assess the use of platelet rich plasma, recombinant human bone morphogenetic protein-2 and different scaffolds (i.e. tricalciumphosphate, polycaprolactone, demineralized bone matrix and anorganic bovine bone mineral) in oral and maxillofacial surgery comparing the relevant literature and own clinical experience. Material and Methods A literature review was conducted using MEDLINE, MEDPILOT and COCHRANE DATABASE OF SY...

  14. Fluorescent Magnesium Nanocomplex in Protein Scaffold for Cell Nuclei Imaging Application

    Energy Technology Data Exchange (ETDEWEB)

    Pandya, Alok [Ahmedabad Univ. (India); Tripathi, Apritam [Ahmedabad Univ. (India); Purohit, Rahul [Ahmedabad Univ. (India); Singh, Sanjay [Ahmedabad Univ. (India); Nandasiri, Manjula I. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Karakoti, Ajay S. [Ahmedabad Univ. (India); Singh, Surinder P. [National Physical Lab., New Delhi (India); Shanker, Rishi [Ahmedabad Univ. (India)

    2015-10-27

    Here in, we report a facile strategy for the synthesis of water-soluble ultra-fine blue emitting fluorescent Magnesium nanoparticles-protein complex (MgNC). This MgNC is demonstrated to exhibit excellent photo stability and biocompatibility. It was also observed that MgNC stain cell nuclei with high specifcity.

  15. Scaffolding during the cell cycle by A-kinase anchoring proteins

    NARCIS (Netherlands)

    Han, B; Poppinga, W J; Schmidt, M

    2015-01-01

    Cell division relies on coordinated regulation of the cell cycle. A process including a well-defined series of strictly regulated molecular mechanisms involving cyclin-dependent kinases, retinoblastoma protein, and polo-like kinases. Dysfunctions in cell cycle regulation are associated with disease

  16. Structural insights into yeast histone chaperone Hif1: a scaffold protein recruiting protein complexes to core histones.

    Science.gov (United States)

    Liu, Hejun; Zhang, Mengying; He, Wei; Zhu, Zhongliang; Teng, Maikun; Gao, Yongxiang; Niu, Liwen

    2014-09-15

    Yeast Hif1 [Hat1 (histone acetyltransferase 1)-interacting factor], a homologue of human NASP (nuclear autoantigenic sperm protein), is a histone chaperone that is involved in various protein complexes which modify histones during telomeric silencing and chromatin reassembly. For elucidating the structural basis of Hif1, in the present paper we demonstrate the crystal structure of Hif1 consisting of a superhelixed TPR (tetratricopeptide repeat) domain and an extended acid loop covering the rear of TPR domain, which represent typical characteristics of SHNi-TPR [Sim3 (start independent of mitosis 3)-Hif1-NASP interrupted TPR] proteins. Our binding assay indicates that Hif1 could bind to the histone octamer via histones H3 and H4. The acid loop is shown to be crucial for the binding of histones and may also change the conformation of the TPR groove. By binding to the core histone complex Hif1 may recruit functional protein complexes to modify histones during chromatin reassembly.

  17. Distinct and overlapping functions of the cullin E3 ligase scaffolding proteins CUL4A and CUL4B.

    Science.gov (United States)

    Hannah, Jeffrey; Zhou, Pengbo

    2015-11-15

    The cullin 4 subfamily of genes includes CUL4A and CUL4B, which share a mostly identical amino acid sequence aside from the elongated N-terminal region in CUL4B. Both act as scaffolding proteins for modular cullin RING ligase 4 (CRL4) complexes which promote the ubiquitination of a variety of substrates. CRL4 function is vital to cells as loss of both genes or their shared substrate adaptor protein DDB1 halts proliferation and eventually leads to cell death. Due to their high structural similarity, CUL4A and CUL4B share a substantial overlap in function. However, in some cases, differences in subcellular localization, spatiotemporal expression patterns and stress-inducibility preclude functional compensation. In this review, we highlight the most essential functions of the CUL4 genes in: DNA repair and replication, chromatin-remodeling, cell cycle regulation, embryogenesis, hematopoiesis and spermatogenesis. CUL4 genes are also clinically relevant as dysregulation can contribute to the onset of cancer and CRL4 complexes are often hijacked by certain viruses to promote viral replication and survival. Also, mutations in CUL4B have been implicated in a subset of patients suffering from syndromic X-linked intellectual disability (AKA mental retardation). Interestingly, the antitumor effects of immunomodulatory drugs are caused by their binding to the CRL4CRBN complex and re-directing the E3 ligase towards the Ikaros transcription factors IKZF1 and IKZF3. Because of their influence over key cellular functions and relevance to human disease, CRL4s are considered promising targets for therapeutic intervention. PMID:26344709

  18. Phthalocyanines as Molecular Scaffolds to Block Disease-Associated Protein Aggregation.

    Science.gov (United States)

    Valiente-Gabioud, Ariel A; Miotto, Marco C; Chesta, María E; Lombardo, Verónica; Binolfi, Andres; Fernández, Claudio O

    2016-05-17

    The aggregation of proteins into toxic conformations plays a critical role in the development of different neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Creutzfled-Jakob's disease (CJD). These disorders share a common pathological mechanism that involves the formation of aggregated protein species including toxic oligomers and amyloid fibrils. The aggregation of alpha-synuclein (αS) in PD and the amyloid beta peptide (Aβ) and tau protein in AD results in neuronal death and disease onset. In the case of CJD, the misfolding of the physiological prion protein (PrP) induces a chain reaction that results in accumulation of particles that elicit brain damage. Currently, there is no preventive therapy for these diseases and the available therapeutic approaches are based on the treatment of the symptoms rather than the underlying causes of the disease. Accordingly, the aggregation pathway of these proteins represents a useful target for therapeutic intervention. Therefore, understanding the mechanism of amyloid formation and its inhibition is of high clinical importance. The design of small molecules that efficiently inhibit the aggregation process and/or neutralize its associated toxicity constitutes a promising tool for the development of therapeutic strategies against these disorders. In this accounts, we discuss current knowledge on the anti-amyloid activity of phthalocyanines and their potential use as drug candidates in neurodegeneration. These tetrapyrrolic compounds modulate the amyloid assembly of αS, tau, Aβ, and the PrP in vitro, and protect cells from the toxic effects of amyloid aggregates. In addition, in scrapie-infected mice, these compounds showed important prophylactic antiscrapie properties. The structural basis for the inhibitory effect of phthalocyanines on amyloid filament assembly relies on specific π-π interactions between the aromatic ring system of these molecules and aromatic residues in the

  19. Incorporation of fibronectin to enhance cytocompatibility in multilayer elastin-like protein scaffolds for tissue engineering

    OpenAIRE

    Ravi, Swathi; Caves, Jeffrey M.; Martinez, Adam W.; Haller, Carolyn A.; Chaikof, Elliot L.

    2012-01-01

    Recombinant, elastin-like protein (ELP) polymers are of significant interest for the engineering of compliant, resilient soft tissues due to a wide range of tunable mechanical properties, biostability, and biocompatibility. Here, we enhance endothelial cell (EC) and mesenchymal stem cell compatibility with ELP constructs by addition of fibronectin (Fn) to the surface or bulk of ELP hydrogels. We find that cell adhesion, proliferation, and migration can be modulated by Fn addition. Adsorption ...

  20. Scaffolding during the cell cycle by A-kinase anchoring proteins

    OpenAIRE

    Han, B.; Poppinga, W J; Schmidt, M.

    2015-01-01

    Cell division relies on coordinated regulation of the cell cycle. A process including a well-defined series of strictly regulated molecular mechanisms involving cyclin-dependent kinases, retinoblastoma protein, and polo-like kinases. Dysfunctions in cell cycle regulation are associated with disease such as cancer, diabetes, and neurodegeneration. Compartmentalization of cellular signaling is a common strategy used to ensure the accuracy and efficiency of cellular responses. Compartmentalizati...

  1. Scaffold Proteins Regulating Extracellular Regulated Kinase Function in Cardiac Hypertrophy and Disease

    OpenAIRE

    Liang, Yan; Sheikh, Farah

    2016-01-01

    The mitogen activated protein kinase (MAPK)-extracellular regulated kinase 1/2 (ERK1/2) pathway is a central downstream signaling pathway that is activated in cardiac muscle cells during mechanical and agonist-mediated hypertrophy. Studies in genetic mouse models deficient in ERK-associated MAPK components pathway have further reinforced a direct role for this pathway in stress-induced cardiac hypertrophy and disease. However, more recent studies have highlighted that these signaling pathways...

  2. The promotion of osteochondral repair by combined intra-articular injection of parathyroid hormone-related protein and implantation of a bi-layer collagen-silk scaffold.

    Science.gov (United States)

    Zhang, Wei; Chen, Jialin; Tao, Jiadong; Hu, Changchang; Chen, Longkun; Zhao, Hongshi; Xu, Guowei; Heng, Boon C; Ouyang, Hong Wei

    2013-08-01

    The repair of osteochondral defects can be enhanced with scaffolds but is often accompanied with undesirable terminal differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Parathyroid hormone-related protein (PTHrP) has been shown to inhibit aberrant differentiation, but administration at inappropriate time points would have adverse effects on chondrogenesis. This study aims to develop an effective tissue engineering strategy by combining PTHrP and collagen-silk scaffold for osteochondral defect repair. The underlying mechanisms of the synergistic effect of combining PTHrP administration with collagen-silk scaffold implantation for rabbit knee joint osteochondral defect repair were investigated. In vitro studies showed that PTHrP treatment significantly reduced Alizarin Red staining and expression of terminal differentiation-related markers. This is achieved in part through blocking activation of the canonical Wnt/β-catenin signaling pathway. For the in vivo repair study, intra-articular injection of PTHrP was carried out at three different time windows (4-6, 7-9 and 10-12 weeks) together with implantation of a bi-layer collagen-silk scaffold. Defects treated with PTHrP at the 4-6 weeks time window exhibited better regeneration (reconstitution of cartilage and subchondral bone) with minimal terminal differentiation (hypertrophy, ossification and matrix degradation), as well as enhanced chondrogenesis (cell shape, Col2 and GAG accumulation) compared with treatment at other time windows. Furthermore, the timing of PTHrP administration also influenced PTHrP receptor expression, thus affecting the treatment outcome. Our results demonstrated that intra-articular injection of PTHrP at 4-6 weeks post-injury together with collagen-silk scaffold implantation is an effective strategy for inhibiting terminal differentiation and enhancing chondrogenesis, thus improving cartilage repair and regeneration in a rabbit model. PMID:23702148

  3. Comparative study of osteogenic potential of a composite scaffold incorporating either endogenous bone morphogenetic protein-2 or exogenous phytomolecule icaritin: an in vitro efficacy study.

    Science.gov (United States)

    Chen, S-H; Wang, X-L; Xie, X-H; Zheng, L-Z; Yao, D; Wang, D-P; Leng, Y; Zhang, G; Qin, L

    2012-08-01

    A local delivery system with sustained and efficient release of therapeutic agents from an appropriate carrier is desirable for orthopedic applications. Novel composite scaffolds made of poly (lactic-co-glycolic acid) with tricalcium phosphate (PLGA/TCP) were fabricated by an advanced low-temperature rapid prototyping technique, which incorporated either endogenous bone morphogenetic protein-2 (BMP-2) (PLGA/TCP/BMP-2) or phytomolecule icaritin (ICT) (PLGA/TCP/ICT) at low, middle and high doses. PLGA/TCP served as control. In vitro degradation, osteogenesis and release tests showed statistical differences among PLGA/TCP/ICT, PLGA/TCP and PLGA/TCP/BMP-2 groups, where PLGA/TCP/ICT had the desired slow release of bioactive icaritin in a dose-dependent manner, whereas there was almost no BMP-2 release from the PLGA/TCP/BMP-2 scaffolds. PLGA/TCP/ICT significantly increased more ALP activity, upregulated mRNA expression of osteogenic genes and enhanced calcium deposition and mineralization in rabbit bone marrow stem cells cultured on scaffolds compared with the other two groups. These results indicate the desired degradation rate, osteogenic capability and release property in PLGA/TCP/ICT composite scaffold, as icaritin preserved its bioactivity and structure after incorporation, while PLGA/TCP/BMP-2 did not show an initially expected osteogenic potential, owing to loss of the original bioactivity of BMP-2 during its incorporation and fabrication procedure. The results suggest that PLGA/TCP composite scaffolds incorporating osteogenic ICT might be a promising approach for bone tissue bioengineering and regeneration. PMID:22543006

  4. Actin-organising properties of the muscular dystrophy protein myotilin.

    Science.gov (United States)

    von Nandelstadh, Pernilla; Grönholm, Mikaela; Moza, Monica; Lamberg, Arja; Savilahti, Harri; Carpén, Olli

    2005-10-15

    Myotilin is a sarcomeric Z-disc protein that binds F-actin directly and bundles actin filaments, although it does not contain a conventional actin-binding domain. Expression of mutant myotilin leads to sarcomeric alterations in the dominantly inherited limb-girdle muscular dystrophy 1A and in myofibrillar myopathy/desmin-related myopathy. Together, with previous in vitro studies, this indicates that myotilin has an important function in the assembly and maintenance of Z-discs. This study characterises further the interaction between myotilin and actin. Functionally important regions in myotilin were identified by actin pull-down and yeast two-hybrid assays and with a novel strategy that combines in vitro DNA transposition-based peptide insertion mutagenesis with phenotype analysis in yeast cells. The shortest fragment to bind actin was the second Ig domain together with a short C-terminal sequence. Concerted action of the first and second Ig domain was, however, necessary for the functional activity of myotilin, as verified by analysis of transposon mutants, actin binding and phenotypic effect in mammalian cells. Furthermore, the Ig domains flanked with N- and C-terminal regions were needed for actin-bundling, indicating that the mere actin-binding sequence was insufficient for the actin-regulating activity. None of the four known disease-associated mutations altered the actin-organising ability. These results, together with previous studies in titin and kettin, identify the Ig domain as an actin-binding unit.

  5. Sla1p Is a Functionally Modular Component of the Yeast Cortical Actin Cytoskeleton Required for Correct Localization of Both Rho1p-GTPase and Sla2p, a Protein with Talin Homology

    OpenAIRE

    Ayscough, Kathryn R.; Eby, Jennifer J.; Lila, Thomas; Dewar, Hilary; Kozminski, Keith G.; Drubin, David G.

    1999-01-01

    SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization. Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches. Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated wi...

  6. Development of Scaffolds for Light Harvesting and Photocatalysis from the Coat Protein of Tobacco Mosaic Virus

    Science.gov (United States)

    Dedeo, Michel Toussaint

    The utility of a previously developed TMV-based light harvesting system has been dramatically expanded through the introduction of reactive handles for the site-specific modification of the interior and exterior surfaces. Further experiments to reengineer the coat protein have produced structures with unique, unexpected, and useful assembly properties that complement the newly available surface modifications. Energy transfer from chromophores in the RNA channel of self-assembled TMV structures to the exterior was made possible by conjugation of acceptor dyes and porphyrins to the N-terminus. By repositioning the N-terminus to the pore through circular permutation, this process was repeated to create structures that mimic the light harvesting 1 complex of photosynthetic bacteria. To study and improve upon natural photosynthesis, closely packed chromophore arrays and gold nanoparticles were tethered to the pore of stabilized TMV disks through introduction of a uniquely reactive lysine. Finally, a dimeric TMV coat protein was produced to control the distribution and arrangement of synthetic groups with synergistic activity.

  7. Design of pH sensitive binding proteins from the hyperthermophilic Sso7d scaffold.

    Directory of Open Access Journals (Sweden)

    Nimish Gera

    Full Text Available We have engineered pH sensitive binding proteins for the Fc portion of human immunoglobulin G (hIgG (hFc using two different strategies - histidine scanning and random mutagenesis. We obtained an hFc-binding protein, Sso7d-hFc, through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus; Sso7d-hFc was isolated from a combinatorial library of Sso7d mutants using yeast surface display. Subsequently, we identified a pH sensitive mutant, Sso7d-his-hFc, through systematic evaluation of Sso7d-hFc mutants containing single histidine substitutions. In parallel, we also developed a yeast display screening strategy to isolate a different pH sensitive hFc binder, Sso7d-ev-hFc, from a library of mutants obtained by random mutagenesis of a pool of hFc binders. In contrast to Sso7d-hFc, both Sso7d-his-hFc and Sso7d-ev-hFc have a higher binding affinity for hFc at pH 7.4 than at pH 4.5. The Sso7d-mutant hFc binders can be recombinantly expressed at high yield in E. coli and are monomeric in solution. They bind an epitope in the CH3 domain of hFc that has high sequence homology in all four hIgG isotypes (hIgG(1-4, and recognize hIgG(1-4 as well as deglycosylated hIgG in western blotting assays. pH sensitive hFc binders are attractive candidates for use in chromatography, to achieve elution of IgG under milder pH conditions. However, the surface density of immobilized hFc binders, as well as the avidity effect arising from the multivalent interaction of dimeric hFc with the capture surface, influences the pH dependence of dissociation from the capture surface. Therefore, further studies are needed to evaluate if the Sso7d mutants identified in this study are indeed useful as affinity ligands in chromatography.

  8. Carbon nanotube interaction with extracellular matrix proteins producing scaffolds for tissue engineering

    Directory of Open Access Journals (Sweden)

    Tonelli FM

    2012-08-01

    Full Text Available Fernanda MP Tonelli,1 Anderson K Santos,1 Katia N Gomes,2 Eudes Lorençon,2 Silvia Guatimosim,3 Luiz O Ladeira,2 Rodrigo R Resende11Cell Signaling and Nanobiotechnology Laboratory, Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, Brazil; 2Nanomaterials Laboratory, Department of Physics, Federal University of Minas Gerais, Belo Horizonte, Brazil; 3Intracellular Cardiomiocyte Signaling Laboratory, Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, BrazilAbstract: In recent years, significant progress has been made in organ transplantation, surgical reconstruction, and the use of artificial prostheses to treat the loss or failure of an organ or bone tissue. In recent years, considerable attention has been given to carbon nanotubes and collagen composite materials and their applications in the field of tissue engineering due to their minimal foreign-body reactions, an intrinsic antibacterial nature, biocompatibility, biodegradability, and the ability to be molded into various geometries and forms such as porous structures, suitable for cell ingrowth, proliferation, and differentiation. Recently, grafted collagen and some other natural and synthetic polymers with carbon nanotubes have been incorporated to increase the mechanical strength of these composites. Carbon nanotube composites are thus emerging as potential materials for artificial bone and bone regeneration in tissue engineering.Keywords: carbon nanotubes, tissue engineering, extracellular matrix proteins, collagen, hyaluronic acid, stem cells

  9. Composites containing albumin protein or cyanoacrylate adhesives and biodegradable scaffolds: I. Acute wound closure study in a rat model

    Science.gov (United States)

    Hoffman, Grant T.; Soller, Eric C.; Heintzelman, Douglas L.; Duffy, Mark T.; Bloom, Jeffrey N.; Gilmour, Travis M.; Gonnerman, Krista N.; McNally-Heintzelman, Karen M.

    2004-07-01

    Composite adhesives composed of biodegradable scaffolds impregnated with a biological or synthetic adhesive were investigated for use in wound closure as an alternative to using either one of the adhesives alone. Two different scaffold materials were investigated: (i) a synthetic biodegradable material fabricated from poly(L-lactic-co-glycolic acid); and (ii) a biological material, small intestinal sub mucosa, manufactured by Cook BioTech. The biological adhesive was composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser. The synthetic adhesive was Ethicon's Dermabond, a 2-octyl-cyanoacrylate. The tensile strength of skin incisions repaired ex vivo in a rat model, by adhesive alone or in combination with a scaffold, as well as the time-to-failure, were measured and compared. The tensile strength of repairs formed using the scaffold-enhanced biological adhesives were on average, 80% stronger than their non-enhanced counterparts, with an accompanying increase in the time-to-failure of the repairs. These results support the theory that a scaffold material with an irregular surface that bridges the wound provides a stronger, more durable and consistent adhesion, due to the distribution of the tensile stress forces over the many micro-adhesions provided by the irregular surface, rather than the one large continuous adhesive contact. This theory is also supported by several previous ex vivo experiments demonstrating enhanced tensile strength of irregular versus smooth scaffold surfaces in identical tissue repairs performed on bovine thoracic aorta, liver, spleen, small intestine and lung tissue.

  10. Designs for the self-assembly of open and closed macromolecular structures and a molecular switch using DNA methyltransferases to order proteins on nucleic acid scaffolds

    Science.gov (United States)

    Smith, Steven S.

    2002-06-01

    The methyltransferase-directed addressing of fusion proteins to DNA scaffolds offers an approach to the construction of protein/nucleic acid biostructures with potential in a variety of applications. The technology is currently only limited by the yield of high occupancy structures. However, current evidence shows that DNA scaffolds that contain three or four targeted proteins can be reliably constructed. This permits a variety of macromolecular designs, several of which are given in this paper. Designs for open and closed two-dimensional and three-dimensional assemblies and a design for a molecular switch are discussed. The closed two-dimensional assembly takes the form of a square, and could find application as a component of other systems including a macromolecular rotaxane. The closed three-dimensional system takes the form of a trigonal bipyramid and could find application as a macromolecular carcerand. The molecular switch could find application as a peptide biosensor. Guidelines for the construction and structural verification of these designs are reported.

  11. The Use of Platelet Rich Plasma, Bone Morphogenetic Protein-2 and Different Scaffolds in Oral and Maxillofacial Surgery - Literature Review in Comparison with Own Clinical Experience

    Directory of Open Access Journals (Sweden)

    Karl-Heinz Schuckert

    2011-01-01

    Full Text Available Objectives: The purpose of this article was to review and critically assess the use of platelet rich plasma, recombinant human bone morphogenetic protein-2 and different scaffolds (i.e. tricalciumphosphate, polycaprolactone, demineralized bone matrix and anorganic bovine bone mineral in oral and maxillofacial surgery comparing the relevant literature and own clinical experience.Material and Methods: A literature review was conducted using MEDLINE, MEDPILOT and COCHRANE DATABASE OF SYSTEMATIC REVIEWS. It concentrated on manuscripts and overviews published in the last five years (2006-2010. The key terms employed were platelet rich plasma, bone morphogenetic proteins and their combinations with the above mentioned scaffolds. The results of clinical studies and animal trials were especially emphasized. The statements from the literature were compared with authors’ own clinical data.Results: New publications and overviews demonstrate the advantages of platelet rich plasma in bone regeneration. The results from the literature review were discussed and compared with the publications detailing authors’ own experiences.Conclusions: A favourable outcome concerning newly grown bone was achieved combining platelet rich plasma in addition to optimal matrices with or without recombinant human bone morphogenetic protein-2, depending on the clinical case. As a consequence, the paradigm shift from transplantation of autogenous bone to bone tissue engineering appears promising.

  12. Microgravity as a means to incorporate HepG2 aggregates in polysaccharide-protein hybrid scaffold.

    Science.gov (United States)

    Sarika, P R; James, Nirmala Rachel; Anilkumar, P R; Raj, Deepa K; Kumary, T V

    2016-02-01

    Tissue culture under microgravity provides a venue which promotes cell-cell association while avoiding the detrimental effects of high shear stress. Hepatocytes cultured on carriers or entrapped within matrices under simulated microgravity conditions showed improved cell function and proliferation. In the present study, a new approach was adopted where a non-cell adherent scaffold was incorporated with hepatospheroids (HepG2) under microgravity. Gum arabic (GA) was cross-linked with gelatin (GA-Gel) and collagen (GA-Col) to prepare non-cell adherent scaffolds. Microgravity experiments with GA-Gel and GA-Col indicated that GA-Col is a better substrate compared to GA-Gel. Microgravity experiments of GA-Col scaffolds with HepG2 cells confirmed that the non-adherent surface with porous architecture can incorporate hepatocyte spheroids and maintain liver specific functions. Albumin and urea synthesis of hepatocytes was sustained up to 6 days under microgravity conditions in the presence of GA-Col scaffold. This new approach of using non-cell adherent matrix and microgravity environment for developing biological substitutes will be beneficial in tissue engineering, bioartificial liver devices and in vitro safety assessment of drugs. PMID:26704544

  13. Induction of bone formation in biphasic calcium phosphate scaffolds by bone morphogenetic protein-2 and primary osteoblasts.

    Science.gov (United States)

    Strobel, L A; Rath, S N; Maier, A K; Beier, J P; Arkudas, A; Greil, P; Horch, R E; Kneser, U

    2014-03-01

    Bone tissue engineering strategies mainly depend on porous scaffold materials. In this study, novel biphasic calcium phosphate (BCP) matrices were generated by 3D-printing. High porosity was achieved by starch consolidation. This study aimed to characterise the porous BCP-scaffold properties and interactions of osteogenic cells and growth factors under in vivo conditions. Five differently treated constructs were implanted subcutaneously in syngeneic rats: plain BCP constructs (group A), constructs pre-treated with BMP-2 (group B; 1.6 µg BMP-2 per scaffold), seeded with primary osteoblasts (OB) (group C), seeded with OB and BMP-2 (group D) and constructs seeded with OB and pre-cultivated in a flow bioreactor for 6 weeks (group E). After 2, 4 and 6 weeks, specimens were explanted and subjected to histological and molecular biological analyses. Explanted scaffolds were invaded by fibrovascular tissue without significant foreign body reactions. Morphometric analysis demonstrated significantly increased bone formation in samples from group D (OB + BMP-2) compared to all other groups. Samples from groups B-E displayed significant mRNA expression of bone-specific genes after 6 weeks. Pre-cultivation in the flow bioreactor (group E) induced bone formation comparable with group B. In this study, differences in bone distribution between samples with BMP-2 or osteoblasts could be observed. In conclusion, combination of osteoblasts and BMP-2 synergistically enhanced bone formation in novel ceramic scaffolds. These results provide the basis for further experiments in orthotopic defect models with a focus on future applications in orthopaedic and reconstructive surgery.

  14. Determination of the critical residues responsible for cardiac myosin binding protein C's interactions.

    Science.gov (United States)

    Bhuiyan, Md Shenuarin; Gulick, James; Osinska, Hanna; Gupta, Manish; Robbins, Jeffrey

    2012-12-01

    Despite early demonstrations of myosin binding protein C's (MyBP-C) interaction with actin, different investigators have reached different conclusions regarding the relevant and necessary domains mediating this binding. Establishing the detailed structure-function relationships is needed to fully understand cMyBP-C's ability to impact on myofilament contraction as mutations in different domains are causative for familial hypertrophic cardiomyopathy. We defined cMyBP-C's N-terminal structural domains that are necessary or sufficient to mediate interactions with actin and/or the head region of the myosin heavy chain (S2-MyHC). Using a combination of genetics and functional assays, we defined the actin binding site(s) present in cMyBP-C. We confirmed that cMyBP-C's C1 and m domains productively interact with actin, while S2-MyHC interactions are restricted to the m domain. Using residue-specific mutagenesis, we identified the critical actin binding residues and distinguished them from the residues that were critical for S2-MyHC binding. To validate the structural and functional significance of these residues, we silenced the endogenous cMyBP-C in neonatal rat cardiomyocytes (NRC) using cMyBP-C siRNA, and replaced the endogenous cMyBP-C with normal or actin binding-ablated cMyBP-C. Replacement with actin binding-ablated cMyBP-C showed that the mutated protein did not incorporate into the sarcomere normally. Residues responsible for actin and S2-MyHC binding are partially present in overlapping domains but are unique. Expression of an actin binding-deficient cMyBP-C resulted in abnormal cytosolic distribution of the protein, indicating that interaction with actin is essential for the formation and/or maintenance of normal cMyBP-C sarcomeric distribution.

  15. Mechanical properties, biological activity and protein controlled release by poly(vinyl alcohol)–bioglass/chitosan–collagen composite scaffolds: A bone tissue engineering applications

    Energy Technology Data Exchange (ETDEWEB)

    Pon-On, Weeraphat, E-mail: fsciwpp@ku.ac.th [Department of Physics, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand); Charoenphandhu, Narattaphol; Teerapornpuntakit, Jarinthorn; Thongbunchoo, Jirawan; Krishnamra, Nateetip [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University (Thailand); Department of Physiology, Faculty of Science, Mahidol University (Thailand); Tang, I-Ming [ThEP Center, Commission of Higher Education, 328 Si Ayutthaya Rd. (Thailand); Department of Materials Science, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand)

    2014-05-01

    In the present study, composite scaffolds made with different weight ratios (0.5:1, 1:1 and 2:1) of bioactive glass (15Ca:80Si:5P) (BG)/polyvinyl alcohol (PVA) (PVABG) and chitosan (Chi)/collagen (Col) (ChiCol) were prepared by three mechanical freeze–thaw followed by freeze-drying to obtain the porous scaffolds. The mechanical properties and the in vitro biocompatibility of the composite scaffolds to simulated body fluid (SBF) and to rat osteoblast-like UMR-106 cells were investigated. The results from the studies indicated that the porosity and compressive strength were controlled by the weight ratio of PVABG:ChiCol. The highest compressive modulus of the composites made was 214.64 MPa which was for the 1:1 weight ratio PVABG:ChiCol. Mineralization study in SBF showed the formation of apatite crystals on the PVABG:ChiCol surface after 7 days of incubation. In vitro cell availability and proliferation tests confirmed the osteoblast attachment and growth on the PVABG:ChiCol surface. MTT and ALP tests on the 1:1 weight ratio PVABG:ChiCol composite indicated that the UMR-106 cells were viable. Alkaline phosphatase activity was found to increase with increasing culturing time. In addition, we showed the potential of PVABG:ChiCol drug delivery through PBS solution studies. 81.14% of BSA loading had been achieved and controlled release for over four weeks was observed. Our results indicated that the PVABG:ChiCol composites, especially the 1:1 weight ratio composite exhibited significantly improved mechanical, mineral deposition, biological properties and controlled release. This made them potential candidates for bone tissue engineering applications. - Graphical abstract: Mechanical properties, biological activity and protein controlled release by poly(vinyl alcohol)–bioglass/chitosan–collagen composite scaffolds: A bone tissue engineering applications. - Highlights: • Preparation of PVABG:ChiCol hybrid composites and their bioactivities • Mechanical

  16. Mechanical properties, biological activity and protein controlled release by poly(vinyl alcohol)–bioglass/chitosan–collagen composite scaffolds: A bone tissue engineering applications

    International Nuclear Information System (INIS)

    In the present study, composite scaffolds made with different weight ratios (0.5:1, 1:1 and 2:1) of bioactive glass (15Ca:80Si:5P) (BG)/polyvinyl alcohol (PVA) (PVABG) and chitosan (Chi)/collagen (Col) (ChiCol) were prepared by three mechanical freeze–thaw followed by freeze-drying to obtain the porous scaffolds. The mechanical properties and the in vitro biocompatibility of the composite scaffolds to simulated body fluid (SBF) and to rat osteoblast-like UMR-106 cells were investigated. The results from the studies indicated that the porosity and compressive strength were controlled by the weight ratio of PVABG:ChiCol. The highest compressive modulus of the composites made was 214.64 MPa which was for the 1:1 weight ratio PVABG:ChiCol. Mineralization study in SBF showed the formation of apatite crystals on the PVABG:ChiCol surface after 7 days of incubation. In vitro cell availability and proliferation tests confirmed the osteoblast attachment and growth on the PVABG:ChiCol surface. MTT and ALP tests on the 1:1 weight ratio PVABG:ChiCol composite indicated that the UMR-106 cells were viable. Alkaline phosphatase activity was found to increase with increasing culturing time. In addition, we showed the potential of PVABG:ChiCol drug delivery through PBS solution studies. 81.14% of BSA loading had been achieved and controlled release for over four weeks was observed. Our results indicated that the PVABG:ChiCol composites, especially the 1:1 weight ratio composite exhibited significantly improved mechanical, mineral deposition, biological properties and controlled release. This made them potential candidates for bone tissue engineering applications. - Graphical abstract: Mechanical properties, biological activity and protein controlled release by poly(vinyl alcohol)–bioglass/chitosan–collagen composite scaffolds: A bone tissue engineering applications. - Highlights: • Preparation of PVABG:ChiCol hybrid composites and their bioactivities • Mechanical

  17. A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission.

    Science.gov (United States)

    Bardwell, A J; Flatauer, L J; Matsukuma, K; Thorner, J; Bardwell, L

    2001-03-30

    The recognition of mitogen-activated protein kinases (MAPKs) by their upstream activators, MAPK/ERK kinases (MEKs), is crucial for the effective and accurate transmission of many signals. We demonstrated previously that the yeast MAPKs Kss1 and Fus3 bind with high affinity to the N terminus of the MEK Ste7, and proposed that a conserved motif in Ste7, the MAPK-docking site, mediates this interaction. Here we show that the corresponding sequences in human MEK1 and MEK2 are necessary and sufficient for the direct binding of the MAPKs ERK1 and ERK2. Mutations in MEK1, MEK2, or Ste7 that altered conserved residues in the docking site diminished binding of the cognate MAPKs. Furthermore, short peptides corresponding to the docking sites in these MEKs inhibited MEK1-mediated phosphorylation of ERK2 in vitro. In yeast cells, docking-defective alleles of Ste7 were modestly compromised in their ability to transmit the mating pheromone signal. This deficiency was dramatically enhanced when the ability of the Ste5 scaffold protein to associate with components of the MAPK cascade was also compromised. Thus, both the MEK-MAPK docking interaction and binding to the Ste5 scaffold make mutually reinforcing contributions to the efficiency of signaling by this MAPK cascade in vivo. PMID:11134045

  18. Locomotor proteins in tissues of primary tumors and metastases of ovarian and breast cancer

    Science.gov (United States)

    Kondakova, I. V.; Yunusova, N. V.; Spirina, L. V.; Shashova, E. E.; Kolegova, E. S.; Kolomiets, L. A.; Slonimskaya, E. M.; Villert, A. B.

    2016-08-01

    The paper discusses the capability for active movement in an extracellular matrix, wherein remodeling of the cytoskeleton by actin binding proteins plays a significant role in metastases formation. We studied the expression of actin binding proteins and β-catenin in tissues of primary tumors and metastases of ovarian and breast cancer. Contents of p45 Ser β-catenin and the actin severing protein gelsolin were decreased in metastases of ovarian cancer relative to primary tumors. The level of the cofilin, functionally similar to gelsolin, was significantly higher in metastases compared to primary ovarian and breast tumor tissue. In breast cancer, significant increase in the number of an actin monomer binder protein thymosin-β4 was observed in metastases as compared to primary tumors. The data obtained suggest the involvement of locomotor proteins in metastases formation in ovarian and breast cancer.

  19. Semiotic scaffolding

    DEFF Research Database (Denmark)

    Hoffmeyer, Jesper

    2015-01-01

    Life processes at all levels (from the genetic to the behavioral) are coordinated by semiotic interactions between cells, tissues, membranes, organs, or individuals and tuned through evolution to stabilize important functions. A stabilizing dynamics based on a system of semiotic scaffoldings...... implies that genes do not control the life of organisms, they merely scaffold it. The nature-nurture dynamics is thus far more complex and open than is often claimed. Contrary to physically based interactions, semiotic interactions do not depend on any direct causal connection between the sign vehicle...... (the representamen) and the effect. Semiotic interaction patterns therefore provide fast and versatile mechanisms for adaptations, mechanisms that depend on communication and “learning” rather than on genetic preformation. Seen as a stabilizing agency supporting the emergence of higher-order structure...

  20. Structural and biochemical characterization of human PR70 in isolation and in complex with the scaffolding subunit of protein phosphatase 2A.

    Directory of Open Access Journals (Sweden)

    Rebecca Dovega

    Full Text Available Protein Phosphatase 2A (PP2A is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A subunit, a catalytic (C subunit and various regulatory (B subunits. Here we report a 2.0 Å crystal structure of the free B''/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B''/PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B''/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B'' containing holoenzymes.

  1. Testosterone delivered with a scaffold is as effective as bone morphologic protein-2 in promoting the repair of critical-size segmental defect of femoral bone in mice.

    Directory of Open Access Journals (Sweden)

    Bi-Hua Cheng

    Full Text Available Loss of large bone segments due to fracture resulting from trauma or tumor removal is a common clinical problem. The goal of this study was to evaluate the use of scaffolds containing testosterone, bone morphogenetic protein-2 (BMP-2, or a combination of both for treatment of critical-size segmental bone defects in mice. A 2.5-mm wide osteotomy was created on the left femur of wildtype and androgen receptor knockout (ARKO mice. Testosterone, BMP-2, or both were delivered locally using a scaffold that bridged the fracture. Results of X-ray imaging showed that in both wildtype and ARKO mice, BMP-2 treatment induced callus formation within 14 days after initiation of the treatment. Testosterone treatment also induced callus formation within 14 days in wildtype but not in ARKO mice. Micro-computed tomography and histological examinations revealed that testosterone treatment caused similar degrees of callus formation as BMP-2 treatment in wildtype mice, but had no such effect in ARKO mice, suggesting that the androgen receptor is required for testosterone to initiate fracture healing. These results demonstrate that testosterone is as effective as BMP-2 in promoting the healing of critical-size segmental defects and that combination therapy with testosterone and BMP-2 is superior to single therapy. Results of this study may provide a foundation to develop a cost effective and efficient therapeutic modality for treatment of bone fractures with segmental defects.

  2. Tetrameric far-red fluorescent protein as a scaffold to assemble an octavalent peptide nanoprobe for enhanced tumor targeting and intracellular uptake in vivo.

    Science.gov (United States)

    Luo, Haiming; Yang, Jie; Jin, Honglin; Huang, Chuan; Fu, Jianwei; Yang, Fei; Gong, Hui; Zeng, Shaoqun; Luo, Qingming; Zhang, Zhihong

    2011-06-01

    Relatively weak tumor affinities and short retention time in vivo hinder the application of targeting peptides in tumor molecular imaging. Multivalent strategies based on various scaffolds have been utilized to improve the ability of peptide-receptor binding or extend the clearance time of peptide-based probes. Here, we use a tetrameric far-red fluorescent protein (tfRFP) as a scaffold to create a self-assembled octavalent peptide fluorescent nanoprobe (Octa-FNP) using a genetic engineering approach. The multiligand connecting, fluorophore labeling and nanostructure formation of Octa-FNP were performed in one step. In vitro studies showed Octa-FNP is a 10-nm fluorescent probe with excellent serum stability. Cellular uptake of Octa-FNP by human nasopharyngeal cancer 5-8F cells is 15-fold of tetravalent probe, ∼80-fold of monovalent probe and ∼600-fold of nulvalent tfRFP. In vivo enhanced tumor targeting and intracellular uptake of Octa-FNP were confirmed using optical imaging and Western blot analysis. It achieved extremely high contrast of Octa-FNP signal between tumor tissue and normal organs, especially seldom Octa-FNP detected in liver and spleen. Owing to easy preparation, precise structural and functional control, and multivalent effect, Octa-FNP provides a powerful tool for tumor optical molecular imaging and evaluating the targeting ability of numerous peptides in vivo.

  3. Neuronal Networks on Nanocellulose Scaffolds.

    Science.gov (United States)

    Jonsson, Malin; Brackmann, Christian; Puchades, Maja; Brattås, Karoline; Ewing, Andrew; Gatenholm, Paul; Enejder, Annika

    2015-11-01

    Proliferation, integration, and neurite extension of PC12 cells, a widely used culture model for cholinergic neurons, were studied in nanocellulose scaffolds biosynthesized by Gluconacetobacter xylinus to allow a three-dimensional (3D) extension of neurites better mimicking neuronal networks in tissue. The interaction with control scaffolds was compared with cationized nanocellulose (trimethyl ammonium betahydroxy propyl [TMAHP] cellulose) to investigate the impact of surface charges on the cell interaction mechanisms. Furthermore, coatings with extracellular matrix proteins (collagen, fibronectin, and laminin) were investigated to determine the importance of integrin-mediated cell attachment. Cell proliferation was evaluated by a cellular proliferation assay, while cell integration and neurite propagation were studied by simultaneous label-free Coherent anti-Stokes Raman Scattering and second harmonic generation microscopy, providing 3D images of PC12 cells and arrangement of nanocellulose fibrils, respectively. Cell attachment and proliferation were enhanced by TMAHP modification, but not by protein coating. Protein coating instead promoted active interaction between the cells and the scaffold, hence lateral cell migration and integration. Irrespective of surface modification, deepest cell integration measured was one to two cell layers, whereas neurites have a capacity to integrate deeper than the cell bodies in the scaffold due to their fine dimensions and amoeba-like migration pattern. Neurites with lengths of >50 μm were observed, successfully connecting individual cells and cell clusters. In conclusion, TMAHP-modified nanocellulose scaffolds promote initial cellular scaffold adhesion, which combined with additional cell-scaffold treatments enables further formation of 3D neuronal networks. PMID:26398224

  4. Neuronal Networks on Nanocellulose Scaffolds.

    Science.gov (United States)

    Jonsson, Malin; Brackmann, Christian; Puchades, Maja; Brattås, Karoline; Ewing, Andrew; Gatenholm, Paul; Enejder, Annika

    2015-11-01

    Proliferation, integration, and neurite extension of PC12 cells, a widely used culture model for cholinergic neurons, were studied in nanocellulose scaffolds biosynthesized by Gluconacetobacter xylinus to allow a three-dimensional (3D) extension of neurites better mimicking neuronal networks in tissue. The interaction with control scaffolds was compared with cationized nanocellulose (trimethyl ammonium betahydroxy propyl [TMAHP] cellulose) to investigate the impact of surface charges on the cell interaction mechanisms. Furthermore, coatings with extracellular matrix proteins (collagen, fibronectin, and laminin) were investigated to determine the importance of integrin-mediated cell attachment. Cell proliferation was evaluated by a cellular proliferation assay, while cell integration and neurite propagation were studied by simultaneous label-free Coherent anti-Stokes Raman Scattering and second harmonic generation microscopy, providing 3D images of PC12 cells and arrangement of nanocellulose fibrils, respectively. Cell attachment and proliferation were enhanced by TMAHP modification, but not by protein coating. Protein coating instead promoted active interaction between the cells and the scaffold, hence lateral cell migration and integration. Irrespective of surface modification, deepest cell integration measured was one to two cell layers, whereas neurites have a capacity to integrate deeper than the cell bodies in the scaffold due to their fine dimensions and amoeba-like migration pattern. Neurites with lengths of >50 μm were observed, successfully connecting individual cells and cell clusters. In conclusion, TMAHP-modified nanocellulose scaffolds promote initial cellular scaffold adhesion, which combined with additional cell-scaffold treatments enables further formation of 3D neuronal networks.

  5. Compound soft regenerated skull material for repairing dog skull defects using bone morphogenetic protein as an inductor and nanohydroxyapatite as a scaffold

    Institute of Scientific and Technical Information of China (English)

    Zhidong Shi; Mingwang Liu; Zhongzong Qin; Qinmei Wang; Ying Guo; Haiyong He; Zhonghe Yu

    2008-01-01

    BACKGROUND: In previous studies of skull defects and regeneration, bone morphogenetic protein as an inductor and nanohydroxyapatite as a scaffold have been cocultured with osteoblasts.OBJECTIVE: To verify the characteristics of the new skull regenerated material after compound soft regenerated skull material implantatiom.DESIGN, TIME AND SETTING: The self-control and inter-group control animal experiment was perfurmed at the Sun Yat-sen University, China from February to July 2007.MATERIALS: Twenty-tour healthy adult dogs of both genders weighing 15-20 kg were used in this study. Nanohydroxyapatite as a scaffold was cocultured with osteoblasts. Using demineralized canine bone matrix as a carrier, recombinant human bone morphogenetic protein-2 was employed to prepare compound soft regenerated skull material. Self-designed compound soft regenerated skull material was implanted in models of skull defects.METHODS: Animals were randomly assigned into two groups, Group A (n = 16) and Group B (n = 8).Bilateral 2.5-cm-diameter full-thickness parietal skull defects were made in all animals. In Group A, the right side was reconstructed with calcium alginate gel, osteoblasts, and nanomcter bone meal composite;the left side was reconstructed with calcium alginate gel, osteoblasts, nanometer bone meal and recombinant human bone morphogenetic protein-2 composite. In Group B, the right side was kept as a simple skull detect, and the left side was reconstructed with calcium alginate gel, osteoblasts, nanometer bone meal and recombinant human bone morphogenetic protein-2 composite.MAIN OUTCOME MEASURES: Bone regeneration and histopathological changes at the site of the skull defect were observed with an optical microscope and a scanning electron microscope after surgery.The ability to form bone was measured by alizarin red S staining. In vitro cultured osteoblasts were observed for morphology.RESULTS: One month following surgery, newly formed bone trabeculae mostly covered the

  6. Developmental Scaffolding

    DEFF Research Database (Denmark)

    Giorgi, Franco; Bruni, Luis Emilio

    2015-01-01

    The concept of scaffolding has wide resonance in several scientific fields. Here we attempt to adopt it for the study of development. In this perspective, the embryo is conceived as an integral whole, comprised of several hierarchical modules as in a recurrent circularity of emerging patterns...... molecular signalling to the complexity of sign recognition proper of a cellular community. In this semiotic perspective, the apparent goal directness of any developmental strategy should no longer be accounted for by a predetermined genetic program, but by the gradual definition of the relationships...

  7. The αRep artificial repeat protein scaffold: a new tool for crystallization and live cell applications.

    Science.gov (United States)

    Valerio-Lepiniec, Marie; Urvoas, Agathe; Chevrel, Anne; Guellouz, Asma; Ferrandez, Yann; Mesneau, Agnès; de la Sierra-Gallay, Ines Li; Aumont-Nicaise, Magali; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe

    2015-10-01

    We have designed a new family of artificial proteins, named αRep, based on HEAT (acronym for Huntingtin, elongation factor 3 (EF3), protein pphosphatase 2A (PP2A), yeast kinase Tor1) repeat proteins containing an α-helical repeated motif. The sequence of the repeated motifs, first identified in a thermostable archae protein was optimized using a consensus design strategy and used for the construction of a library of artificial proteins. All proteins from this library share the same general fold but differ both in the number of repeats and in five highly randomized amino acid positions within each repeat. The randomized side chains altogether provide a hypervariable surface on αRep variants. Sequences from this library are efficiently expressed as soluble, folded and very stable proteins. αRep binders with high affinity for various protein targets were selected by phage display. Low micromolar to nanomolar dissociation constants between partners were measured and the structures of several complexes (specific αRep/protein target) were solved by X-ray crystallography. Using GFP as a model target, it was demonstrated that αReps can be used as bait in pull-down experiments. αReps can be expressed in eukaryotic cells and specifically interact with their target addressed to different cell compartments.

  8. The αRep artificial repeat protein scaffold: a new tool for crystallization and live cell applications.

    Science.gov (United States)

    Valerio-Lepiniec, Marie; Urvoas, Agathe; Chevrel, Anne; Guellouz, Asma; Ferrandez, Yann; Mesneau, Agnès; de la Sierra-Gallay, Ines Li; Aumont-Nicaise, Magali; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe

    2015-10-01

    We have designed a new family of artificial proteins, named αRep, based on HEAT (acronym for Huntingtin, elongation factor 3 (EF3), protein pphosphatase 2A (PP2A), yeast kinase Tor1) repeat proteins containing an α-helical repeated motif. The sequence of the repeated motifs, first identified in a thermostable archae protein was optimized using a consensus design strategy and used for the construction of a library of artificial proteins. All proteins from this library share the same general fold but differ both in the number of repeats and in five highly randomized amino acid positions within each repeat. The randomized side chains altogether provide a hypervariable surface on αRep variants. Sequences from this library are efficiently expressed as soluble, folded and very stable proteins. αRep binders with high affinity for various protein targets were selected by phage display. Low micromolar to nanomolar dissociation constants between partners were measured and the structures of several complexes (specific αRep/protein target) were solved by X-ray crystallography. Using GFP as a model target, it was demonstrated that αReps can be used as bait in pull-down experiments. αReps can be expressed in eukaryotic cells and specifically interact with their target addressed to different cell compartments. PMID:26517888

  9. The Role of Actin-Capping Protein and Src signalling in tissue growth and apoptosis during Drosophila wing development

    OpenAIRE

    Jezowska, Barbara Zofia

    2012-01-01

    Dissertation presented to obtain the Ph.D degree in Developmental Biology The actin cytoskeleton controls numerous cellular processes, including cell morphology and polarity, endocytosis, intracellular trafficking, contractility and cell division. Actin filament growth, stability and disassembly are controlled by a plethora of actin-binding proteins. Among them Capping Protein is a highly conserved αβ heterodimer, which binds the barbed ends of actin filaments, inhibiting addit...

  10. VPS29 is not an active metallo-phosphatase but is a rigid scaffold required for retromer interaction with accessory proteins.

    Directory of Open Access Journals (Sweden)

    James D Swarbrick

    Full Text Available VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn(2+ and Zn(2+ in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies.

  11. Two distinct domains of protein 4.1 critical for assembly offunctional nuclei in Vitro

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Sharon Wald; Heald, Rebecca; Lee, Gloria; Nunomura, Wataru; Gimm,J. Aura; Mohandas, Narla; Chasis, Joel AnneJ. Aura; Mohandas, Narla; Chasis, Joel Anne

    2002-11-15

    Protein 4.1R, a multifunctional structural protein, acts asan adaptor in mature red cell membrane skeletons linking spectrin-actincomplexes to plasma membrane-associated proteins. In nucleated cellsprotein 4.1 is not associated exclusively with plasma membrane but isalso detected at several important subcellular locations crucial for celldivision. To identify 4.1 domains having critical functions in nuclearassembly, 4.1 domain peptides were added to Xenopus egg extract nuclearreconstitution reactions. Morphologically disorganized, replicationdeficient nuclei assembled when spectrin-actin binding domain orNuMA-binding C-terminal domain peptides were present. However, controlvariant spectrin-actin binding domain peptides incapable of bindingactin, or mutant C-terminal domain peptides with reduced NuMA binding,had no deleterious effects on nuclear reconstitution. To test if 4.1 isrequired for proper nuclear assembly, 4.1 isoforms were depleted withspectrin-actin binding or C-terminal domain-specific antibodies. Nucleiassembled in depleted extracts ha d deranged phenotypes. However, nuclearassembly could be rescued by addition of recombinant 4.1R. Our dataestablishes that protein 4.1 is essential for nuclear assembly andidentifies two distinct 4.1 domains, initially characterized incytoskeletal interactions, that have crucial and versatile functions innuclear assembly.

  12. Scaffolder - software for manual genome scaffolding

    Directory of Open Access Journals (Sweden)

    Barton Michael D

    2012-05-01

    Full Text Available Abstract Background The assembly of next-generation short-read sequencing data can result in a fragmented non-contiguous set of genomic sequences. Therefore a common step in a genome project is to join neighbouring sequence regions together and fill gaps. This scaffolding step is non-trivial and requires manually editing large blocks of nucleotide sequence. Joining these sequences together also hides the source of each region in the final genome sequence. Taken together these considerations may make reproducing or editing an existing genome scaffold difficult. Methods The software outlined here, “Scaffolder,” is implemented in the Ruby programming language and can be installed via the RubyGems software management system. Genome scaffolds are defined using YAML - a data format which is both human and machine-readable. Command line binaries and extensive documentation are available. Results This software allows a genome build to be defined in terms of the constituent sequences using a relatively simple syntax. This syntax further allows unknown regions to be specified and additional sequence to be used to fill known gaps in the scaffold. Defining the genome construction in a file makes the scaffolding process reproducible and easier to edit compared with large FASTA nucleotide sequences. Conclusions Scaffolder is easy-to-use genome scaffolding software which promotes reproducibility and continuous development in a genome project. Scaffolder can be found at http://next.gs.

  13. Assembly of the novel five-component apicomplexan multi-aminoacyl-tRNA synthetase complex is driven by the hybrid scaffold protein Tg-p43.

    Directory of Open Access Journals (Sweden)

    Jason M van Rooyen

    Full Text Available In Toxoplasma gondii, as in other eukaryotes, a subset of the amino-acyl-tRNA synthetases are arranged into an abundant cytoplasmic multi-aminoacyl-tRNA synthetase (MARS complex. Through a series of genetic pull-down assays, we have identified the enzymes of this complex as: methionyl-, glutaminyl-, glutamyl-, and tyrosyl-tRNA synthetases, and we show that the N-terminal GST-like domain of a partially disordered hybrid scaffold protein, Tg-p43, is sufficient for assembly of the intact complex. Our gel filtration studies revealed significant heterogeneity in the size and composition of isolated MARS complexes. By targeting the tyrosyl-tRNA synthetases subunit, which was found exclusively in the complete 1 MDa complex, we were able to directly visualize MARS particles in the electron microscope. Image analyses of the negative stain data revealed the observed heterogeneity and instability of these complexes to be driven by the intrinsic flexibility of the domain arrangements within the MARS complex. These studies provide unique insights into the assembly of these ubiquitous but poorly understood eukaryotic complexes.

  14. Molecular Recognition within Synaptic Scaffolds

    DEFF Research Database (Denmark)

    Erlendsson, Simon

    domains, responsible for tethering their respective synaptic protein ligands. Therefore, understanding the specificity and binding mechanisms of PDZ domain proteins is essential to understand regulation of synaptic plasticity. PICK1 is a PDZ domain-containing scaffolding protein predominantly expressed...... and characterized in the postsynaptic neurons, where it is involved in regulating processes underlying LTP and LTD. However, PICK1 has also been found to interact with a wide range of other regulatory proteins, receptors and transporters, which implicates PICK1 in several processes important for proper synaptic...

  15. Inactivation of lmpA, Encoding a LIMPII-related Endosomal Protein, Suppresses the Internalization and Endosomal Trafficking Defects in Profilin-null Mutants

    OpenAIRE

    Temesvari, Lesly; Zhang, Linyi; Fodera, Brent; Janssen, Klaus-Peter; Schleicher, Michael; Cardelli, James A.

    2000-01-01

    Profilin is a key phosphoinositide and actin-binding protein connecting and coordinating changes in signal transduction pathways with alterations in the actin cytoskeleton. Using biochemical assays and microscopic approaches, we demonstrate that profilin-null cells are defective in macropinocytosis, fluid phase efflux, and secretion of lysosomal enzymes but are unexpectedly more efficient in phagocytosis than wild-type cells. Disruption of the lmpA gene encoding a protein (DdLIMP) belonging t...

  16. A dimer of the Toll-like receptor 4 cytoplasmic domain provides a specific scaffold for the recruitment of signalling adaptor proteins.

    Directory of Open Access Journals (Sweden)

    Ricardo Núñez Miguel

    Full Text Available The Toll-like receptor 4 (TLR4 is a class I transmembrane receptor expressed on the surface of immune system cells. TLR4 is activated by exposure to lipopolysaccharides derived from the outer membrane of Gram negative bacteria and forms part of the innate immune response in mammals. Like other class 1 receptors, TLR4 is activated by ligand induced dimerization, and recent studies suggest that this causes concerted conformational changes in the receptor leading to self association of the cytoplasmic Toll/Interleukin 1 receptor (TIR signalling domain. This homodimerization event is proposed to provide a new scaffold that is able to bind downstream signalling adaptor proteins. TLR4 uses two different sets of adaptors; TRAM and TRIF, and Mal and MyD88. These adaptor pairs couple two distinct signalling pathways leading to the activation of interferon response factor 3 (IRF-3 and nuclear factor kappaB (NFkappaB respectively. In this paper we have generated a structural model of the TLR4 TIR dimer and used molecular docking to probe for potential sites of interaction between the receptor homodimer and the adaptor molecules. Remarkably, both the Mal and TRAM adaptors are strongly predicted to bind at two symmetry-related sites at the homodimer interface. This model of TLR4 activation is supported by extensive functional studies involving site directed mutagenesis, inhibition by cell permeable peptides and stable protein phosphorylation of receptor and adaptor TIR domains. Our results also suggest a molecular mechanism for two recent findings, the caspase 1 dependence of Mal signalling and the protective effects conferred by the Mal polymorphism Ser180Leu.

  17. Downstream-of-FGFR Is a Fibroblast Growth Factor-Specific Scaffolding Protein and Recruits Corkscrew upon Receptor Activation

    OpenAIRE

    Petit, Valérie; Nussbaumer, Ute; Dossenbach, Caroline; Affolter, Markus

    2004-01-01

    Fibroblast growth factor (FGF) receptor (FGFR) signaling controls the migration of glial, mesodermal, and tracheal cells in Drosophila melanogaster. Little is known about the molecular events linking receptor activation to cytoskeletal rearrangements during cell migration. We have performed a functional characterization of Downstream-of-FGFR (Dof), a putative adapter protein that acts specifically in FGFR signal transduction in Drosophila. By combining reverse genetic, cell culture, and bioch...

  18. Biphasic Alteration of the Inhibitory Synapse Scaffold Protein Gephyrin in Early and Late Stages of an Alzheimer Disease Model.

    Science.gov (United States)

    Kiss, Eva; Gorgas, Karin; Schlicksupp, Andrea; Groß, Dagmar; Kins, Stefan; Kirsch, Joachim; Kuhse, Jochen

    2016-09-01

    The pathogenesis of Alzheimer disease (AD) is thought to begin many years before the diagnosis of dementia. Accumulating evidence indicates the involvement of GABAergic neurotransmission in the physiopathology of AD. However, in comparison to excitatory synapses, the structural and functional alterations of inhibitory synapses in AD are less well characterized. We studied the expression and distribution of proteins specific for inhibitory synapses in hippocampal areas of APPPS1 mice at different ages. Interestingly, by immunoblotting and confocal fluorescence microscopy, we disclosed a robust increase in the expression of gephyrin, an organizer of ligand-gated ion channels at inhibitory synapses in hippocampus CA1 and dentate gyrus of young presymptomatic APPPS1 mice (1 to 3 months) as compared to controls. The postsynaptic γ2-GABA(A)-receptor subunit and the presynaptic vesicular inhibitory amino acid transporter protein showed similar expression patterns. In contrast, adult transgenic animals (12 months) displayed decreased levels of these proteins in comparison to wild type in hippocampus areas devoid of amyloid plaques. Within most plaques, strong gephyrin immunoreactivity was detected, partially colocalizing with vesicular amino acid transporter and GABA(A)-receptor γ2 subunit immunoreactivities. Our results indicate a biphasic alteration in expression of hippocampal inhibitory synapse components in AD. Altered inhibition of neurotransmission might be an early prognostic marker and might even be involved in the pathogenesis of AD.

  19. Biphasic Alteration of the Inhibitory Synapse Scaffold Protein Gephyrin in Early and Late Stages of an Alzheimer Disease Model.

    Science.gov (United States)

    Kiss, Eva; Gorgas, Karin; Schlicksupp, Andrea; Groß, Dagmar; Kins, Stefan; Kirsch, Joachim; Kuhse, Jochen

    2016-09-01

    The pathogenesis of Alzheimer disease (AD) is thought to begin many years before the diagnosis of dementia. Accumulating evidence indicates the involvement of GABAergic neurotransmission in the physiopathology of AD. However, in comparison to excitatory synapses, the structural and functional alterations of inhibitory synapses in AD are less well characterized. We studied the expression and distribution of proteins specific for inhibitory synapses in hippocampal areas of APPPS1 mice at different ages. Interestingly, by immunoblotting and confocal fluorescence microscopy, we disclosed a robust increase in the expression of gephyrin, an organizer of ligand-gated ion channels at inhibitory synapses in hippocampus CA1 and dentate gyrus of young presymptomatic APPPS1 mice (1 to 3 months) as compared to controls. The postsynaptic γ2-GABA(A)-receptor subunit and the presynaptic vesicular inhibitory amino acid transporter protein showed similar expression patterns. In contrast, adult transgenic animals (12 months) displayed decreased levels of these proteins in comparison to wild type in hippocampus areas devoid of amyloid plaques. Within most plaques, strong gephyrin immunoreactivity was detected, partially colocalizing with vesicular amino acid transporter and GABA(A)-receptor γ2 subunit immunoreactivities. Our results indicate a biphasic alteration in expression of hippocampal inhibitory synapse components in AD. Altered inhibition of neurotransmission might be an early prognostic marker and might even be involved in the pathogenesis of AD. PMID:27423698

  20. The scaffolding protein Dlg1 is a negative regulator of cell-free virus infectivity but not of cell-to-cell HIV-1 transmission in T cells.

    Directory of Open Access Journals (Sweden)

    Patrycja Nzounza

    Full Text Available BACKGROUND: Cell-to-cell virus transmission of Human immunodeficiency virus type-1 (HIV-1 is predominantly mediated by cellular structures such as the virological synapse (VS. The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunological synapse (IS. We have previously identified the human homologue of the Drosophila Discs Large (Dlg1 protein as a new cellular partner for the HIV-1 Gag protein and a negative regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before. METHODOLOGY/PRINCIPAL FINDINGS: Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quantitative cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA. CONCLUSION: Despite its role in the IS formation, Dlg1 does not affect the VS and cell-to-cell spread of HIV-1, but plays a role in HIV-1 cell-free virus transmission. We propose that the effect of Dlg1 on HIV-1 infectivity is at the stage of virus entry.

  1. Biodegradable synthetic polymer scaffolds for reinforcement of albumin protein solders used for laser-assisted tissue repair.

    Science.gov (United States)

    Hoffman, Grant T; Soller, Eric C; McNally-Heintzelman, Karen M

    2002-01-01

    Laser tissue soldering has been investigated for several years by researchers in our laboratory as an alternative to conventional tissue fasteners, including sutures, staples and clips. Laser tissue soldering is a bonding technique in which protein solder is applied to the tissue surfaces to be joined, and laser energy is used to bond the solder to the tissue surfaces. Over the past four years we have been investigating the use of synthetic polymer membranes as a means for reinforcing the strength of tissue repairs formed using traditional laser tissue soldering techniques. The purpose of this study was to assess the influence of various processing parameters on the strength of tissue repairs formed using the reinforced solder. Biodegradable polymer membranes of specific porosity were fabricated by means of a solvent-casting and particulate-leaching technique, using three different poly(alpha ester)s: polyglycolic acid (PGA), polylactic acid (PLA) and poly(L-lactic-co-glycolic acid) (PLGA). In addition, several membranes were also prepared with poly(ethylene glycol) (PEG). The membranes were then doped with the traditional protein solder mixture of serum albumin and indocyanine green dye. Varied processing parameters included the polymer type, the PLGA copolymer blend ratio, the polymer/PEG blend ratio, the porosity of the polymer membrane and the initial albumin weight fraction. Variation of the polymer type had negligible effect on the strength of the repairs. Although it is known that alteration of the copolymer blend ratio of PLGA influences the degradation rate of the polymer, this variation also had no significant effect on the strength of the repairs formed. Increased membrane flexibility was observed when PEG was added during the casting stage. An increase in the porosity of the polymer membranes led to a subsequent increase in the final concentration of protein contained within the membranes, hence aiding in strengthening the resultant repairs. Likewise

  2. The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins [v3; ref status: indexed, http://f1000r.es/51m

    Directory of Open Access Journals (Sweden)

    Sandeep Chakraborty

    2015-01-01

    Full Text Available The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4 inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237 and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff with known structures using serine protease (SPASE motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of

  3. Dipeptidyl peptidase-IV inhibitors used in type-2 diabetes inhibit a phospholipase C: a case of promiscuous scaffolds in proteins [v2; ref status: indexed, http://f1000r.es/4wz

    Directory of Open Access Journals (Sweden)

    Sandeep Chakraborty

    2015-01-01

    Full Text Available The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4 inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237 and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff with known structures using serine protease (SPASE motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of

  4. The scaffolding protein NHERF1 sensitizes EGFR-dependent tumor growth, motility and invadopodia function to gefitinib treatment in breast cancer cells.

    Science.gov (United States)

    Bellizzi, Antonia; Greco, Maria Raffaella; Rubino, Rosa; Paradiso, Angelo; Forciniti, Stefania; Zeeberg, Katrine; Cardone, Rosa Angela; Reshkin, Stephan Joel

    2015-03-01

    Triple negative breast cancer (TNBC) patients cannot be treated with endocrine therapy or targeted therapies due to lack of related receptors. These patients overexpress the epidermal growth factor receptor (EGFR), but are resistant to tyrosine kinase inhibitors (TKIs) and anti-EGFR therapies. Mechanisms suggested for resistance to TKIs include EGFR independence, mutations and alterations in EGFR and in its downstream signalling pathways. Ligand-induced endocytosis and degradation of EGFR play important roles in the downregulation of the EGFR signal suggesting that its activity could be regulated by targeting its trafficking. Evidence in normal cells showing that the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF1) can associate with EGFR to regulate its trafficking, led us to hypothesize that NHERF1 expression levels could regulate EGFR trafficking and functional expression in TNBC cells and, in this way, modulate its role in progression and response to treatment. We investigated the subcellular localization of NHERF1 and its interaction with EGFR in a metastatic basal like TNBC cell model, MDA-MB‑231, and the role of forced NHERF1 overexpression and/or stimulation with EGF on the sensitivity to EGFR specific TKI treatment with gefitinib. Stimulation with EGF induces an interaction of NHERF1 with EGFR to regulate its localization, degradation and function. NHERF1 overexpression is sufficient to drive its interaction with EGFR in non-stimulated conditions, inhibits EGFR degradation and increases its retention time in the plasma membrane. Importantly, NHERF1 overexpression strongly sensitized the cell to the pharmacological inhibition by gefitinib of EGFR-driven growth, motility and invadopodia-dependent ECM proteolysis. The further determination of how the NHERF1‑EGFR interaction is regulated may improve our understanding of TNBC resistance to the action of existing anticancer drugs.

  5. Scaffold preferences of mesenchymal stromal cells and adipose-derived stem cells from green fluorescent protein transgenic mice influence the tissue engineering of bone.

    Science.gov (United States)

    Wittenburg, Gretel; Flade, Viktoria; Garbe, Annette I; Lauer, Günter; Labudde, Dirk

    2014-05-01

    We have analysed the growth and differentiation of mesenchymal stromal cells (MSC) from bone marrow, and of adipose derived stem cells (ASC) from murine abdominal fat tissue, of green fluorescent protein (GFP) transgenic animals grown directly on two types of hydroxyapatite ceramic bone substitutes. BONITmatrix® and NanoBone® have specific mechanical and physiochemical properties such as porosity and an inner surface that influence cellular growth. Both MSC and ASC were separately seeded on 200mg of each biomaterial and cultured for 3 weeks under osteogenic differentiation conditions. The degree of mineralisation was assessed by alizarin red dye and the specific alkaline phosphatase activity of the differentiated cells. The morphology of the cells was examined by scanning electron microscopy and confocal microscopy. The osteoblastic phenotype of the cells was confirmed by analysing the expression of bone-specific genes (Runx2, osteocalcin, osteopontin, and osteonectin) by semiquantitative reverse transcriptase polymerase chain reaction (PCR). Comparison of BONITmatrix® and NanoBone® showed cell type-specific preferences in terms of osteogenic differentiation. MSC-derived osteoblast-like cells spread optimally on the surface of NanoBone® but not BONITmatrix® granules. In contrast BONITmatrix® granules conditioned the growth of osteoblast-like cells derived from ASC. The osteoblastic phenotype of the cultured cells on all matrices was confirmed by specific gene expression. Our results show that the in vitro growth and osteogenic differentiation of murine MSC or ASC of GFP transgenic mice are distinctly influenced by the ceramic substratum. While NanoBone® granules support the proliferation and differentiation of murine MSC isolated from bone marrow, the growth of murine ASC is supported by BONITmatrix® granules. NanoBone® is therefore recommended for use as scaffold in tissue engineering that requires MSC, whereas ASC can be combined with BONITmatrix® for

  6. Self-Assembly of Synthetic Metabolons through Synthetic Protein Scaffolds: One-Step Purification, Co-immobilization, and Substrate Channeling

    Energy Technology Data Exchange (ETDEWEB)

    You, C; Zhang, YHP

    2013-02-01

    One-step purification of a multi-enzyme complex was developed based on a mixture of cell extracts containing three dockerin-containing enzymes and one family 3 cellulose-binding module (CBM3)-containing scaffoldin through high-affinity adsorption on low-cost solid regenerated amorphous cellulose (RAC). The three-enzyme complex, called synthetic metabolon, was self-assembled through the high-affinity interaction between the dockerin in each enzyme and three cohesins in the synthetic scaffoldin. The metabolons were either immobilized on the external surface of RAC or free when the scaffoldin contained an intein between the CBM3 and three cohesins. The immobilized and free metabolons containing triosephosphate isomerase, aldolase, and fructose 1,6-biphosphatase exhibited initial reaction rates 48 and 38 times, respectively, that of the non-complexed three-enzyme mixture at the same enzyme loading. Such reaction rate enhancements indicated strong substrate channeling among synthetic metabolons due to the close spatial organization among cascade enzymes. These results suggested that the construction of synthetic metabolons by using cohesins, dockerins, and cellulose-binding modules from cellulosomes not only decreased protein purification labor and cost for in vitro synthetic biology projects but also accelerated reaction rates by 1 order of magnitude compared to non-complexed enzymes. Synthetic metabolons would be an important biocatalytic module for in vitro and in vivo synthetic biology projects.

  7. Engineering a Biocompatible Scaffold with Either Micrometre or Nanometre Scale Surface Topography for Promoting Protein Adsorption and Cellular Response

    Directory of Open Access Journals (Sweden)

    Xuan Le

    2013-01-01

    Full Text Available Surface topographical features on biomaterials, both at the submicrometre and nanometre scales, are known to influence the physicochemical interactions between biological processes involving proteins and cells. The nanometre-structured surface features tend to resemble the extracellular matrix, the natural environment in which cells live, communicate, and work together. It is believed that by engineering a well-defined nanometre scale surface topography, it should be possible to induce appropriate surface signals that can be used to manipulate cell function in a similar manner to the extracellular matrix. Therefore, there is a need to investigate, understand, and ultimately have the ability to produce tailor-made nanometre scale surface topographies with suitable surface chemistry to promote favourable biological interactions similar to those of the extracellular matrix. Recent advances in nanoscience and nanotechnology have produced many new nanomaterials and numerous manufacturing techniques that have the potential to significantly improve several fields such as biological sensing, cell culture technology, surgical implants, and medical devices. For these fields to progress, there is a definite need to develop a detailed understanding of the interaction between biological systems and fabricated surface structures at both the micrometre and nanometre scales.

  8. Postsynaptic scaffolds for nicotinic receptors on neurons

    Institute of Scientific and Technical Information of China (English)

    Robert A NEFF III; David GOMEZ-VARELA; Catarina C FERNANDES; Darwin K BERG

    2009-01-01

    Complex postsynaptic scaffolds determine the structure and signaling capabilities of glutamatergic synapses. Recent studies indicate that some of the same scaffold components contribute to the formation and function of nicotinic synapses on neurons. PDZ-containing proteins comprising the PSD-95 family co-localize with nicotinic acetylcholine receptors (nAChRs) and mediate downstream signaling in the neurons. The PDZ-proteins also promote functional nicotinic innerva- tion of the neurons, as does the scaffold protein APC and transmembrane proteins such as neuroligin and the EphB2 recep- tor. In addition, specific chaperones have been shown to facilitate nAChR assembly and transport to the cell surface. This review summarizes recent results in these areas and raises questions for the future about the mechanism and synaptic role of nAChR trafficking.

  9. Sbi00515, a Protein of Unknown Function from Streptomyces bingchenggensis, Highlights the Functional Versatility of the Acetoacetate Decarboxylase Scaffold.

    Science.gov (United States)

    Mueller, Lisa S; Hoppe, Robert W; Ochsenwald, Jenna M; Berndt, Robert T; Severin, Geoffrey B; Schwabacher, Alan W; Silvaggi, Nicholas R

    2015-06-30

    The acetoacetate decarboxylase-like superfamily (ADCSF) is a group of ~4000 enzymes that, until recently, was thought to be homogeneous in terms of the reaction catalyzed. Bioinformatic analysis shows that the ADCSF consists of up to seven families that differ primarily in their active site architectures. The soil-dwelling bacterium Streptomyces bingchenggensis BCW-1 produces an ADCSF enzyme of unknown function that shares a low level of sequence identity (~20%) with known acetoacetate decarboxylases (ADCs). This enzyme, Sbi00515, belongs to the MppR-like family of the ADCSF because of its similarity to the mannopeptimycin biosynthetic protein MppR from Streptomyces hygroscopicus. Herein, we present steady state kinetic data that show Sbi00515 does not catalyze the decarboxylation of any α- or β-keto acid tested. Rather, we show that Sbi00515 catalyzes the condensation of pyruvate with a number of aldehydes, followed by dehydration of the presumed aldol intermediate. Thus, Sbi00515 is a pyruvate aldolase-dehydratase and not an acetoacetate decarboxylase. We have also determined the X-ray crystal structures of Sbi00515 in complexes with formate and pyruvate. The structures show that the overall fold of Sbi00515 is nearly identical to those of both ADC and MppR. The pyruvate complex is trapped as the Schiff base, providing evidence that the Schiff base chemistry that drives the acetoacetate decarboxylases has been co-opted to perform a new function, and that this core chemistry may be conserved across the superfamily. The structures also suggest possible catalytic roles for several active site residues. PMID:26039798

  10. Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyoung-Dong; Chung, Woo-Hyun; Kim, Hyo-Jin; Lee, Kyung-Chang [Laboratory of Molecular Microbiology, School of Biological Sciences, Institute of Microbiology, Seoul National University, Seoul 151-742 (Korea, Republic of); Roe, Jung-Hye, E-mail: jhroe@snu.ac.kr [Laboratory of Molecular Microbiology, School of Biological Sciences, Institute of Microbiology, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2010-02-12

    Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The {Delta}grx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1{sup +} gene encoding a putative A-type Fe-S scaffold, in addition to mas5{sup +} and hsc1{sup +} genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2{sup +}, but not isu1{sup +} and ssc1{sup +}, complemented the growth phenotype of {Delta}grx5 mutant as isa1{sup +} did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.

  11. Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

    International Nuclear Information System (INIS)

    Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The Δgrx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1+ gene encoding a putative A-type Fe-S scaffold, in addition to mas5+ and hsc1+ genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2+, but not isu1+ and ssc1+, complemented the growth phenotype of Δgrx5 mutant as isa1+ did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.

  12. Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN.

    Science.gov (United States)

    Takayanagi, Hiroki; Yuzawa, Satoru; Sumimoto, Hideki

    2015-02-01

    The adaptor protein LGN interacts via the N-terminal domain comprising eight tetratricopeptide-repeat (TPR) motifs with its partner proteins mInsc, NuMA, Frmpd1 and Frmpd4 in a mutually exclusive manner. Here, the crystal structure of the LGN TPR domain in complex with human Frmpd4 is described at 1.5 Å resolution. In the complex, the LGN-binding region of Frmpd4 (amino-acid residues 990-1011) adopts an extended structure that runs antiparallel to LGN along the concave surface of the superhelix formed by the TPR motifs. Comparison with the previously determined structures of the LGN-Frmpd1, LGN-mInsc and LGN-NuMA complexes reveals that these partner proteins interact with LGN TPR1-6 via a common core binding region with consensus sequence (E/Q)XEX4-5(E/D/Q)X1-2(K/R)X0-1(V/I). In contrast to Frmpd1, Frmpd4 makes additional contacts with LGN via regions N- and C-terminal to the core sequence. The N-terminal extension is replaced by a specific α-helix in mInsc, which drastically increases the direct contacts with LGN TPR7/8, consistent with the higher affinity of mInsc for LGN. A crystal structure of Frmpd4-bound LGN in an oxidized form is also reported, although oxidation does not appear to strongly affect the interaction with Frmpd4.

  13. Towards biomimetic scaffolds: anhydrous scaffold fabrication from biodegradable amine-reactive diblock copolymers.

    Science.gov (United States)

    Hacker, Michael; Tessmar, Jörg; Neubauer, Markus; Blaimer, Andrea; Blunk, Torsten; Göpferich, Achim; Schulz, Michaela B

    2003-11-01

    The development of biomimetic materials and their processing into three-dimensional cell carrying scaffolds is one promising tissue engineering strategy to improve cell adhesion, growth and differentiation on polymeric constructs developing mature and viable tissue. This study was concerned with the fabrication of scaffolds made from amine-reactive diblock copolymers, N-succinimidyl tartrate monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid), which are able to suppress unspecific protein adsorption and to covalently bind proteins or peptides. An appropriate technique for their processing had to be both anhydrous, to avoid hydrolysis of the active ester, and suitable for the generation of interconnected porous structures. Attempts to fabricate scaffolds utilizing hard paraffin microparticles as hexane-extractable porogens failed. Consequently, a technique was developed involving lipid microparticles, which served as biocompatible porogens on which the scaffold forming polymer was precipitated in the porogen extraction media (n-hexane). Porogen melting during the extraction and polymer precipitation step led to an interconnected network of pores. Suitable lipid mixtures and their melting points, extraction conditions (temperature and time) and a low-toxic polymer solvent system were determined for their use in processing diblock copolymers of different molecular weights (22 and 42 kDa) into highly porous off-the-shelf cell carriers ready for easy surface modification towards biomimetic scaffolds. Insulin was employed to demonstrate the principal of instant protein coupling to a prefabricated scaffold. PMID:12922156

  14. The dynamics of scaffolding

    NARCIS (Netherlands)

    Van Geert, P. L. C.; Steenbeek, H.W.

    2005-01-01

    In this article we have reinterpreted a relatively standard definition of scaffolding in the context of dynamic systems theory. Our main point is that scaffolding cannot be understood outside the context of a dynamic approach of learning and (formal or informal) teaching. We provide a dynamic system

  15. Gelatin–PMVE/MA composite scaffold promotes expansion of embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chhabra, Hemlata [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); Gupta, Priyanka [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India); IITB-Monash Research Academy, Mumbai (India); Department of Chemical Engineering, Monash University, Melbourne (Australia); Verma, Paul J. [Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, South Australia (Australia); Jadhav, Sameer; Bellare, Jayesh R. [Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai (India)

    2014-04-01

    We introduce a new composite scaffold of gelatin and polymethyl vinyl ether-alt-maleic anhydride (PMVE/MA) for expansion of embryonic stem cells (ESCs) in an in vitro environment. To optimize the scaffold, we prepared a gelatin scaffold (G) and three composite scaffolds namely GP-1, GP-2, and GP-3 with varying PMVE/MA concentrations (0.2–1%) and characterized them by scanning electron microscopy (SEM), swelling study, compression testing and FTIR. SEM micrographs revealed interconnected porous structure in all the scaffolds. The permissible hemolysis ratio and activation of platelets by scaffolds confirmed the hemocompatibility of scaffolds. Initial biocompatibility assessment of scaffolds was conducted using hepatocarcinoma (Hep G2) cells and adhesion, proliferation and infiltration of Hep G2 cells in depth of scaffolds were observed, proving the scaffold's biocompatibility. Further Oct4B2 mouse embryonic stem cells (mESCs), which harbor a green fluorescence protein transgene under regulatory control of the Oct4 promotor, were examined for expansion on scaffolds with MTT assay. The GP-2 scaffold demonstrated the best cell proliferation and was further explored for ESC adherence and infiltration in depth (SEM and confocal), and pluripotent state of mESCs was assessed with the expression of Oct4-GFP and stage-specific embryonic antigen-1 (SSEA-1). This study reports the first demonstration of biocompatibility of gelatin–PMVE/MA composite scaffold and presents this scaffold as a promising candidate for embryonic stem cell based tissue engineering. - Highlights: • Composite scaffolds of gelatin and PMVE/MA were prepared by freeze-drying method. • SEM micrographs showed porous structure in all scaffolds of varying pore dimension. • GP-2 composite exhibited better cellular response in comparison to other scaffolds. • mESCs proliferated and expressed Oct-4 and SSEA-1, when cultured on GP-2 scaffold.

  16. Mechanical properties, biological activity and protein controlled release by poly(vinyl alcohol)-bioglass/chitosan-collagen composite scaffolds: a bone tissue engineering applications.

    Science.gov (United States)

    Pon-On, Weeraphat; Charoenphandhu, Narattaphol; Teerapornpuntakit, Jarinthorn; Thongbunchoo, Jirawan; Krishnamra, Nateetip; Tang, I-Ming

    2014-05-01

    In the present study, composite scaffolds made with different weight ratios (0.5:1, 1:1 and 2:1) of bioactive glass (15Ca:80Si:5P) (BG)/polyvinyl alcohol (PVA) (PVABG) and chitosan (Chi)/collagen (Col) (ChiCol) were prepared by three mechanical freeze-thaw followed by freeze-drying to obtain the porous scaffolds. The mechanical properties and the in vitro biocompatibility of the composite scaffolds to simulated body fluid (SBF) and to rat osteoblast-like UMR-106 cells were investigated. The results from the studies indicated that the porosity and compressive strength were controlled by the weight ratio of PVABG:ChiCol. The highest compressive modulus of the composites made was 214.64 MPa which was for the 1:1 weight ratio PVABG:ChiCol. Mineralization study in SBF showed the formation of apatite crystals on the PVABG:ChiCol surface after 7 days of incubation. In vitro cell availability and proliferation tests confirmed the osteoblast attachment and growth on the PVABG:ChiCol surface. MTT and ALP tests on the 1:1 weight ratio PVABG:ChiCol composite indicated that the UMR-106 cells were viable. Alkaline phosphatase activity was found to increase with increasing culturing time. In addition, we showed the potential of PVABG:ChiCol drug delivery through PBS solution studies. 81.14% of BSA loading had been achieved and controlled release for over four weeks was observed. Our results indicated that the PVABG:ChiCol composites, especially the 1:1 weight ratio composite exhibited significantly improved mechanical, mineral deposition, biological properties and controlled release. This made them potential candidates for bone tissue engineering applications.

  17. TIP-1 Has PDZ Scaffold Antagonist Activity

    Science.gov (United States)

    Alewine, Christine; Olsen, Olav; Wade, James B.

    2006-01-01

    PDZ proteins usually contain multiple protein–protein interaction domains and act as molecular scaffolds that are important for the generation and maintenance of cell polarity and cell signaling. Here, we identify and characterize TIP-1 as an atypical PDZ protein that is composed almost entirely of a single PDZ domain and functions as a negative regulator of PDZ-based scaffolding. We found that TIP-1 competes with the basolateral membrane mLin-7/CASK complex for interaction with the potassium channel Kir 2.3 in model renal epithelia. Consequently, polarized plasma membrane expression of Kir 2.3 is disrupted resulting in pronounced endosomal targeting of the channel, similar to the phenotype observed for mutant Kir 2.3 channels lacking the PDZ-binding motif. TIP-1 is ubiquitously expressed, raising the possibility that TIP-1 may play a similar role in regulating the expression of other membrane proteins containing a type I PDZ ligand. PMID:16855024

  18. Hierarchical Scaffolding With Bambus

    OpenAIRE

    Pop, Mihai; Kosack, Daniel S.; Salzberg, Steven L

    2004-01-01

    The output of a genome assembler generally comprises a collection of contiguous DNA sequences (contigs) whose relative placement along the genome is not defined. A procedure called scaffolding is commonly used to order and orient these contigs using paired read information. This ordering of contigs is an essential step when finishing and analyzing the data from a whole-genome shotgun project. Most recent assemblers include a scaffolding module; however, users have little control over the scaf...

  19. Graphene oxide scaffold accelerates cellular proliferative response and alveolar bone healing of tooth extraction socket.

    Science.gov (United States)

    Nishida, Erika; Miyaji, Hirofumi; Kato, Akihito; Takita, Hiroko; Iwanaga, Toshihiko; Momose, Takehito; Ogawa, Kosuke; Murakami, Shusuke; Sugaya, Tsutomu; Kawanami, Masamitsu

    2016-01-01

    Graphene oxide (GO) consisting of a carbon monolayer has been widely investigated for tissue engineering platforms because of its unique properties. For this study, we fabricated a GO-applied scaffold and assessed the cellular and tissue behaviors in the scaffold. A preclinical test was conducted to ascertain whether the GO scaffold promoted bone induction in dog tooth extraction sockets. For this study, GO scaffolds were prepared by coating the surface of a collagen sponge scaffold with 0.1 and 1 µg/mL GO dispersion. Scaffolds were characterized using scanning electron microscopy (SEM), physical testing, cell seeding, and rat subcutaneous implant testing. Then a GO scaffold was implanted into a dog tooth extraction socket. Histological observations were made at 2 weeks postsurgery. SEM observations show that GO attached to the surface of collagen scaffold struts. The GO scaffold exhibited an interconnected structure resembling that of control subjects. GO application improved the physical strength, enzyme resistance, and adsorption of calcium and proteins. Cytocompatibility tests showed that GO application significantly increased osteoblastic MC3T3-E1 cell proliferation. In addition, an assessment of rat subcutaneous tissue response revealed that implantation of 1 µg/mL GO scaffold stimulated cellular ingrowth behavior, suggesting that the GO scaffold exhibited good biocompatibility. The tissue ingrowth area and DNA contents of 1 µg/mL GO scaffold were, respectively, approximately 2.5-fold and 1.4-fold greater than those of the control. Particularly, the infiltration of ED2-positive (M2) macrophages and blood vessels were prominent in the GO scaffold. Dog bone-formation tests showed that 1 µg/mL GO scaffold implantation enhanced bone formation. New bone formation following GO scaffold implantation was enhanced fivefold compared to that in control subjects. These results suggest that GO was biocompatible and had high bone-formation capability for the scaffold

  20. Grafting of protein-protein binding sites

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A strategy for grafting protein-protein binding sites is described. Firstly, key interaction residues at the interface of ligand protein to be grafted are identified and suitable positions in scaffold protein for grafting these key residues are sought. Secondly, the scaffold proteins are superposed onto the ligand protein based on the corresponding Ca and Cb atoms. The complementarity between the scaffold protein and the receptor protein is evaluated and only matches with high score are accepted. The relative position between scaffold and receptor proteins is adjusted so that the interface has a reasonable packing density. Then the scaffold protein is mutated to corresponding residues in ligand protein at each candidate position. And the residues having bad steric contacts with the receptor proteins, or buried charged residues not involved in the formation of any salt bridge are mutated. Finally, the mutated scaffold protein in complex with receptor protein is co-minimized by Charmm. In addition, we deduce a scoring function to evaluate the affinity between mutated scaffold protein and receptor protein by statistical analysis of rigid binding data sets.

  1. Semiotic Scaffolding in Mathematics

    DEFF Research Database (Denmark)

    Johansen, Mikkel Willum; Misfeldt, Morten

    2015-01-01

    This paper investigates the notion of semiotic scaffolding in relation to mathematics by considering its influence on mathematical activities, and on the evolution of mathematics as a research field. We will do this by analyzing the role different representational forms play in mathematical...... cognition, and more broadly on mathematical activities. In the main part of the paper, we will present and analyze three different cases. For the first case, we investigate the semiotic scaffolding involved in pencil and paper multiplication. For the second case, we investigate how the development of new...... in both mathematical cognition and in the development of mathematics itself, but mathematical cognition cannot itself be reduced to the use of semiotic scaffolding....

  2. Histological and radiographic evaluation of the muscle tissue of rats after implantation of bone morphogenic protein (rhBMP-2 in a scaffold of inorganic bone and after stimulation with low-power laser light

    Directory of Open Access Journals (Sweden)

    Bengtson Antonio

    2010-01-01

    Full Text Available Objective: The present study histologically and radiologically evaluates the muscle tissue of rats after implantation of bone morphogenic protein (rhBMP-2 in a natural inorganic bone mineral scaffold from a bull calf femur and irradiation with low-power light laser. Materials and Methods: The right and left hind limbs of 16 rats were shaved and an incision was made in the muscle on the face corresponding to the median portion of the tibia, into which rhBMP-2 in a scaffold of inorganic bone was implanted. Two groups of limbs were formed: control (G1 and laser irradiation (G2. G2 received diode laser light applied in the direction of the implant, at a dose of 8 J/cm2 for three minutes. On the 7th, 21st, 40th and 112th days after implantation, hind limbs of 4 animals were radiographed and their implants removed together with the surrounding tissue for study under the microscope. The histological results were graded as 0=absence, 1=slight presence, 2=representative and 3=very representative, with regard to the following events: formation of osteoid structure, acute inflammation, chronic inflammation, fibrin deposition, neovascularization, foreign-body granuloma and fibrosis. Results: There were no statistically significant differences in these events at each evaluation times, between the two groups (P > 0.05; Mann-Whitney test. Nevertheless, it could be concluded that the natural inorganic bone matrix with rhBMP-2, from the femur of a bull calf, is a biocompatible combination. Conclusions: Under these conditions, the inductive capacity of rhBMP-2 for cell differentiation was inhibited. There was a slight acceleration in tissue healing in the group that received irradiation with low-power laser light.

  3. Scaffolding students’ assignments

    DEFF Research Database (Denmark)

    Slot, Marie Falkesgaard

    2013-01-01

    , on the shoulders of linguistics research, results of “assignment-didactics” as a useful but also very tentative term in the context of the assessment of design principles for the design of assignments. Based on empirical studies, I discuss cognition, multi-modality and collaboration as crucial types of scaffolding...

  4. Plant-Derived Human Collagen Scaffolds for Skin Tissue Engineering

    Science.gov (United States)

    Willard, James J.; Drexler, Jason W.; Das, Amitava; Roy, Sashwati; Shilo, Shani; Shoseyov, Oded

    2013-01-01

    Tissue engineering scaffolds are commonly formed using proteins extracted from animal tissues, such as bovine hide. Risks associated with the use of these materials include hypersensitivity and pathogenic contamination. Human-derived proteins lower the risk of hypersensitivity, but possess the risk of disease transmission. Methods engineering recombinant human proteins using plant material provide an alternate source of these materials without the risk of disease transmission or concerns regarding variability. To investigate the utility of plant-derived human collagen (PDHC) in the development of engineered skin (ES), PDHC and bovine hide collagen were formed into tissue engineering scaffolds using electrospinning or freeze-drying. Both raw materials were easily formed into two common scaffold types, electrospun nonwoven scaffolds and lyophilized sponges, with similar architectures. The processing time, however, was significantly lower with PDHC. PDHC scaffolds supported primary human cell attachment and proliferation at an equivalent or higher level than the bovine material. Interleukin-1 beta production was significantly lower when activated THP-1 macrophages where exposed to PDHC electrospun scaffolds compared to bovine collagen. Both materials promoted proper maturation and differentiation of ES. These data suggest that PDHC may provide a novel source of raw material for tissue engineering with low risk of allergic response or disease transmission. PMID:23298216

  5. How Scaffolds shape MAPK signalling: What we know and opportunities for systems approaches

    Directory of Open Access Journals (Sweden)

    Franziska eWitzel

    2012-12-01

    Full Text Available Scaffolding proteins add a new layer of complexity to the dynamics of cell signalling. Above their basic function to bring several components of a signalling pathway together, recent experimental research has found that scaffolds influence signalling in a much more complex way: Scaffolds can exert some catalytic function, influence signalling by allosteric mechanisms, are feedback-regulated, localise signalling activity to distinct regions of the cell or increase pathway fidelity. Here we review experimental and theoretical approaches that address the function of two MAPK scaffolds, Ste5, a scaffold of the yeast mating pathway and Ksr1/2, a scaffold of the classical mammalian MAPK signalling pathway. For the yeast scaffold Ste5, detailed mechanistic models have been valuable for the understanding of its function. For scaffolds in mammalian signalling, however, models have been rather generic and sketchy. For example, these models predicted narrow optimal scaffold concentrations, but when revisiting these models by assuming typical concentrations, rather a range of scaffold levels optimally supports signalling. Thus, more realistic models are needed to understand the role of scaffolds in mammalian signal transduction, which opens a big opportunity for systems biology.

  6. Development of porous scaffolds for bone tissue engineering

    Science.gov (United States)

    Ramay, Hassna Rehman

    -liquid phase separation technique with a polyelectrolyte complex of chitosan and alginate as scaffolding material. These scaffolds were used to study sustained delivery of a model protein. The effect of size of ceramic in polymer matrix on mechanical and biological properties of scaffold is also examined by using both nano- and micron-sized ceramic particles.

  7. The Src Homology 3 Domain Is Required for Junctional Adhesion Molecule Binding to the Third PDZ Domain of the Scaffolding Protein ZO-1

    Energy Technology Data Exchange (ETDEWEB)

    Nomme, Julian; Fanning, Alan S.; Caffrey, Michael; Lye, Ming F.; Anderson, James M.; Lavie, Arnon (NIH); (UNC); (UIC)

    2012-01-20

    Tight junctions are cell-cell contacts that regulate the paracellular flux of solutes and prevent pathogen entry across cell layers. The assembly and permeability of this barrier are dependent on the zonula occludens (ZO) membrane-associated guanylate kinase (MAGUK) proteins ZO-1, -2, and -3. MAGUK proteins are characterized by a core motif of protein-binding domains that include a PDZ domain, a Src homology 3 (SH3) domain, and a region of homology to guanylate kinase (GUK); the structure of this core motif has never been determined for any MAGUK. To better understand how ZO proteins organize the assembly of protein complexes we have crystallized the entire PDZ3-SH3-GUK core motif of ZO-1. We have also crystallized this core motif in complex with the cytoplasmic tail of the ZO-1 PDZ3 ligand, junctional adhesion molecule A (JAM-A) to determine how the activity of different domains is coordinated. Our study shows a new feature for PDZ class II ligand binding that implicates the two highly conserved Phe{sup -2} and Ser{sup -3} residues of JAM. Our x-ray structures and NMR experiments also show for the first time a role for adjacent domains in the binding of ligands to PDZ domains in the MAGUK proteins family.

  8. Mineralization Content Alters Osteogenic Responses of Bone Marrow Stromal Cells on Hydroxyapatite/Polycaprolactone Composite Nanofiber Scaffolds

    Directory of Open Access Journals (Sweden)

    Ketul C. Popat

    2012-11-01

    Full Text Available Synthetic tissue scaffolds have a high potential impact for patients experiencing osteogenesis imperfecta. Using electrospinning, tissue scaffolds composed of hydroxyapatite/polycaprolactone (HAp/PCL composite nanofibers were fabricated with two different HAp concentrations—1% and 10% of the solid scaffold weight. After physico-chemical scaffold characterization, rat bone marrow stromal cells were cultured on the composite scaffolds in maintenance medium and then in osteogenic medium. Quantitative PCR, colorimetric assays, immunofluorescent labeling, and electron microscopy measured osteogenic cell responses to the HAp/PCL scaffolds. In maintenance conditions, both Hap/PCL scaffolds and control scaffolds supported cell colonization through seven days with minor differences. In osteogenic conditions, the 10% HAp scaffolds exhibited significantly increased ALP assay levels at week 3, consistent with previous reports. However, qPCR analysis demonstrated an overall decrease in bone matrix-associated genes on Hap/PCL scaffolds. Osteopontin and osteocalcin immunofluorescent microscopy revealed a trend that both mineralized scaffolds had greater amounts of both proteins, though qPCR results indicated the opposite trend for osteopontin. Additionally, type I collagen expression decreased on HAp scaffolds. These results indicate that cells are sensitive to minor changes in mineral content within nanofibers, even at just 1% w/w, and elucidating the sensing mechanism may lead to optimized osteogenic scaffold designs.

  9. Impact of silk fibroin-based scaffold structures on human osteoblast MG63 cell attachment and proliferation

    Directory of Open Access Journals (Sweden)

    Varkey A

    2015-10-01

    Full Text Available Aneesia Varkey,1,2 Elakkiya Venugopal,2 Ponjanani Sugumaran,2 Gopinathan Janarthanan,1 Mamatha M Pillai,2 Selvakumar Rajendran,2 Amitava Bhattacharyya1 1Advanced Textile and Polymer Research Laboratory, 2Tissue Engineering Laboratory, PSG Institute of Advanced Studies, Coimbatore, Tamil Nadu, India Abstract: The present study was carried out to investigate the impact of various types of silk fibroin (SF scaffolds on human osteoblast-like cell (MG63 attachment and proliferation. SF was isolated from Bombyx mori silk worm cocoons after degumming. Protein concentration in the degummed SF solution was estimated using Bradford method. Aqueous SF solution was used to fabricate three different types of scaffolds, viz, electrospun nanofiber mat, sponge, and porous film. The structures of the prepared scaffolds were characterized using optical micro­scopy and field emission scanning electron microscopy. The changes in the secondary structure of the proteins and the thermal behavior of the scaffolds were determined by Fourier transform infrared spectroscopy and thermo-gravimetric analysis, respectively. The biodegradation rate of scaffolds was determined by incubating the scaffolds in simulated body fluid for 4 weeks. MG63 cells were seeded on the scaffolds and their attachment and proliferation onto the scaffolds were studied. The MTT assay was carried out to deduce the toxicity of the developed scaffolds. All the scaffolds were found to be biocompatible. The amount of collagen produced by the osteoblast-like cells growing on different scaffolds was estimated. Keywords: silk fibroin scaffold, electrospun nanofiber, porous film, sponge, osteoblast

  10. Part I. Cobalt thiolate complexes modeling the active site of cobalt nitrile hydratase. Part II. Formation of inorganic nanoparticles on protein scaffolding in Escherichia coli glutamine synthetase

    Science.gov (United States)

    Kung, Irene Yuk Man

    scaffolding for inorganic mineralization. US nanoclusters of discreet size seem to grow in the presence of E165C in aqueous solution spontaneously. Commercially available mono(maleimido)undecagold seem to bind only to E165C through the reactive cysteine side chains. Reduction of Au3+ to elemental gold in solution with E165C, generates long, linear structures of approximately 100-nm diameter.

  11. Taurine chloramine-induced inactivation of cofilin protein through methionine oxidation.

    Science.gov (United States)

    Luo, Shen; Uehara, Hiroshi; Shacter, Emily

    2014-10-01

    Cofilin regulates reorganization of actin filaments (F-actin) in eukaryotes. A recent finding has demonstrated that oxidation of cofilin by taurine chloramine (TnCl), a physiological oxidant derived from neutrophils, causes cofilin to translocate to the mitochondria inducing apoptosis (F. Klamt et al. Nat. Cell Biol.11:1241-1246; 2009). Here we investigated the effect of TnCl on biological activities of cofilin in vitro. Our data show that TnCl-induced oxidation of recombinant human cofilin-1 inhibits its F-actin-binding and depolymerization activities. Native cofilin contains four free Cys and three Met residues. Incubation of oxidized cofilin with DTT does not lead to its reactivation. A double Cys to Ala mutation on the two C-terminal Cys shows similar biological activities as the wild type, but does not prevent the TnCl-induced inactivation. In contrast, incubation of oxidized cofilin with methionine sulfoxide reductases results in its reactivation. Phosphorylation is known to inhibit cofilin activities. We found that Met oxidation also prevents phosphorylation of cofilin, which is reversed by incubating oxidized cofilin with methionine sulfoxide reductases. Interestingly, intact protein mass spectrometry of the oxidized mutant indicated one major oxidation product with an additional mass of 16 Da, consistent with oxidation of one specific Met residue. This residue was identified as Met-115 by peptide mapping and tandem mass spectrometry. It is adjacent to Lys-114, a known residue on globular-actin-binding site, implying that oxidation of Met-115 disrupts the globular-actin-binding site of cofilin, which causes TnCl-induced inactivation. The findings identify Met-115 as a redox switch on cofilin that regulates its biological activity.

  12. Modification on ursodeoxycholic acid (UDCA) scaffold. discovery of bile acid derivatives as selective agonists of cell-surface G-protein coupled bile acid receptor 1 (GP-BAR1).

    Science.gov (United States)

    Sepe, Valentina; Renga, Barbara; Festa, Carmen; D'Amore, Claudio; Masullo, Dario; Cipriani, Sabrina; Di Leva, Francesco Saverio; Monti, Maria Chiara; Novellino, Ettore; Limongelli, Vittorio; Zampella, Angela; Fiorucci, Stefano

    2014-09-25

    Bile acids are signaling molecules interacting with the nuclear receptor FXR and the G-protein coupled receptor 1 (GP-BAR1/TGR5). GP-BAR1 is a promising pharmacological target for the treatment of steatohepatitis, type 2 diabetes, and obesity. Endogenous bile acids and currently available semisynthetic bile acids are poorly selective toward GP-BAR1 and FXR. Thus, in the present study we have investigated around the structure of UDCA, a clinically used bile acid devoid of FXR agonist activity, to develop a large family of side chain modified 3α,7β-dihydroxyl cholanoids that selectively activate GP-BAR1. In vivo and in vitro pharmacological evaluation demonstrated that administration of compound 16 selectively increases the expression of pro-glucagon 1, a GP-BAR1 target, in the small intestine, while it had no effect on FXR target genes in the liver. Further, compound 16 results in a significant reshaping of bile acid pool in a rodent model of cholestasis. These data demonstrate that UDCA is a useful scaffold to generate novel and selective steroidal ligands for GP-BAR1. PMID:25162837

  13. Olfactory receptor signaling is regulated by the post-synaptic density 95, Drosophila discs large, zona-occludens 1 (PDZ) scaffold multi-PDZ domain protein 1.

    LENUS (Irish Health Repository)

    Dooley, Ruth

    2009-12-01

    The unique ability of mammals to detect and discriminate between thousands of different odorant molecules is governed by the diverse array of olfactory receptors expressed by olfactory sensory neurons in the nasal epithelium. Olfactory receptors consist of seven transmembrane domain G protein-coupled receptors and comprise the largest gene superfamily in the mammalian genome. We found that approximately 30% of olfactory receptors possess a classical post-synaptic density 95, Drosophila discs large, zona-occludens 1 (PDZ) domain binding motif in their C-termini. PDZ domains have been established as sites for protein-protein interaction and play a central role in organizing diverse cell signaling assemblies. In the present study, we show that multi-PDZ domain protein 1 (MUPP1) is expressed in the apical compartment of olfactory sensory neurons. Furthermore, on heterologous co-expression with olfactory sensory neurons, MUPP1 was shown to translocate to the plasma membrane. We found direct interaction of PDZ domains 1 + 2 of MUPP1 with the C-terminus of olfactory receptors in vitro. Moreover, the odorant-elicited calcium response of OR2AG1 showed a prolonged decay in MUPP1 small interfering RNA-treated cells. We have therefore elucidated the first building blocks of the putative \\'olfactosome\\

  14. A PDZ-Like Motif in the Biliary Transporter ABCB4 Interacts with the Scaffold Protein EBP50 and Regulates ABCB4 Cell Surface Expression.

    Directory of Open Access Journals (Sweden)

    Quitterie Venot

    Full Text Available ABCB4/MDR3, a member of the ABC superfamily, is an ATP-dependent phosphatidylcholine translocator expressed at the canalicular membrane of hepatocytes. Defects in the ABCB4 gene are associated with rare biliary diseases. It is essential to understand the mechanisms of its canalicular membrane expression in particular for the development of new therapies. The stability of several ABC transporters is regulated through their binding to PDZ (PSD95/DglA/ZO-1 domain-containing proteins. ABCB4 protein ends by the sequence glutamine-asparagine-leucine (QNL, which shows some similarity to PDZ-binding motifs. The aim of our study was to assess the potential role of the QNL motif on the surface expression of ABCB4 and to determine if PDZ domain-containing proteins are involved. We found that truncation of the QNL motif decreased the stability of ABCB4 in HepG2-transfected cells. The deleted mutant ABCB4-ΔQNL also displayed accelerated endocytosis. EBP50, a PDZ protein highly expressed in the liver, strongly colocalized and coimmunoprecipitated with ABCB4, and this interaction required the QNL motif. Down-regulation of EBP50 by siRNA or by expression of an EBP50 dominant-negative mutant caused a significant decrease in the level of ABCB4 protein expression, and in the amount of ABCB4 localized at the canalicular membrane. Interaction of ABCB4 with EBP50 through its PDZ-like motif plays a critical role in the regulation of ABCB4 expression and stability at the canalicular plasma membrane.

  15. Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications

    OpenAIRE

    Tiede, Christian; Tang, Anna A. S.; Deacon, Sarah E.; Mandal, Upasana; Nettleship, Joanne E.; Owen, Robin L.; George, Suja E.; Harrison, David J; Owens, Raymond J.; Tomlinson, Darren C.; McPherson, Michael J.

    2014-01-01

    We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (T m ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Å resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality ...

  16. 调控黑色素瘤转移的新信号蛋白: Syntenin%Syntenin: a novel PDZ domain-containing scaffolding protein associated with human melanoma metastasis

    Institute of Scientific and Technical Information of China (English)

    Jian-bo Yang; James B. McCarthy

    2007-01-01

    Syntenin蛋白在多种肿瘤中表达增强,最近被认为是一个新的黑色素瘤转移调节因子.作为一类支架信号蛋白,Syntenin通过它的两个PDZ 功能基团可与许多细胞膜受体胞内末端或细胞内的信号分子结合,调控多种重要的细胞生理过程和信号传导途径,包括细胞膜受体的聚集,细胞内蛋白质的转运,细胞骨架的重建,转录因子的激活,以增强肿瘤细胞的生长、黏附以及肿瘤的血管生成、侵袭和转移能力.本文简要综述了syntenin的结构和功能,相关的信号途径,及其在黑色素瘤研究领域的最新进展.%Syntenin is overexpressed in multiple human cancers and is newly recognized as a novel regulator in melanoma metastasis. It functions as a scaffolding protein, via its two PDZ domains interacting with multiple transmembrane and cytoplasmic partners to regulate many of the major signaling pathways involved in various cellular processes, such as cell surface receptor clustering, protein trafficking, cytoskeleton remodeling, and activation of transcription factor, and results in the increased abilities for tumor cell growth, adhesion, angiogenesis, invasion and metastasis. The present article attempts to review the structure and functions of syntenin by summarizing our current knowledge on the interacting partners and diverse signaling pathways related to syntenin, and highlight the importance of syntenin as a new potential therapeutic target for the aggressive human melanoma.

  17. A Novel Albumin-Based Tissue Scaffold for Autogenic Tissue Engineering Applications

    Science.gov (United States)

    Li, Pei-Shan; -Liang Lee, I.; Yu, Wei-Lin; Sun, Jui-Sheng; Jane, Wann-Neng; Shen, Hsin-Hsin

    2014-07-01

    Tissue scaffolds provide a framework for living tissue regeneration. However, traditional tissue scaffolds are exogenous, composed of metals, ceramics, polymers, and animal tissues, and have a defined biocompatibility and application. This study presents a new method for obtaining a tissue scaffold from blood albumin, the major protein in mammalian blood. Human, bovine, and porcine albumin was polymerised into albumin polymers by microbial transglutaminase and was then cast by freeze-drying-based moulding to form albumin tissue scaffolds. Scanning electron microscopy and material testing analyses revealed that the albumin tissue scaffold possesses an extremely porous structure, moderate mechanical strength, and resilience. Using a culture of human mesenchymal stem cells (MSCs) as a model, we showed that MSCs can be seeded and grown in the albumin tissue scaffold. Furthermore, the albumin tissue scaffold can support the long-term osteogenic differentiation of MSCs. These results show that the albumin tissue scaffold exhibits favourable material properties and good compatibility with cells. We propose that this novel tissue scaffold can satisfy essential needs in tissue engineering as a general-purpose substrate. The use of this scaffold could lead to the development of new methods of artificial fabrication of autogenic tissue substitutes.

  18. SHOP: receptor-based scaffold hopping by GRID-based similarity searches

    DEFF Research Database (Denmark)

    Bergmann, Rikke; Liljefors, Tommy; Sørensen, Morten D;

    2009-01-01

    A new field-derived 3D method for receptor-based scaffold hopping, implemented in the software SHOP, is presented. Information from a protein-ligand complex is utilized to substitute a fragment of the ligand with another fragment from a database of synthetically accessible scaffolds. A GRID-based...

  19. Biomimetic apatite-coated porous PVA scaffolds promote the growth of breast cancer cells

    International Nuclear Information System (INIS)

    Recapitulating the native environment of bone tissue is essential to develop in vitro models of breast cancer bone metastasis. The bone is a composite material consisting of organic matrix and inorganic mineral phase, primarily hydroxyapatite. In this study, we report the mineralization of porous poly vinyl alcohol (PVA) scaffolds upon incubation in modified Hanks' Balanced Salt Solution (HBSS) for 14 days. Scanning electron microscopy, energy dispersive X-ray analysis, and X-ray diffraction analysis revealed that the deposited minerals have composition similar to hydroxyapatite. The study demonstrated that the rate of nucleation and growth of minerals was faster on surfaces of less porous scaffolds. However, upon prolonged incubation, formation of mineral layer was observed on the surface of all the scaffolds. In addition, the study also demonstrated that 3D mineralization only occurred for scaffolds with highly interconnected porous networks. The mineralization of the scaffolds promoted the adsorption of serum proteins and consequently, the adhesion and proliferation of breast cancer cells. - Highlights: • Porous PVA scaffolds fabricated via mechanical agitation followed by freeze-drying. • Mineralization of the scaffold was carried out by utilizing biomimetic approach. • Mineralization resulted in increased protein adsorption on the scaffold. • Increased breast cancer cell growth was observed on mineralized scaffolds

  20. Biomimetic apatite-coated porous PVA scaffolds promote the growth of breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Mao; Mohanty, Pravansu; Ghosh, Gargi, E-mail: gargi@umich.edu

    2014-11-01

    Recapitulating the native environment of bone tissue is essential to develop in vitro models of breast cancer bone metastasis. The bone is a composite material consisting of organic matrix and inorganic mineral phase, primarily hydroxyapatite. In this study, we report the mineralization of porous poly vinyl alcohol (PVA) scaffolds upon incubation in modified Hanks' Balanced Salt Solution (HBSS) for 14 days. Scanning electron microscopy, energy dispersive X-ray analysis, and X-ray diffraction analysis revealed that the deposited minerals have composition similar to hydroxyapatite. The study demonstrated that the rate of nucleation and growth of minerals was faster on surfaces of less porous scaffolds. However, upon prolonged incubation, formation of mineral layer was observed on the surface of all the scaffolds. In addition, the study also demonstrated that 3D mineralization only occurred for scaffolds with highly interconnected porous networks. The mineralization of the scaffolds promoted the adsorption of serum proteins and consequently, the adhesion and proliferation of breast cancer cells. - Highlights: • Porous PVA scaffolds fabricated via mechanical agitation followed by freeze-drying. • Mineralization of the scaffold was carried out by utilizing biomimetic approach. • Mineralization resulted in increased protein adsorption on the scaffold. • Increased breast cancer cell growth was observed on mineralized scaffolds.

  1. Microporous dermal-mimetic electrospun scaffolds pre-seeded with fibroblasts promote tissue regeneration in full-thickness skin wounds.

    Directory of Open Access Journals (Sweden)

    Paul P Bonvallet

    Full Text Available Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone electrospun scaffold (70:30 col/PCL containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM, and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344 rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14% over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold. Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration

  2. Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells*

    OpenAIRE

    Padányi, Rita; Xiong, Yuning; Antalffy, Géza; Lór, Krisztina; Pászty, Katalin; STREHLER, EMANUEL E.; Enyedi, Ágnes

    2010-01-01

    The membrane localization of the plasma membrane Ca2+-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na+/H+ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expressio...

  3. Boron containing poly-(lactide-co-glycolide) (PLGA) scaffolds for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Doğan, Ayşegül; Demirci, Selami [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University 34755 Istanbul (Turkey); Bayir, Yasin [Department of Biochemistry, Faculty of Pharmacy, Ataturk University, 25240, Erzurum (Turkey); Halici, Zekai [Department of Pharmacology, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Karakus, Emre [Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ataturk University, 25240, Erzurum (Turkey); Aydin, Ali [Department of Orthopedics and Traumatology, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Cadirci, Elif [Department of Pharmacology, Faculty of Pharmacy, Ataturk University, 25240, Erzurum (Turkey); Albayrak, Abdulmecit [Department of Pharmacology, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Demirci, Elif [Department of Pathology, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Karaman, Adem [Department of Radiology, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Ayan, Arif Kursat [Department of Nuclear Medicine, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Gundogdu, Cemal [Department of Pathology, Faculty of Medicine, Ataturk University, 25240, Erzurum (Turkey); Şahin, Fikrettin, E-mail: fsahin@yeditepe.edu.tr [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University 34755 Istanbul (Turkey)

    2014-11-01

    Scaffold-based bone defect reconstructions still face many challenges due to their inadequate osteoinductive and osteoconductive properties. Various biocompatible and biodegradable scaffolds, combined with proper cell type and biochemical signal molecules, have attracted significant interest in hard tissue engineering approaches. In the present study, we have evaluated the effects of boron incorporation into poly-(lactide-co-glycolide-acid) (PLGA) scaffolds, with or without rat adipose-derived stem cells (rADSCs), on bone healing in vitro and in vivo. The results revealed that boron containing scaffolds increased in vitro proliferation, attachment and calcium mineralization of rADSCs. In addition, boron containing scaffold application resulted in increased bone regeneration by enhancing osteocalcin, VEGF and collagen type I protein levels in a femur defect model. Bone mineralization density (BMD) and computed tomography (CT) analysis proved that boron incorporated scaffold administration increased the healing rate of bone defects. Transplanting stem cells into boron containing scaffolds was found to further improve bone-related outcomes compared to control groups. Additional studies are highly warranted for the investigation of the mechanical properties of these scaffolds in order to address their potential use in clinics. The study proposes that boron serves as a promising innovative approach in manufacturing scaffold systems for functional bone tissue engineering. - Highlights: • Boron containing PLGA scaffolds were developed for bone tissue engineering. • Boron incorporation increased cell viability and mineralization of stem cells. • Boron containing scaffolds increased bone-related protein expression in vivo. • Implantation of stem cells on boron containing scaffolds improved bone healing.

  4. Multifunctional Thin Film Biomatrice Biosensor in a Degradable Scaffold Containing Bone Morphogenetic Protein-2 (BMP-2) for Controlled Release in Skeletal Tissue Engineering

    Science.gov (United States)

    McDaniel, Harvey; Lomax, Linda

    2001-03-01

    Bone morphonogenetic proteins (BMP-2) have been under investigation for three decades. Deminerialized bone and extracts of deminerialized bone are o steoinductive with a temporal sequence of bone induction. Native and recombi nant BMP's have shown the ability, thru growth and differentiative factors t o induce de novo bone formation both invitro and invivo. Their principle fun ction is to induce transformation of undifferentiated mesenchymal cells into osteoblasts. Native and recombinant BMP's, when purified and used without carrier disp erse after implantation and exert no effect on bone induction. The delivery system provides the missing component to successsfully applying osteogenic p roteins for clinical need. Biological and physio-chemical properties are str ictly adhered tofor a successful delivery system. The BMP delivery system ca rrier for osteo inductive payload provided; 1)non tumorgenic genecity, 2) no n immunogenecity, 3) water insoluble, 4) biosorbability with predictable enz ymatic degradation, and 5) an optimized surface for compatibility, cell migr ation and attachment with a negative surface change that encouraged target c ell attachment. Being a controlled Release System, it binded the proteins wi th predictible BMP released kinetics. Porosity with interconnecting voids pr otected the BMP from noon specific proteolysis and promoted rapid vascular a nd mesenchymal invasion. Far wide ranging clinical applications of mechanica l and biofunctional requirements were met with the BMP delivery system. Cohe sion and malleability were reqiured forcontour augmentation, and reconstruct ion of the discontinuity defects, prevented dislocation and retained the sha pe and bone replaced the system. Biological systems have elastic activity associated with them. The activi ty was current associated with a time dependant biological/biochemical react ion (enzymic activity). Bioelectric phoenomena associated with charged molec ules in a biologic structure caused

  5. Electrospun multifunctional tissue engineering scaffolds

    Science.gov (United States)

    Wang, Chong; Wang, Min

    2014-03-01

    Tissue engineering holds great promises in providing successful treatments of human body tissue loss that current methods are unable to treat or unable to achieve satisfactory clinical outcomes. In scaffold-based tissue engineering, a highperformance scaffold underpins the success of a tissue engineering strategy and a major direction in the field is to create multifunctional tissue engineering scaffolds for enhanced biological performance and for regenerating complex body tissues. Electrospinning can produce nanofibrous scaffolds that are highly desirable for tissue engineering. The enormous interest in electrospinning and electrospun fibrous structures by the science, engineering and medical communities has led to various developments of the electrospinning technology and wide investigations of electrospun products in many industries, including biomedical engineering, over the past two decades. It is now possible to create novel, multicomponent tissue engineering scaffolds with multiple functions. This article provides a concise review of recent advances in the R & D of electrospun multifunctional tissue engineering scaffolds. It also presents our philosophy and research in the designing and fabrication of electrospun multicomponent scaffolds with multiple functions.

  6. Graphene oxide scaffold accelerates cellular proliferative response and alveolar bone healing of tooth extraction socket

    Directory of Open Access Journals (Sweden)

    Nishida E

    2016-05-01

    Full Text Available Erika Nishida,1 Hirofumi Miyaji,1 Akihito Kato,1 Hiroko Takita,2 Toshihiko Iwanaga,3 Takehito Momose,1 Kosuke Ogawa,1 Shusuke Murakami,1 Tsutomu Sugaya,1 Masamitsu Kawanami11Department of Periodontology and Endodontology, Hokkaido University Graduate School of Dental Medicine, Sapporo, Japan; 2Support Section for Education and Research, Hokkaido University Graduate School of Dental Medicine, Sapporo, Japan; 3Laboratory of Histology and Cytology, Hokkaido University Graduate School of Medicine, Sapporo, JapanAbstract: Graphene oxide (GO consisting of a carbon monolayer has been widely investigated for tissue engineering platforms because of its unique properties. For this study, we fabricated a GO-applied scaffold and assessed the cellular and tissue behaviors in the scaffold. A preclinical test was conducted to ascertain whether the GO scaffold promoted bone induction in dog tooth extraction sockets. For this study, GO scaffolds were prepared by coating the surface of a collagen sponge scaffold with 0.1 and 1 µg/mL GO dispersion. Scaffolds were characterized using scanning electron microscopy (SEM, physical testing, cell seeding, and rat subcutaneous implant testing. Then a GO scaffold was implanted into a dog tooth extraction socket. Histological observations were made at 2 weeks postsurgery. SEM observations show that GO attached to the surface of collagen scaffold struts. The GO scaffold exhibited an interconnected structure resembling that of control subjects. GO application improved the physical strength, enzyme resistance, and adsorption of calcium and proteins. Cytocompatibility tests showed that GO application significantly increased osteoblastic MC3T3-E1 cell proliferation. In addition, an assessment of rat subcutaneous tissue response revealed that implantation of 1 µg/mL GO scaffold stimulated cellular ingrowth behavior, suggesting that the GO scaffold exhibited good biocompatibility. The tissue ingrowth area and DNA contents of 1

  7. Collagen a natural scaffold for biology and engineering

    Science.gov (United States)

    Collagen, the most abundant protein in mammals, constitutes a quarter of the animal's total weight. The unique structure of fibrous collagens, a long triple helix that further associates into fibers, provides an insoluble scaffold that gives strength and form to the skin, tendons, bones, cornea and...

  8. Fabrication of uniformly cell-laden porous scaffolds using a gas-in-liquid templating technique.

    Science.gov (United States)

    Takei, Takayuki; Aokawa, Ryuta; Shigemitsu, Takamasa; Kawakami, Koei; Yoshida, Masahiro

    2015-11-01

    Design of porous scaffolds in tissue engineering field was challenging. Uniform immobilization of cells in the scaffolds with high porosity was essential for homogeneous tissue formation. The present study was aimed at fabricating uniformly cell-laden porous scaffolds with porosity >74% using the gas-in-liquid foam templating technique. To this end, we used gelatin, microbial transglutaminase and argon gas as a scaffold material, cross-linker of the protein and porogen of scaffold, respectively. We confirmed that a porosity of >74% could be achieved by increasing the gas volume delivered to a gelatin solution. Pore size in the scaffold could be controlled by stirring speed, stirring time and the pore size of the filter through which the gas passed. The foaming technique enabled us to uniformly immobilize a human hepatoblastoma cell line in scaffold. Engraftment efficiency of the cell line entrapped within the scaffold in nude mice was higher than that of cells in free-form. These results showed that the uniformly cell-laden porous scaffolds were promising for tissue engineering.

  9. BSA/HSA ratio modulates the properties of Ca(2+)-induced cold gelation scaffolds.

    Science.gov (United States)

    Ribeiro, Artur; Volkov, Vadim; Oliveira, Mariana B; Padrão, Jorge; Mano, João F; Gomes, Andreia C; Cavaco-Paulo, Artur

    2016-08-01

    An effective tissue engineering approach requires adjustment according to the target tissue to be engineered. The possibility of obtaining a protein-based formulation for the development of multivalent tunable scaffolds that can be adapted for several types of cells and tissues is explored in this work. The incremental substitution of bovine serum albumin (BSA) by human serum albumin (HSA), changing the scaffolds' hydrophilic/hydrophobic ratio, on a previously optimized scaffold formulation resulted in a set of uniform porous scaffolds with different physical properties and associated cell proliferation profile along time. There was a general trend towards an increase in hydrophilicity, swelling degree and in vitro degradation of the scaffolds with increasing replacement of BSA by HAS. The set of BSA/HSA scaffolds presented distinct values for the storage (elastic) modulus and loss factor which were similar to those described for different native tissues such as bone, cartilage, muscle, skin and neural tissue. The preferential adhesion and proliferation of skin fibroblasts on the BSA25%HSA75% and HSA100% scaffolds, as predicted by their viscoelastic properties, demonstrate that the BSA/HSA scaffold formulation is promising for the development of scaffolds that can be tuned according to the tissue to be repaired and restored. PMID:27156695

  10. Semiotic scaffolding of multicellularity

    DEFF Research Database (Denmark)

    Hoffmeyer, Jesper

    2015-01-01

    semiotic scaffoldings had to be invented in order to prevent this. While a unicellular self may go on to live practically forever, the multicellular self most often must run through an individuation process ending in the death of the individual. Due to basic differences in cells of plants, fungi......The threshold from unicellularity to multicellularity has been crossed only three times in evolution with any lasting success. The hard problem was to create a multicellular self. Such a self is vulnerable to breakdown due to the unavoidable appearance of mutant anarchistic cells, and stringent...... and animals this individuation process poses very different challenges in the three kingdoms of plants, fungi and animals, and the solutions found to these differences are discussed. In the same time as multicellularity ushered life into the epoch of mortality it logically also led to the appearance...

  11. Influence of collagen concentration and glutaraldehyde on collagen-based scaffold properties.

    Science.gov (United States)

    Perez-Puyana, V; Romero, A; Guerrero, A

    2016-06-01

    Several studies have shown the influence of the physical properties of scaffolds on their mechanical properties. An initial characterization of a type of collagen protein was carried out by studying its composition andits solubility at different pH values and infrared spectroscopy. Subsequently, porosity and scaffold pore size were studied, assessing how varying the composition of the initial solution (increasing the protein concentration or adding glutaraldehyde) changed the properties of the final scaffolds obtained. Lastly, rheological measurements were performed to evaluate the mechanical strength of the scaffolds. The initial characterization revealed that the type I collagen protein used is considerably denatured. In addition, increasing the protein content in the scaffold decreases the porosity, related to an increase in the elastic modulus producing an enhancement of its mechanical strength, while adding glutaraldehyde to the scaffold increases its mechanical strength without lowering its pore size or porosity. The results obtained are useful in that they demonstrate that it is possible to design a scaffold with specific properties, by just controlling the collagen concentration or adding glutaraldehyde to the initial solution. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1462-1468, 2016. PMID:26833811

  12. Evolutionary and functional diversity of coronin proteins.

    Science.gov (United States)

    Xavier, Charles-Peter; Eichinger, Ludwig; Fernandez, M Pilar; Morgan, Reginald O; Clemen, Christoph S

    2008-01-01

    This chapter discusses various aspects of coronin phylogeny, structure and function that are of specific interest. Two subfamilies of ancient coronins of unicellular pathogens such as Entamoeba, Trypanosoma, Leishmania and Acanthamoeba as well as of Plasmodium, Babesia, and Trichomonas are presented in the first two sections. Their coronins generally bind to F-actin and apparently are involved in proliferation, locomotion and phagocytosis. However, there are so far no studies addressing a putative role of coronin in the virulence of these pathogens. The following section delineates genetic anomalies like the chimeric coronin-fusion products with pelckstrin homology and gelsolin domains that are found in amoeba. Moreover, most nonvertebrate metazoa appear to encode CRN8, CRN9 and CRN7 representatives (for these coronin symbols see Chapter 2), but in e.g., Drosophila melanogaster and Caenorhabditis elegans a CRN9 is missing. The forth section deals with the evolutionary expansion of vertebrate coronins. Experimental data on the F-actin binding CRN2 of Xenopus (Xcoronin) including a Cdc42/Rac interactive binding (CRIB) motif that is also present in other members of the coronin protein family are discussed. Xenopus laevis represents a case for the expansion of the seven vertebrate coronins due to tetraploidization events. Other examples for a change in the number of coronin paralogs are zebrafish and birds, but (coronin) gene duplication events also occurred in unicellular protozoa. The fifth section of this chapter briefly summarizes three different cellular processes in which CRN4/CORO1A is involved, namely actin-binding, superoxide generation and Ca(2+)-signaling and refers to the largely unexplored mammalian coronins CRN5/CORO2A and CRN6/CORO2B, the latter binding to vinculin. The final section discusses how, by unveiling the aspects of coronin function in organisms reported so far, one can trace a remarkable evolution and diversity in their individual roles

  13. Multilayered Magnetic Gelatin Membrane Scaffolds

    Science.gov (United States)

    Samal, Sangram K.; Goranov, Vitaly; Dash, Mamoni; Russo, Alessandro; Shelyakova, Tatiana; Graziosi, Patrizio; Lungaro, Lisa; Riminucci, Alberto; Uhlarz, Marc; Bañobre-López, Manuel; Rivas, Jose; Herrmannsdörfer, Thomas; Rajadas, Jayakumar; De Smedt, Stefaan; Braeckmans, Kevin; Kaplan, David L.; Dediu, V. Alek

    2016-01-01

    A versatile approach for the design and fabrication of multilayer magnetic scaffolds with tunable magnetic gradients is described. Multilayer magnetic gelatin membrane scaffolds with intrinsic magnetic gradients were designed to encapsulate magnetized bioagents under an externally applied magnetic field for use in magnetic-field-assisted tissue engineering. The temperature of the individual membranes increased up to 43.7 °C under an applied oscillating magnetic field for 70 s by magnetic hyperthermia, enabling the possibility of inducing a thermal gradient inside the final 3D multilayer magnetic scaffolds. On the basis of finite element method simulations, magnetic gelatin membranes with different concentrations of magnetic nanoparticles were assembled into 3D multilayered scaffolds. A magnetic-gradient-controlled distribution of magnetically labeled stem cells was demonstrated in vitro. This magnetic biomaterial–magnetic cell strategy can be expanded to a number of different magnetic biomaterials for various tissue engineering applications. PMID:26451743

  14. ScaffoldSeq: Software for characterization of directed evolution populations.

    Science.gov (United States)

    Woldring, Daniel R; Holec, Patrick V; Hackel, Benjamin J

    2016-07-01

    ScaffoldSeq is software designed for the numerous applications-including directed evolution analysis-in which a user generates a population of DNA sequences encoding for partially diverse proteins with related functions and would like to characterize the single site and pairwise amino acid frequencies across the population. A common scenario for enzyme maturation, antibody screening, and alternative scaffold engineering involves naïve and evolved populations that contain diversified regions, varying in both sequence and length, within a conserved framework. Analyzing the diversified regions of such populations is facilitated by high-throughput sequencing platforms; however, length variability within these regions (e.g., antibody CDRs) encumbers the alignment process. To overcome this challenge, the ScaffoldSeq algorithm takes advantage of conserved framework sequences to quickly identify diverse regions. Beyond this, unintended biases in sequence frequency are generated throughout the experimental workflow required to evolve and isolate clones of interest prior to DNA sequencing. ScaffoldSeq software uniquely handles this issue by providing tools to quantify and remove background sequences, cluster similar protein families, and dampen the impact of dominant clones. The software produces graphical and tabular summaries for each region of interest, allowing users to evaluate diversity in a site-specific manner as well as identify epistatic pairwise interactions. The code and detailed information are freely available at http://research.cems.umn.edu/hackel. Proteins 2016; 84:869-874. © 2016 Wiley Periodicals, Inc.

  15. Studies on the mitotic chromosome scaffold of Allium sativum

    Institute of Scientific and Technical Information of China (English)

    ZHAOJIAN; SHAOBOJIN; 等

    1995-01-01

    An argentophilic structure is present in the metaphase chromosomes of garlic(Allium sativum),Cytochemical studies indicate that the main component of the structure is non-histone proteins(NHPs).The results of light and electron microscopic observations reveal that the chromosme NHP scaffold is a network which is composed of fibres and granules and distributed throughout the chromosomes.In the NHP network,there are many condensed regions that are connected by redlatively looser regions.The distribution of the condensed regions varies in individual chromosomes.In some of the chromosomes the condensed regions are lognitudinally situsted in the central part of a chromatid while in others these regions appear as coillike transverse bands.At early metaphase.scaffolds of the sister chromatids of a chromosome are linked to each other in the centromeric region,meanwhile,they are connected by scafold materials along the whole length of the chromosome.At late metaphase,however,the connective scaffold materials between the two sister chromatids disappear gradually and the chromatids begin to separate from one another at their ends.but the chromatids are linked together in the centromeric region until anaphase.This connection seems to be related to the special structure of the NHP scaffold formed in the centromeric region.The morphological features and dynamic changes of the chromosome scaffold are discussed.

  16. Smart scaffolds in bone tissue engineering: A systematicreview of literature

    Institute of Scientific and Technical Information of China (English)

    Saeed Reza Motamedian; Sepanta Hosseinpour; Mitra Ghazizadeh Ahsaie; Arash Khojasteh

    2015-01-01

    AIM To improve osteogenic differentiation and attachmentof cells.METHODS: An electronic search was conducted inPubMed from January 2004 to December 2013. Studieswhich performed smart modifications on conventionalbone scaffold materials were included. Scaffoldswith controlled release or encapsulation of bioactivemolecules were not included. Experiments which did notinvestigate response of cells toward the scaffold (cellattachment, proliferation or osteoblastic differentiation)were excluded.RESULTS: Among 1458 studies, 38 met the inclusion andexclusion criteria. The main scaffold varied extensivelyamong the included studies. Smart modificationsincluded addition of growth factors (group Ⅰ-11 studies),extracellular matrix-like molecules (group Ⅱ-13 studies)and nanoparticles (nano-HA) (group Ⅲ-17 studies). In allgroups, surface coating was the most commonly appliedapproach for smart modification of scaffolds. In group I,bone morphogenetic proteins were mainly used as growthfactor stabilized on polycaprolactone (PCL). In groupⅡ, collagen 1 in combination with PCL, hydroxyapatite(HA) and tricalcium phosphate were the most frequentscaffolds used. In the third group, nano-HA with PCL andchitosan were used the most. As variable methods wereused, a thorough and comprehensible compare betweenthe results and approaches was unattainable.CONCLUSION: Regarding the variability in methodologyof these in vitro studies it was demonstrated that smartmodification of scaffolds can improve tissue properties.

  17. Solvent/Non-Solvent Sintering To Make Microsphere Scaffolds

    Science.gov (United States)

    Laurencin, Cato T.; Brown, Justin L.; Nair, Lakshmi

    2011-01-01

    A solvent/non-solvent sintering technique has been devised for joining polymeric microspheres to make porous matrices for use as drug-delivery devices or scaffolds that could be seeded with cells for growing tissues. Unlike traditional sintering at elevated temperature and pressure, this technique is practiced at room temperature and pressure and, therefore, does not cause thermal degradation of any drug, protein, or other biochemical with which the microspheres might be loaded to impart properties desired in a specific application. Also, properties of scaffolds made by this technique are more reproducible than are properties of comparable scaffolds made by traditional sintering. The technique involves the use of two miscible organic liquids: one that is and one that is not a solvent for the affected polymer. The polymeric microspheres are placed in a mold having the size and shape of the desired scaffold, then the solvent/non-solvent mixture is poured into the mold to fill the void volume between the microspheres, then the liquid mixture is allowed to evaporate. Some of the properties of the resulting scaffold can be tailored through choice of the proportions of the liquids and the diameter of the microspheres.

  18. Composite Scaffolds for Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Min Wang

    2006-01-01

    Full Text Available Biomaterial and scaffold development underpins the advancement of tissue engineering. Traditional scaffolds based on biodegradable polymers such as poly(lactic acid and poly(lactic acid-co-glycolic acid are weak and non-osteoconductive. For bone tissue engineering, polymer-based composite scaffolds containing bioceramics such as hydroxyapatite can be produced and used. The bioceramics can be either incorporated in the scaffolds as a dispersed secondary phase or form a thin coating on the pore surface of polymer scaffolds. This bioceramic phase renders the scaffolds bioactive and also strengthens the scaffolds. There are a number of methods that can be used to produce bioceramic-polymer composite scaffolds. This paper gives an overview of our efforts in developing composite scaffolds for bone tissue engineering.

  19. Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells.

    Science.gov (United States)

    Finoli, Anthony; Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C

    2016-01-01

    Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

  20. Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

    Directory of Open Access Journals (Sweden)

    Anthony Finoli

    2016-01-01

    Full Text Available Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

  1. Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

    Science.gov (United States)

    Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C.

    2016-01-01

    Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

  2. Nanostructured gel scaffolds for osteogenesis through biological assembly of biopolymers via specific nucleobase pairing.

    Science.gov (United States)

    Fan, Ming; Yan, Jingxuan; Tan, Huaping; Ben, Dandan; He, Qiuling; Huang, Zhongwei; Hu, Xiaohong

    2014-11-01

    Biopolymer-based gel scaffolds have great potential in the field of tissue regenerative medicine. In this work, a nanostructured biopolymer gel scaffold via specific pairing of functionalized nucleobases was developed for specifically targeted drug delivery and in vitro osteogenesis. The biopolymer gel system was established by the Watson-Crick base pairing between thymine and adenine via the hydrogen bonding. As gel scaffold precursors, opposite charged polysaccharide derivatives, e.g. quaternized cellulose and heparin, could be additionally crosslinked by extra electrostatic interactions. The potential application of this gel scaffold in bone tissue engineering was confirmed by encapsulation behavior of osteoblasts. In combination with cell growth factor, e.g. bone morphogenetic protein, the nanostructured gel scaffold exhibited beneficial effects on osteoblast activity and differentiation, which suggested a promising future for local treatment of pathologies involving bone loss.

  3. Collagen/chitosan porous bone tissue engineering composite scaffold incorporated with Ginseng compound K.

    Science.gov (United States)

    Muthukumar, Thangavelu; Aravinthan, Adithan; Sharmila, Judith; Kim, Nam Soo; Kim, Jong-Hoon

    2016-11-01

    In this study, suitable scaffold materials for bone tissue engineering were successfully prepared using fish scale collagen, hydroxyapatite, chitosan, and beta-tricalcium phosphate. Porous composite scaffolds were prepared by freeze drying method. The Korean traditional medicinal ginseng compound K, a therapeutic agent for the treatment of osteoporosis that reduces inflammation and enhances production of bone morphogenetic protein-2, was incorporated into the composite scaffold. The scaffold was characterized for pore size, swelling, density, degradation, mineralization, cell viability and attachment, and its morphological features were examined using scanning electron microscopy. This characterization and in vitro analysis showed that the prepared scaffold was biocompatible and supported the growth of MG-63 cells, and therefore has potential as an alternative approach for bone regeneration. PMID:27516305

  4. Electrospun biomimetic scaffold of hydroxyapatite/chitosan supports enhanced osteogenic differentiation of mMSCs

    Science.gov (United States)

    Peng, Hongju; Yin, Zi; Liu, Huanhuan; Chen, Xiao; Feng, Bei; Yuan, Huihua; Su, Bo; Ouyang, Hongwei; Zhang, Yanzhong

    2012-12-01

    Engaging functional biomaterial scaffolds to regulate stem cell differentiation has drawn a great deal of attention in the tissue engineering and regenerative medicine community. In this study, biomimetic composite nanofibrous scaffolds of hydroxyapatite/chitosan (HAp/CTS) were prepared to investigate their capacity for inducing murine mesenchymal stem cells (mMSCs) to differentiate into the osteogenic lineage, in the absence and presence of an osteogenic supplementation (i.e., ascorbic acid, β-glycerol phosphate, and dexamethasone), respectively. Using electrospun chitosan (CTS) nanofibrous scaffolds as the control, cell morphology, growth, specific osteogenic genes expression, and quantified proteins secretion on the HAp/CTS scaffolds were sequentially examined and assessed. It appeared that the HAp/CTS scaffolds supported better attachment and proliferation of the mMSCs. Most noteworthy was that in the absence of the osteogenic supplementation, expression of osteogenic genes including collagen I (Col I), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were significantly upregulated in mMSCs cultured on the HAp/CTS nanofibrous scaffolds. Also increased secretion of the osteogenesis protein markers of alkaline phosphatase and collagen confirmed that the HAp/CTS nanofibrous scaffold markedly promoted the osteogenic commitment in the mMSCs. Moreover, the presence of osteogenic supplementation proved an enhanced efficacy of mMSC osteogenesis on the HAp/CTS nanofibrous scaffolds. Collectively, this study demonstrated that the biomimetic nanofibrous HAp/CTS scaffolds could support and enhance the adhesion, proliferation, and particularly osteogenic differentiation of the mMSCs. It also substantiated the potential of using biomimetic nanofibrous scaffolds of HAp/CTS for functional bone repair and regeneration applications.

  5. Water-stable three-dimensional ultrafine fibrous scaffolds from keratin for cartilage tissue engineering.

    Science.gov (United States)

    Xu, Helan; Cai, Shaobo; Xu, Lan; Yang, Yiqi

    2014-07-22

    Intrinsically water-stable scaffolds composed of ultrafine keratin fibers oriented randomly and evenly in three dimensions were electrospun for cartilage tissue engineering. Keratin has been recognized as a biomaterial that could substantially support the growth and development of multiple cell lines. Besides, three-dimensional (3D) ultrafine fibrous structures were preferred in tissue engineering due to their structural similarity to native extracellular matrices in soft tissues. Recently, we have developed a nontraditional approach to developing 3D fibrous scaffolds from alcohol-soluble corn protein, zein, and verified their structural advantages in tissue engineering. However, keratin with highly cross-linked molecular structures could not be readily dissolved in common solvents for fiber spinning, which required the remarkable drawability of solution. So far, 3D fibrous scaffolds from pure keratin for biomedical applications have not been reported. In this research, the highly cross-linked keratin from chicken feathers was de-cross-linked and disentangled into linear and aligned molecules with preserved molecular weights, forming highly stretchable spinning dope. The solution was readily electrospun into scaffolds with ultrafine keratin fibers oriented randomly in three dimensions. Due to the highly cross-linked molecular structures, keratin scaffolds showed intrinsic water stability. Adipose-derived mesenchymal stem cells could penetrate much deeper, proliferate, and chondrogenically differentiate remarkably better on the 3D keratin scaffolds than on 2D PLA fibrous scaffolds, 3D soy protein fibrous scaffolds, or 3D commercial nonfibrous scaffolds. In summary, the electrospun 3D ultrafine fibrous scaffolds from keratin could be promising candidates for cartilage tissue engineering. PMID:25010870

  6. Silk-Hydroxyapatite Nanoscale Scaffolds with Programmable Growth Factor Delivery for Bone Repair.

    Science.gov (United States)

    Ding, Zhaozhao; Fan, Zhihai; Huang, Xiaowei; Lu, Qiang; Xu, Weian; Kaplan, David L

    2016-09-21

    Osteoinductive biomaterials are attractive for repairing a variety of bone defects, and biomimetic strategies are useful toward developing bone scaffolds with such capacity. Here, a multiple biomimetic design was developed to improve the osteogenesis capacity of composite scaffolds consisting of hydroxyapatite nanoparticles (HA) and silk fibroin (SF). SF nanofibers and water-dispersible HA nanoparticles were blended to prepare the nanoscaled composite scaffolds with a uniform distribution of HA with a high HA content (40%), imitating the extracellular matrix (ECM) of bone. Bone morphogenetic protein-2 (BMP-2) was loaded in the SF scaffolds and HA to tune BMP-2 release. In vitro studies showed the preservation of BMP-2 bioactivity in the composite scaffolds, and programmable sustained release was achieved through adjusting the ratio of BMP-2 loaded on SF and HA. In vitro and in vivo osteogenesis studies demonstrated that the composite scaffolds showed improved osteogenesis capacity under suitable BMP-2 release conditions, significantly better than that of BMP-2 loaded SF-HA composite scaffolds reported previously. Therefore, these biomimetic SF-HA nanoscaled scaffolds with tunable BMP-2 delivery provide preferable microenvironments for bone regeneration.

  7. Mesoporous bioactive glass nanolayer-functionalized 3D-printed scaffolds for accelerating osteogenesis and angiogenesis

    Science.gov (United States)

    Zhang, Yali; Xia, Lunguo; Zhai, Dong; Shi, Mengchao; Luo, Yongxiang; Feng, Chun; Fang, Bing; Yin, Jingbo; Chang, Jiang; Wu, Chengtie

    2015-11-01

    The hierarchical microstructure, surface and interface of biomaterials are important factors influencing their bioactivity. Porous bioceramic scaffolds have been widely used for bone tissue engineering by optimizing their chemical composition and large-pore structure. However, the surface and interface of struts in bioceramic scaffolds are often ignored. The aim of this study is to incorporate hierarchical pores and bioactive components into the bioceramic scaffolds by constructing nanopores and bioactive elements on the struts of scaffolds and further improve their bone-forming activity. Mesoporous bioactive glass (MBG) modified β-tricalcium phosphate (MBG-β-TCP) scaffolds with a hierarchical pore structure and a functional strut surface (~100 nm of MBG nanolayer) were successfully prepared via 3D printing and spin coating. The compressive strength and apatite-mineralization ability of MBG-β-TCP scaffolds were significantly enhanced as compared to β-TCP scaffolds without the MBG nanolayer. The attachment, viability, alkaline phosphatase (ALP) activity, osteogenic gene expression (Runx2, BMP2, OPN and Col I) and protein expression (OPN, Col I, VEGF, HIF-1α) of rabbit bone marrow stromal cells (rBMSCs) as well as the attachment, viability and angiogenic gene expression (VEGF and HIF-1α) of human umbilical vein endothelial cells (HUVECs) in MBG-β-TCP scaffolds were significantly upregulated compared with conventional bioactive glass (BG)-modified β-TCP (BG-β-TCP) and pure β-TCP scaffolds. Furthermore, MBG-β-TCP scaffolds significantly enhanced the formation of new bone in vivo as compared to BG-β-TCP and β-TCP scaffolds. The results suggest that application of the MBG nanolayer to modify 3D-printed bioceramic scaffolds offers a new strategy to construct hierarchically porous scaffolds with significantly improved physicochemical and biological properties, such as mechanical properties, osteogenesis, angiogenesis and protein expression for bone tissue

  8. Novel chitosan/collagen scaffold containing transforming growth factor-β1 DNA for periodontal tissue engineering

    International Nuclear Information System (INIS)

    The current rapid progression in tissue engineering and local gene delivery system has enhanced our applications to periodontal tissue engineering. In this study, porous chitosan/collagen scaffolds were prepared through a freeze-drying process, and loaded with plasmid and adenoviral vector encoding human transforming growth factor-β1 (TGF-β1). These scaffolds were evaluated in vitro by analysis of microscopic structure, porosity, and cytocompatibility. Human periodontal ligament cells (HPLCs) were seeded in this scaffold, and gene transfection could be traced by green fluorescent protein (GFP). The expression of type I and type III collagen was detected with RT-PCR, and then these scaffolds were implanted subcutaneously into athymic mice. Results indicated that the pore diameter of the gene-combined scaffolds was lower than that of pure chitosan/collagen scaffold. The scaffold containing Ad-TGF-β1 exhibited the highest proliferation rate, and the expression of type I and type III collagen up-regulated in Ad-TGF-β1 scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferated but also recruited surrounding tissue to grow in the scaffold. This study demonstrated the potential of chitosan/collagen scaffold combined Ad-TGF-β1 as a good substrate candidate in periodontal tissue engineering

  9. Osteogenic Capacity of Human Adipose-Derived Stem Cells is Preserved Following Triggering of Shape Memory Scaffolds.

    Science.gov (United States)

    Tseng, Ling-Fang; Wang, Jing; Baker, Richard M; Wang, Guirong; Mather, Patrick T; Henderson, James H

    2016-08-01

    Recent advances in shape memory polymers have enabled the study of programmable, shape-changing, cytocompatible tissue engineering scaffolds. For treatment of bone defects, scaffolds with shape memory functionality have been studied for their potential for minimally invasive delivery, conformal fitting to defect margins, and defect stabilization. However, the extent to which the osteogenic differentiation capacity of stem cells resident in shape memory scaffolds is preserved following programmed shape change has not yet been determined. As a result, the feasibility of shape memory polymer scaffolds being employed in stem cell-based treatment strategies remains unclear. To test the hypothesis that stem cell osteogenic differentiation can be preserved during and following triggering of programmed architectural changes in shape memory polymer scaffolds, human adipose-derived stem cells were seeded in shape memory polymer foam scaffolds or in shape memory polymer fibrous scaffolds programmed to expand or contract, respectively, when warmed to body temperature. Osteogenic differentiation in shape-changing and control scaffolds was compared using mineral deposition, protein production, and gene expression assays. For both shape-changing and control scaffolds, qualitatively and quantitatively comparable amounts of mineral deposition were observed; comparable levels of alkaline phosphatase activity were measured; and no significant differences in the expression of genetic markers of osteogenesis were detected. These findings support the feasibility of employing shape memory in scaffolds for stem cell-based therapies for bone repair.

  10. Laser 3D printing with sub-microscale resolution of porous elastomeric scaffolds for supporting human bone stem cells.

    Science.gov (United States)

    Petrochenko, Peter E; Torgersen, Jan; Gruber, Peter; Hicks, Lucas A; Zheng, Jiwen; Kumar, Girish; Narayan, Roger J; Goering, Peter L; Liska, Robert; Stampfl, Jürgen; Ovsianikov, Aleksandr

    2015-04-01

    A reproducible method is needed to fabricate 3D scaffold constructs that results in periodic and uniform structures with precise control at sub-micrometer and micrometer length scales. In this study, fabrication of scaffolds by two-photon polymerization (2PP) of a biodegradable urethane and acrylate-based photoelastomer is demonstrated. This material supports 2PP processing with sub-micrometer spatial resolution. The high photoreactivity of the biophotoelastomer permits 2PP processing at a scanning speed of 1000 mm s(-1), facilitating rapid fabrication of relatively large structures (>5 mm(3)). These structures are custom printed for in vitro assay screening in 96-well plates and are sufficiently flexible to enable facile handling and transplantation. These results indicate that stable scaffolds with porosities of greater than 60% can be produced using 2PP. Human bone marrow stromal cells grown on 3D scaffolds exhibit increased growth and proliferation compared to smooth 2D scaffold controls. 3D scaffolds adsorb larger amounts of protein than smooth 2D scaffolds due to their larger surface area; the scaffolds also allow cells to attach in multiple planes and to completely infiltrate the porous scaffolds. The flexible photoelastomer material is biocompatible in vitro and is associated with facile handling, making it a viable candidate for further study of complex 3D-printed scaffolds.

  11. Mesoporous bioactive glass surface modified poly(lactic-co-glycolic acid) electrospun fibrous scaffold for bone regeneration.

    Science.gov (United States)

    Chen, Shijie; Jian, Zhiyuan; Huang, Linsheng; Xu, Wei; Liu, Shaohua; Song, Dajiang; Wan, Zongmiao; Vaughn, Amanda; Zhan, Ruisen; Zhang, Chaoyue; Wu, Song; Hu, Minghua; Li, Jinsong

    2015-01-01

    A mesoporous bioactive glass (MBG) surface modified with poly(lactic-co-glycolic acid) (PLGA) electrospun fibrous scaffold for bone regeneration was prepared by dip-coating a PLGA electrospun fibrous scaffold into MBG precursor solution. Different surface structures and properties were acquired by different coating times. Surface morphology, chemical composition, microstructure, pore size distribution, and hydrophilicity of the PLGA-MBG scaffold were characterized. Results of scanning electron microscopy indicated that MBG surface coating made the scaffold rougher with the increase of MBG content. Scaffolds after MBG modification possessed mesoporous architecture on the surface. The measurements of the water contact angles suggested that the incorporation of MBG into the PLGA scaffold improved the surface hydrophilicity. An energy dispersive spectrometer evidenced that calcium-deficient carbonated hydroxyapatite formed on the PLGA-MBG scaffolds after a 7-day immersion in simulated body fluid. In vitro studies showed that the incorporation of MBG favored cell proliferation and osteogenic differentiation of human mesenchymal stem cells on the PLGA scaffolds. Moreover, the MBG surface-modified PLGA (PLGA-MBG) scaffolds were shown to be capable of providing the improved adsorption/release behaviors of bone morphogenetic protein-2 (BMP-2). It is very significant that PLGA-MBG scaffolds could be effective for BMP-2 delivery and bone regeneration. PMID:26082632

  12. Mesoporous bioactive glass surface modified poly(lactic-co-glycolic acid) electrospun fibrous scaffold for bone regeneration.

    Science.gov (United States)

    Chen, Shijie; Jian, Zhiyuan; Huang, Linsheng; Xu, Wei; Liu, Shaohua; Song, Dajiang; Wan, Zongmiao; Vaughn, Amanda; Zhan, Ruisen; Zhang, Chaoyue; Wu, Song; Hu, Minghua; Li, Jinsong

    2015-01-01

    A mesoporous bioactive glass (MBG) surface modified with poly(lactic-co-glycolic acid) (PLGA) electrospun fibrous scaffold for bone regeneration was prepared by dip-coating a PLGA electrospun fibrous scaffold into MBG precursor solution. Different surface structures and properties were acquired by different coating times. Surface morphology, chemical composition, microstructure, pore size distribution, and hydrophilicity of the PLGA-MBG scaffold were characterized. Results of scanning electron microscopy indicated that MBG surface coating made the scaffold rougher with the increase of MBG content. Scaffolds after MBG modification possessed mesoporous architecture on the surface. The measurements of the water contact angles suggested that the incorporation of MBG into the PLGA scaffold improved the surface hydrophilicity. An energy dispersive spectrometer evidenced that calcium-deficient carbonated hydroxyapatite formed on the PLGA-MBG scaffolds after a 7-day immersion in simulated body fluid. In vitro studies showed that the incorporation of MBG favored cell proliferation and osteogenic differentiation of human mesenchymal stem cells on the PLGA scaffolds. Moreover, the MBG surface-modified PLGA (PLGA-MBG) scaffolds were shown to be capable of providing the improved adsorption/release behaviors of bone morphogenetic protein-2 (BMP-2). It is very significant that PLGA-MBG scaffolds could be effective for BMP-2 delivery and bone regeneration.

  13. The Gas2 family protein Pigs is a microtubule +TIP that affects cytoskeleton organisation.

    Science.gov (United States)

    Girdler, Gemma C; Applewhite, Derek A; Perry, Wick M G; Rogers, Stephen L; Röper, Katja

    2016-01-01

    Coordination between different cytoskeletal systems is crucial for many cell biological functions, including cell migration and mitosis, and also plays an important role during tissue morphogenesis. Proteins of the class of cytoskeletal crosslinkers, or cytolinkers, have the ability to interact with more than one cytoskeletal system at a time and are prime candidates to mediate any coordination. One such class comprises the Gas2-like proteins, combining a conserved calponin-homology-type actin-binding domain and a Gas2 domain predicted to bind microtubules (MTs). This domain combination is also found in spectraplakins, huge cytolinkers that play important roles in many tissues in both invertebrates and vertebrates. Here, we dissect the ability of the single Drosophila Gas2-like protein Pigs to interact with both actin and MT cytoskeletons, both in vitro and in vivo, and illustrate complex regulatory interactions that determine the localisation of Pigs to and its effects on the cytoskeleton.

  14. Mutation of single hydrophobic residue I27, L35, F39, L58, L65, L67, or L71 in the N terminus of VP5 abolishes interaction with the scaffold protein and prevents closure of herpes simplex virus type 1 capsid shells.

    Science.gov (United States)

    Walters, Jewell N; Sexton, Gerry L; McCaffery, J Michael; Desai, Prashant

    2003-04-01

    Protein-protein interactions drive the assembly of the herpes simplex virus type 1 (HSV-1) capsid. A key interaction occurs between the C-terminal tail of the scaffold protein (pre-22a) and the major capsid protein (VP5). Previously (Z. Hong, M. Beaudet-Miller, J. Durkin, R. Zhang, and A. D. Kwong, J. Virol. 70:533-540, 1996) it was shown that the minimal domain in the scaffold protein necessary for this interaction was composed of a hydrophobic amphipathic helix. The goal of this study was to identify the hydrophobic residues in VP5 important for this bimolecular interaction. Results from the genetic analysis of second-site revertant virus mutants identified the importance of the N terminus of VP5 for the interaction with the scaffold protein. This allowed us to focus our efforts on a small region of this large polypeptide. Twenty-four hydrophobic residues, starting at L23 and ending at F84, were mutated to alanine. All the mutants were first screened for interaction with pre-22a in the yeast two-hybrid assay. From this in vitro assay, seven residues, I27, L35, F39, L58, L65, L67, and L71, that eliminated the interaction when mutated were identified. All 24 mutants were introduced into the virus genome with a genetic marker rescue/marker transfer system. For this system, viruses and cell lines that greatly facilitated the introduction of the mutants into the genome were made. The same seven mutants that abolished interaction of VP5 with pre-22a resulted in an absolute requirement for wild-type VP5 for growth of the viruses. The viruses encoding these mutations in VP5 were capable of forming capsid shells comprised of VP5, VP19C, VP23, and VP26, but the closure of these shells into an icosahedral structure was prevented. Mutation at L75 did not affect the ability of this protein to interact with pre-22a, as judged from the in vitro assay, but this mutation specified a lethal effect for virus growth and abolished the formation of any detectable assembled structure

  15. Binding assay and preliminary X-ray crystallographic analysis of ACTIBIND, a protein with anticarcinogenic and antiangiogenic activities

    International Nuclear Information System (INIS)

    Native ACTIBIND was successfully crystallized and it was shown that the interaction between ACTIBIND and actin is in a molar ratio of 1:2, with a binding constant of 16.17 × 104 M−1. ACTIBIND is a T2 RNase extracellular glycoprotein produced by the mould Aspergillus niger B1 (CMI CC 324626) that possesses anticarcinogenic and antiangiogenic activities. ACTIBIND was found to be an actin-binding protein that interacts with rabbit muscle actin in a 1:2 molar ratio (ACTIBIND:actin) with a binding constant of 16.17 × 104 M−1. Autoclave-treated ACTIBIND (EI-ACTIBIND) lost its RNase activity, but its actin-binding ability was conserved. ACTIBIND crystals were grown using 20% PEG 3350, 0.2 M ammonium dihydrogen phosphate solution at room temperature (293 K). One to four single crystals appeared in each droplet within a few days and grew to approximate dimensions of 0.5 × 0.5 × 0.5 mm after about two weeks. Diffraction studies of these crystals at low temperature (100 K) indicated that they belong to the P3121 space group, with unit-cell parameters a = 78, b = 78, c = 104 Å

  16. Binding assay and preliminary X-ray crystallographic analysis of ACTIBIND, a protein with anticarcinogenic and antiangiogenic activities

    Energy Technology Data Exchange (ETDEWEB)

    Leeuw, Marina de [Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva 84105 (Israel); Roiz, Levava [The Institute of Plant Sciences and Genetics in Agriculture, The Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, PO Box 12, Rehovot 76100 (Israel); Smirnoff, Patricia; Schwartz, Betty [The Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem (Israel); Shoseyov, Oded [The Institute of Plant Sciences and Genetics in Agriculture, The Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, PO Box 12, Rehovot 76100 (Israel); Almog, Orna, E-mail: almogo@bgu.ac.il [Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva 84105 (Israel)

    2007-08-01

    Native ACTIBIND was successfully crystallized and it was shown that the interaction between ACTIBIND and actin is in a molar ratio of 1:2, with a binding constant of 16.17 × 10{sup 4} M{sup −1}. ACTIBIND is a T2 RNase extracellular glycoprotein produced by the mould Aspergillus niger B1 (CMI CC 324626) that possesses anticarcinogenic and antiangiogenic activities. ACTIBIND was found to be an actin-binding protein that interacts with rabbit muscle actin in a 1:2 molar ratio (ACTIBIND:actin) with a binding constant of 16.17 × 10{sup 4} M{sup −1}. Autoclave-treated ACTIBIND (EI-ACTIBIND) lost its RNase activity, but its actin-binding ability was conserved. ACTIBIND crystals were grown using 20% PEG 3350, 0.2 M ammonium dihydrogen phosphate solution at room temperature (293 K). One to four single crystals appeared in each droplet within a few days and grew to approximate dimensions of 0.5 × 0.5 × 0.5 mm after about two weeks. Diffraction studies of these crystals at low temperature (100 K) indicated that they belong to the P3{sub 1}21 space group, with unit-cell parameters a = 78, b = 78, c = 104 Å.

  17. Binding assay and preliminary X-ray crystallographic analysis of ACTIBIND, a protein with anticarcinogenic and antiangiogenic activities

    Science.gov (United States)

    de Leeuw, Marina; Roiz, Levava; Smirnoff, Patricia; Schwartz, Betty; Shoseyov, Oded; Almog, Orna

    2007-01-01

    ACTIBIND is a T2 RNase extracellular glycoprotein produced by the mould Aspergillus niger B1 (CMI CC 324626) that possesses anticarcinogenic and antiangiogenic activities. ACTIBIND was found to be an actin-binding protein that interacts with rabbit muscle actin in a 1:2 molar ratio (ACTIBIND:actin) with a binding constant of 16.17 × 104  M −1. Autoclave-treated ACTIBIND (EI-ACTIBIND) lost its RNase activity, but its actin-binding ability was conserved. ACTIBIND crystals were grown using 20% PEG 3350, 0.2 M ammonium dihydrogen phosphate solution at room temperature (293 K). One to four single crystals appeared in each droplet within a few days and grew to approximate dimensions of 0.5 × 0.5 × 0.5 mm after about two weeks. Diffraction studies of these crystals at low temperature (100 K) indicated that they belong to the P3121 space group, with unit-cell parameters a = 78, b = 78, c = 104 Å. PMID:17671376

  18. Factorial Study of Compressive Mechanical Properties and Primary In Vitro Osteoblast Response of PHBV/PLLA Scaffolds

    Directory of Open Access Journals (Sweden)

    Naznin Sultana

    2012-01-01

    Full Text Available For bone tissue regeneration, composite scaffolds containing biodegradable polymers and nanosized osteoconductive bioceramics have been regarded as promising biomimetic systems. Polymer blends of poly(hydroxybutyrate-co-hydroxyvalerate (PHBV and poly(L-lactic acid (PLLA can be used as the polymer matrix to control the degradation rate. In order to render the scaffolds osteoconductive, nano-sized hydroxyapatite (nHA particles can be incorporated into the polymer matrix. In the first part of this study, a factorial design approach to investigate the influence of materials on the initial compressive mechanical properties of the scaffolds was studied. In the second part, the protein adsorption behavior and the attachment and morphology of osteoblast-like cells (Saos-2 of the scaffolds in vitro were also studied. It was observed that nHA incorporated PHBV/PLLA composite scaffolds adsorbed more bovine serum albumin (BSA protein than PHBV or PHBV/PLLA scaffolds. In vitro studies also revealed that the attachment of human osteoblastic cells (SaOS-2 was significantly higher in nHA incorporated PHBV/PLLA composite scaffolds. From the SEM micrographs of nHA incorporated PHBV/PLLA composite scaffolds seeded with SaOS-2 cells after a 7-day cell culture period, it was observed that the cells were well expanded and spread in all directions on the scaffolds.

  19. Geometrical versus Random β-TCP Scaffolds: Exploring the Effects on Schwann Cell Growth and Behavior

    Science.gov (United States)

    Czisch, Christopher; Witek, Lukasz; Shi, Yang; Smay, Jim; Plant, Giles W.; Yang, Yunzhi

    2015-01-01

    Numerous studies have demonstrated that Schwann cells (SCs) play a role in nerve regeneration; however, their role in innervating a bioceramic scaffold for potential application in bone regeneration is still unknown. Here we report the cell growth and functional behavior of SCs on β-tricalcium phosphate (β-TCP) scaffolds arranged in 3D printed-lattice (P-β-TCP) and randomly-porous, template-casted (N-β-TCP) structures. Our results indicate that SCs proliferated well and expressed the phenotypic markers p75LNGFR and the S100-β subunit of SCs as well as displayed growth morphology on both scaffolds, but SCs showed spindle-shaped morphology with a significant degree of SCs alignment on the P-β-TCP scaffolds, seen to a lesser degree in the N-β-TCP scaffold. The gene expressions of nerve growth factor (β-ngf), neutrophin–3 (nt–3), platelet-derived growth factor (pdgf-bb), and vascular endothelial growth factor (vegf-a) were higher at day 7 than at day 14. While no significant differences in protein secretion were measured between these last two time points, the scaffolds promoted the protein secretion at day 3 compared to that on the cell culture plates. These results together imply that the β-TCP scaffolds can support SC cell growth and that the 3D-printed scaffold appeared to significantly promote the alignment of SCs along the struts. Further studies are needed to investigate the early and late stage relationship between gene expression and protein secretion of SCs on the scaffolds with refined characteristics, thus better exploring the potential of SCs to support vascularization and innervation in synthetic bone grafts. PMID:26444999

  20. Functionally graded electrospun scaffolds with tunable mechanical properties for vascular tissue regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, Vinoy [Center for Nanoscale Materials and Biointegration (CNMB), Department of Physics, University of Alabama at Birmingham (UAB), AL 35294 (United States); Zhang Xing [Department of Biomedical Engineering, School of Engineering, University of Alabama at Birmingham (UAB), AL 35294 (United States); Catledge, Shane A [Center for Nanoscale Materials and Biointegration (CNMB), Department of Physics, University of Alabama at Birmingham (UAB), AL 35294 (United States); Vohra, Yogesh K [Center for Nanoscale Materials and Biointegration (CNMB), Department of Physics, University of Alabama at Birmingham (UAB), AL 35294 (United States)

    2007-12-15

    Electrospun tubular scaffolds (4 mm inner diameter) based on bio-artificial blends of polyglyconate (Maxon (registered) ) and proteins such as gelatin and elastin having a spatially designed multilayer structure were prepared for use as vascular tissue scaffolds. Scanning electron microscopy analysis of scaffolds showed a random nanofibrous morphology with fiber diameter in the range of 200-400 nm for protein-blended Maxon, which mimics the nanoscale dimensions of collagen (50-500 nm). The scaffolds have a well interconnected pore structure and porosity up to 82%, with protein blending and multi-layering in contrast to electrospun Maxon (registered) scaffolds (67%). Fourier-transform infrared spectroscopy, x-ray diffraction and differential scanning calorimetry results confirmed the blended composition and crystallinity of fibers. Uniaxial tensile testing revealed a strength of 14.46 {+-} 0.42 MPa and a modulus of 15.44 {+-} 2.53 MPa with a failure strain of 322.5 {+-} 10% for a pure Maxon (registered) scaffold. The blending of polyglyconate with biopolymers decreased the tensile properties in general, with an exception of the tensile modulus (48.38 {+-} 2 MPa) of gelatin/Maxon mesh, which was higher than that of the pure Maxon (registered) scaffold. Trilayered tubular scaffolds of gelatin/elastin, gelatin/elastin/Maxon and gelatin/Maxon (GE-GEM-GM) that mimic the complex trilayer matrix structure of natural artery have been prepared by sequential electrospinning. Tensile testing under dry conditions revealed a tensile strength of 2.71 {+-} 0.2 MPa and a modulus of 20.4 {+-} 3 MPa with a failure strain of 140 {+-} 10%. However, GE-GEM-GM scaffolds tested under wet conditions after soaking in a phosphate buffered saline medium at 37 {sup 0}C for 24 h exhibited mechanical properties (2.5 MPa tensile strength and 9 MPa tensile modulus) comparable to those of native femoral artery.

  1. Geometrical versus Random β-TCP Scaffolds: Exploring the Effects on Schwann Cell Growth and Behavior.

    Directory of Open Access Journals (Sweden)

    Lauren Sweet

    Full Text Available Numerous studies have demonstrated that Schwann cells (SCs play a role in nerve regeneration; however, their role in innervating a bioceramic scaffold for potential application in bone regeneration is still unknown. Here we report the cell growth and functional behavior of SCs on β-tricalcium phosphate (β-TCP scaffolds arranged in 3D printed-lattice (P-β-TCP and randomly-porous, template-casted (N-β-TCP structures. Our results indicate that SCs proliferated well and expressed the phenotypic markers p75LNGFR and the S100-β subunit of SCs as well as displayed growth morphology on both scaffolds, but SCs showed spindle-shaped morphology with a significant degree of SCs alignment on the P-β-TCP scaffolds, seen to a lesser degree in the N-β-TCP scaffold. The gene expressions of nerve growth factor (β-ngf, neutrophin-3 (nt-3, platelet-derived growth factor (pdgf-bb, and vascular endothelial growth factor (vegf-a were higher at day 7 than at day 14. While no significant differences in protein secretion were measured between these last two time points, the scaffolds promoted the protein secretion at day 3 compared to that on the cell culture plates. These results together imply that the β-TCP scaffolds can support SC cell growth and that the 3D-printed scaffold appeared to significantly promote the alignment of SCs along the struts. Further studies are needed to investigate the early and late stage relationship between gene expression and protein secretion of SCs on the scaffolds with refined characteristics, thus better exploring the potential of SCs to support vascularization and innervation in synthetic bone grafts.

  2. Microwave-assisted synthesis of porous chitosan-modified montmorillonite-hydroxyapatite composite scaffolds.

    Science.gov (United States)

    Kar, Sumanta; Kaur, Tejinder; Thirugnanam, A

    2016-01-01

    In this study, a porous chitosan-organically modified montmorillonite-hydroxyapatite (CS-OM-HA) composite scaffold was developed by combining microwave irradiation and gas foaming method. Hydroxyapatite (HA) particles of size ∼ 65 nm were synthesized and characterized by X-ray diffraction (XRD) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. The prepared composite scaffolds were characterized using ATR-FTIR, XRD, mercury intrusion porosimeter (MIP) and scanning electron microscopy (SEM) studies. The synergistic effect of HA and OM on the mechanical and in vitro biological properties (swelling, degradation, protein adsorption and bioactivity) of the composite scaffolds were evaluated. Swelling, degradation, mechanical property, bioactivity and protein adsorption studies of CS-OM-HA composite scaffolds have shown desirable results in comparison with the pure CS and CS-OM composite scaffolds. CS-OM-HA composite scaffolds were also found to be non-cytotoxic to MG 63 osteoblast cell lines. From the study, it can be concluded that the novel CS-OM-HA composite scaffold with improved mechanical and in vitro biological properties has wide potential in non-load bearing bone tissue engineering applications.

  3. The peripheral chromosome scaffold, a novel structural component of mitotic chromosomes.

    Science.gov (United States)

    Sheval, Eugene V; Polyakov, Vladimir Y

    2008-06-01

    Using an original high-salt extraction protocol, we observed a novel chromosome substructure, referred to as the peripheral chromosome scaffold. This chromosome domain contained the perichromosomal layer proteins pKi-67, B23/nucleophosmin and fibrillarin, but no DNA fragments (i.e., the loop domain bases were not associated with the peripheral scaffold). Modern models of chromosome organization do not predict the existence of a peripheral chromosome scaffold domain, and thus our observations have conceptual implications for understanding chromosome architecture. PMID:18337132

  4. Biomaterials & scaffolds for tissue engineering

    Directory of Open Access Journals (Sweden)

    Fergal J. O'Brien

    2011-03-01

    Full Text Available Every day thousands of surgical procedures are performed to replace or repair tissue that has been damaged through disease or trauma. The developing field of tissue engineering (TE aims to regenerate damaged tissues by combining cells from the body with highly porous scaffold biomaterials, which act as templates for tissue regeneration, to guide the growth of new tissue. This article describes the functional requirements, and types, of materials used in developing state of the art of scaffolds for tissue engineering applications. Furthermore, it describes the challenges and where future research and direction is required in this rapidly advancing field.

  5. MCR synthesis of a tetracyclic tetrazole scaffold

    NARCIS (Netherlands)

    Patil, Pravin; Khoury, Kareem; Herdtweck, Eberhardt; Dömling, Alexander

    2015-01-01

    Scaffold diversity is key in the ongoing exercise of discovery of novel bioactive compounds using high throughput screening (HTS). Based on the Ugi tetrazole synthesis we have designed novel bi- and tri-cyclic scaffolds featuring interesting pharmacophore properties. The compounds of the scaffold (B

  6. Rethinking Scaffolding in the Information Age

    Science.gov (United States)

    Yelland, Nicola; Masters, Jennifer

    2007-01-01

    This paper addresses the use of scaffolding in learning contexts that incorporate technologically based novel problems. We suggest that in computer contexts extended conceptualisations of scaffolding are needed in order to gain greater insights into teaching and learning processes. Our work has revealed that traditional forms of scaffolding, based…

  7. A new heterologous fibrin sealant as scaffold to recombinant human bone morphogenetic protein-2 (rhBMP-2) and natural latex proteins for the repair of tibial bone defects.

    Science.gov (United States)

    Machado, Eduardo Gomes; Issa, João Paulo Mardegan; Figueiredo, Fellipe Augusto Tocchini de; Santos, Geovane Ribeiro Dos; Galdeano, Ewerton Alexandre; Alves, Mariana Carla; Chacon, Erivelto Luis; Ferreira Junior, Rui Seabra; Barraviera, Benedito; Cunha, Marcelo Rodrigues da

    2015-04-01

    Tissue engineering has special interest in bone tissue aiming at future medical applications Studies have focused on recombinant human bone morphogenetic protein-2 (rhBMP-2) and natural latex proteins due to the osteogenic properties of rhBMP-2 and the angiogenic characteristic of fraction 1 protein (P-1) extracted from the rubber tree Hevea brasiliensis. Furthermore, heterologous fibrin sealant (FS) has been shown as a promising alternative in regenerative therapies. The aim of this study was to evaluate these substances for the repair of bone defects in rats. A bone defect measuring 3mm in diameter was created in the proximal metaphysis of the left tibia of 60 rats and was implanted with rhBMP-2 or P-1 in combination with a new heterologous FS derived from snake venom. The animals were divided into six groups: control (unfilled bone defect), rhBMP-2 (defect filled with 5μg rhBMP-2), P-1 (defect filled with 5μg P-1), FS (defect filled with 8μg FS), FS/rhBMP-2 (defect filled with 8μg FS and 5μg rhBMP-2), FS/P-1 (defect filled with 8μg FS and 5μg P-1). The animals were sacrificed 2 and 6 weeks after surgery. The newly formed bone projected from the margins of the original bone and exhibited trabecular morphology and a disorganized arrangement of osteocyte lacunae. Immunohistochemical analysis showed intense expression of osteocalcin in all groups. Histometric analysis revealed a significant difference in all groups after 2 weeks (p0.05). A statistically significant difference (p<0.05) was observed in all groups after 6 weeks in relation to the volume of newly formed bone in the surgical area. In conclusion, the new heterologous fibrin sealant was found to be biocompatible and the combination with rhBMP-2 showed the highest osteogenic and osteoconductive capacity for bone healing. These findings suggest a promising application of this combination in the regeneration surgery. PMID:25825118

  8. Inactivation of Tor proteins affects the dynamics of endocytic proteins in early stage of endocytosis

    Indian Academy of Sciences (India)

    Brandon Tenay; Evin Kimberlin; Michelle Williams; Juliette Denise; Joshua Fakilahyel; Kyoungtae Kim

    2013-06-01

    Tor2 is an activator of the Rom2/Rho1 pathway that regulates -factor internalization. Since the recruitment of endocytic proteins such as actin-binding proteins and the amphiphysins precedes the internalization of -factor, we hypothesized that loss of Tor function leads to an alteration in the dynamics of the endocytic proteins. We report here that endocytic proteins, Abp1 and Rvs167, are less recruited to endocytic sites not only in tor2 but also tor1 mutants. Furthermore, we found that the endocytic proteins Rvs167 and Sjl2 are completely mistargeted to the cytoplasm in tor1tor2ts double mutant cells. We also demonstrate here that the efficiency of endocytic internalization or scission in all tor mutants was drastically decreased. In agreement with the Sjl2 mislocalization, we found that in tor1tor2ts double mutant cells, as well as other tor mutant cells, the overall PIP2 level was dramatically increased. Finally, the cell wall chitin content in tor2ts and tor1tor2ts mutant cells was also significantly increased. Taken together, both functional Tor proteins, Tor1 and Tor2, are essentially required for proper endocytic protein dynamics at the early stage of endocytosis.

  9. Strategic Scaffolding for Scientific Inquiry

    Science.gov (United States)

    Shelton, Angela; Natarajan, Uma; Willard, Catherine; Kane, Tera; Ketelhut, Diane Jass; Schifter, Catherine

    2013-01-01

    Though many national and international science organizations stress the importance of integrating scientific inquiry into classroom instruction, this is often difficult for teachers. Moreover, assessing and scaffolding inquiry skills for students can be even more of a challenge. This paper investigated the student performances in an inquiry-based,…

  10. Membrane Domain Formation on Nanostructured Scaffolds

    Science.gov (United States)

    Collier, Charles; Liu, Fangjie; Srijanto, Bernadeta

    The spatial organization of lipids and proteins in biological membranes seems to have a functional role in the life of a cell. Separation of the lipids into distinct domains of greater order and anchoring to the cytoskeleton are two main mechanisms for organizing the membrane in cells. We propose a novel model membrane consisting of a lipid bilayer suspended over a nanostructured scaffold consisting of arrays of fabricated nanopillars. Unlike traditional model membranes, our model will have well-defined lateral structure and distributed substrate attachments that will emulate the connections of cellular membranes to the underlying cytoskeleton. Membranes will be characterized using neutron reflectometry, atomic force microscopy and fluorescence to verify a suspended, planar geometry with restricted diffusion at suspension points, and free diffusion in between. This architecture will allow the controlled study of lipid domain reorganization, viral infection and signal transduction that depend on the lateral structure of the membrane.

  11. Electrospun Scaffolds for Corneal Tissue Engineering: A Review

    Directory of Open Access Journals (Sweden)

    Bin Kong

    2016-07-01

    Full Text Available Corneal diseases constitute the second leading cause of vision loss and affect more than 10 million people globally. As there is a severe shortage of fresh donated corneas and an unknown risk of immune rejection with traditional heterografts, it is very important and urgent to construct a corneal equivalent to replace pathologic corneal tissue. Corneal tissue engineering has emerged as a practical strategy to develop corneal tissue substitutes, and the design of a scaffold with mechanical properties and transparency similar to that of natural cornea is paramount for the regeneration of corneal tissues. Nanofibrous scaffolds produced by electrospinning have high surface area–to-volume ratios and porosity that simulate the structure of protein fibers in native extra cellular matrix (ECM. The versatilities of electrospinning of polymer components, fiber structures, and functionalization have made the fabrication of nanofibrous scaffolds with suitable mechanical strength, transparency and biological properties for corneal tissue engineering feasible. In this paper, we review the recent developments of electrospun scaffolds for engineering corneal tissues, mainly including electrospun materials (single and blended polymers, fiber structures (isotropic or anisotropic, functionalization (improved mechanical properties and transparency, applications (corneal cell survival, maintenance of phenotype and formation of corneal tissue and future development perspectives.

  12. Polyurethane-based scaffolds for myocardial tissue engineering.

    Science.gov (United States)

    Chiono, Valeria; Mozetic, Pamela; Boffito, Monica; Sartori, Susanna; Gioffredi, Emilia; Silvestri, Antonella; Rainer, Alberto; Giannitelli, Sara Maria; Trombetta, Marcella; Nurzynska, Daria; Di Meglio, Franca; Castaldo, Clotilde; Miraglia, Rita; Montagnani, Stefania; Ciardelli, Gianluca

    2014-02-01

    Bi-layered scaffolds with a 0°/90° lay-down pattern were prepared by melt-extrusion additive manufacturing (AM) using a poly(ester urethane) (PU) synthesized from poly(ε-caprolactone) diol, 1,4-butandiisocyanate and l-lysine ethyl ester dihydrochloride chain extender. Rheological analysis and differential scanning calorimetry of the starting material showed that compression moulded PU films were in the molten state at a higher temperature than 155°C. The AM processing temperature was set at 155°C after verifying the absence of PU thermal degradation phenomena by isothermal thermogravimetry analysis and rheological characterization performed at 165°C. Scaffolds highly reproduced computer-aided design geometry and showed an elastomeric-like behaviour which is promising for applications in myocardial regeneration. PU scaffolds supported the adhesion and spreading of human cardiac progenitor cells (CPCs), whereas they did not stimulate CPC proliferation after 1-14 days culture time. In the future, scaffold surface functionalization with bioactive peptides/proteins will be performed to specifically guide CPC behaviour.

  13. Building bone tissue: matrices and scaffolds in physiology and biotechnology

    Directory of Open Access Journals (Sweden)

    Riminucci M.

    2003-01-01

    Full Text Available Deposition of bone in physiology involves timed secretion, deposition and removal of a complex array of extracellular matrix proteins which appear in a defined temporal and spatial sequence. Mineralization itself plays a role in dictating and spatially orienting the deposition of matrix. Many aspects of the physiological process are recapitulated in systems of autologous or xenogeneic transplantation of osteogenic precursor cells developed for tissue engineering or modeling. For example, deposition of bone sialoprotein, a member of the small integrin-binding ligand, N-linked glycoprotein family, represents the first step of bone formation in ectopic transplantation systems in vivo. The use of mineralized scaffolds for guiding bone tissue engineering has revealed unexpected manners in which the scaffold and cells interact with each other, so that a complex interplay of integration and disintegration of the scaffold ultimately results in efficient and desirable, although unpredictable, effects. Likewise, the manner in which biomaterial scaffolds are "resorbed" by osteoclasts in vitro and in vivo highlights more complex scenarios than predicted from knowledge of physiological bone resorption per se. Investigation of novel biomaterials for bone engineering represents an essential area for the design of tissue engineering strategies.

  14. Polyurethane-based scaffolds for myocardial tissue engineering.

    Science.gov (United States)

    Chiono, Valeria; Mozetic, Pamela; Boffito, Monica; Sartori, Susanna; Gioffredi, Emilia; Silvestri, Antonella; Rainer, Alberto; Giannitelli, Sara Maria; Trombetta, Marcella; Nurzynska, Daria; Di Meglio, Franca; Castaldo, Clotilde; Miraglia, Rita; Montagnani, Stefania; Ciardelli, Gianluca

    2014-02-01

    Bi-layered scaffolds with a 0°/90° lay-down pattern were prepared by melt-extrusion additive manufacturing (AM) using a poly(ester urethane) (PU) synthesized from poly(ε-caprolactone) diol, 1,4-butandiisocyanate and l-lysine ethyl ester dihydrochloride chain extender. Rheological analysis and differential scanning calorimetry of the starting material showed that compression moulded PU films were in the molten state at a higher temperature than 155°C. The AM processing temperature was set at 155°C after verifying the absence of PU thermal degradation phenomena by isothermal thermogravimetry analysis and rheological characterization performed at 165°C. Scaffolds highly reproduced computer-aided design geometry and showed an elastomeric-like behaviour which is promising for applications in myocardial regeneration. PU scaffolds supported the adhesion and spreading of human cardiac progenitor cells (CPCs), whereas they did not stimulate CPC proliferation after 1-14 days culture time. In the future, scaffold surface functionalization with bioactive peptides/proteins will be performed to specifically guide CPC behaviour. PMID:24501673

  15. Selective laser sintering fabrication of nano-hydroxyapatite/poly-ε-caprolactone scaffolds for bone tissue engineering applications

    Directory of Open Access Journals (Sweden)

    Xia Y

    2013-11-01

    Full Text Available Yan Xia,1,* Panyu Zhou,1,* Xiaosong Cheng,1,* Yang Xie,1,* Chong Liang,2 Chao Li,1 Shuogui Xu1,2 1Department of Orthopedics, Changhai Hospital, Second Military Medical University, Shanghai, People's Republic of China; 2Department of Neurosurgery, The 81 Hospital of People's Liberation Army of China, Nanjing, People's Republic of China *These authors contributed equally to this work Abstract: The regeneration of functional tissue in osseous defects is a formidable challenge in orthopedic surgery. In the present study, a novel biomimetic composite scaffold, here called nano-hydroxyapatite (HA/poly-ε-caprolactone (PCL was fabricated using a selective laser sintering technique. The macrostructure, morphology, and mechanical strength of the scaffolds were characterized. Scanning electronic microscopy (SEM showed that the nano-HA/PCL scaffolds exhibited predesigned, well-ordered macropores and interconnected micropores. The scaffolds have a range of porosity from 78.54% to 70.31%, and a corresponding compressive strength of 1.38 MPa to 3.17 MPa. Human bone marrow stromal cells were seeded onto the nano-HA/PCL or PCL scaffolds and cultured for 28 days in vitro. As indicated by the level of cell attachment and proliferation, the nano-HA/PCL showed excellent biocompatibility, comparable to that of PCL scaffolds. The hydrophilicity, mineralization, alkaline phosphatase activity, and Alizarin Red S staining indicated that the nano-HA/PCL scaffolds are more bioactive than the PCL scaffolds in vitro. Measurements of recombinant human bone morphogenetic protein-2 (rhBMP-2 release kinetics showed that after nano-HA was added, the material increased the rate of rhBMP-2 release. To investigate the in vivo biocompatibility and osteogenesis of the composite scaffolds, both nano-HA/PCL scaffolds and PCL scaffolds were implanted in rabbit femur defects for 3, 6, and 9 weeks. The wounds were studied radiographically and histologically. The in vivo results showed

  16. Proteomic analysis of ACTN4-interacting proteins reveals it's a putative involvement in mRNA metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Khotin, Mikhail, E-mail: h_mg@mail.ru [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Turoverova, Lidia [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Aksenova, Vasilisa [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Department of Genetics, St. Petersburg State University, Universitetskaya nab., 7/9, 199034 St. Petersburg (Russian Federation); Barlev, Nikolai [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN (United Kingdom); Borutinskaite, Veronika Viktorija [Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Department of Developmental Biology, Institute of Biochemistry, LT-08662 Vilnius (Lithuania); Vener, Alexander [Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Bajenova, Olga [Department of Genetics, St. Petersburg State University, Universitetskaya nab., 7/9, 199034 St. Petersburg (Russian Federation); Magnusson, Karl-Eric [Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linkoeping University, SE-581 85 Linkoeping (Sweden); Pinaev, George P. [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation); Tentler, Dmitri, E-mail: dtentler@mail.cytspb.rssi.ru [Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky av., 4, 194064 St. Petersburg (Russian Federation)

    2010-06-25

    Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

  17. Biomimetic Mineralized Hierarchical Graphene Oxide/Chitosan Scaffolds with Adsorbability for Immobilization of Nanoparticles for Biomedical Applications.

    Science.gov (United States)

    Xie, Chaoming; Lu, Xiong; Han, Lu; Xu, Jielong; Wang, Zhenming; Jiang, Lili; Wang, Kefeng; Zhang, Hongping; Ren, Fuzeng; Tang, Youhong

    2016-01-27

    Biomimetic calcium phosphate mineralized graphene oxide/chitosan (GO/CS) scaffolds with hierarchical structures were developed. First, GO/CS scaffolds with large micropores (∼300 μm) showed high mechanical strength due to the electrostatic interaction between the oxygen-containing functional groups of GO and the amine groups of CS. Second, octacalcuim phosphate (OCP) with porous structures (∼1 μm) was biomimetically mineralized on the surfaces of the GO/CS scaffolds (OCP-GO/CS). The hierarchical microporous structures of OCP-GO/CS scaffolds provide a suitable environment for cell adhesion and growth. The scaffolds have exceptional adsorbability of nanoparticles. Bone morphogenetic protein-2 (BMP-2)-encapsulated bovine serum albumin (BSA) nanoparticles and Ag nanoparticles (Ag-NPs) were adsorbed in the scaffolds for enhancement of osteoinductivity and antibacterial properties, respectively. Antibacterial tests showed that the scaffolds exhibited high antibacterial properties against both Escherichia coli and Staphylococcus epidermidis. In vitro and in vivo experiments revealed that the scaffolds have good biocompatibility, enhanced bone marrow stromal cells proliferation and differentiation, and induced bone tissue regeneration. Thus, the biomimetic OCP-GO/CS scaffolds with immobilized growth factors and antibacterial agents might be excellent candidates for bone tissue engineering. PMID:26710937

  18. The Selective Autophagy Receptor p62 Forms a Flexible Filamentous Helical Scaffold

    Directory of Open Access Journals (Sweden)

    Rodolfo Ciuffa

    2015-05-01

    Full Text Available The scaffold protein p62/SQSTM1 is involved in protein turnover and signaling and is commonly found in dense protein bodies in eukaryotic cells. In autophagy, p62 acts as a selective autophagy receptor that recognizes and shuttles ubiquitinated proteins to the autophagosome for degradation. The structural organization of p62 in cellular bodies and the interplay of these assemblies with ubiquitin and the autophagic marker LC3 remain to be elucidated. Here, we present a cryo-EM structural analysis of p62. Together with structures of assemblies from the PB1 domain, we show that p62 is organized in flexible polymers with the PB1 domain constituting a helical scaffold. Filamentous p62 is capable of binding LC3 and addition of long ubiquitin chains induces disassembly and shortening of filaments. These studies explain how p62 assemblies provide a large molecular scaffold for the nascent autophagosome and reveal how they can bind ubiquitinated cargo.

  19. A scaffold-enhanced light-activated surgical adhesive technique: surface selection for enhanced tensile strength in wound repair

    Science.gov (United States)

    Soller, Eric C.; Hoffman, Grant T.; Heintzelman, Douglas L.; Duffy, Mark T.; Bloom, Jeffrey N.; McNally-Heintzelman, Karen M.

    2004-07-01

    An ex vivo study was conducted to determine the effect of the irregularity of the scaffold surface on the tensile strength of repairs formed using our Scaffold-Enhanced Biological Adhesive (SEBA). Two different scaffold materials were investigated: (i) a synthetic biodegradable material fabricated from poly(L-lactic-co-glycolic acid); and (ii) a biological material, small intestinal submucosa, manufactured by Cook BioTech. The scaffolds were doped with protein solder composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser. The tensile strength of repairs performed on bovine thoracic aorta, liver, spleen, small intestine and lung, using the smooth and irregular surfaces of the above scaffold-enhanced materials were measured and the time-to-failure was recorded. The tensile strength of repairs formed using the irregular surfaces of the scaffolds were consistently higher than those formed using the smooth surfaces of the scaffolds. The largest difference was observed on repairs formed on the aorta and small intestine, where the repairs were, on average, 50% stronger using the irregular versus the smooth scaffold surfaces. In addition, the time-to-failure of repairs formed using the irregular surfaces of the scaffolds were between 50% and 100% longer than that achieved using the smooth surfaces of the scaffolds. It has previously been shown that distributing or dispersing the adhesive forces over the increased surface area of the scaffold, either smooth or irregular, produces stronger repairs than albumin solder alone. The increase in the absolute strength and longevity of repairs seen in this new study when the irregular surfaces of the scaffolds are used is thought to be due to the distribution of forces between the many independent micro-adhesions provided by the irregular surfaces.

  20. 双层蛛丝蛋白血管支架的体外降解研究%The study on in vitro degradation of bilayer spider silk protein vascular scaffold

    Institute of Scientific and Technical Information of China (English)

    赵亮; 徐艳丽; 何孟; 李敏; 陈雨晴

    2013-01-01

    血管支架的制备成为治疗心血管疾病的必需条件。应用静电纺丝技术制备了(pNSR16/PCL/CS)/(pNSR16/PCL/Gt)双层血管支架,并研究其在磷酸盐缓冲液(pH 值=7.4)和多酶降解液中浸泡不同时间的体外降解情况。于2、4、8、12周分别取材,测试失重率、吸水率、降解液的 pH 值、力学性能和分子量的变化,并进行扫描电镜观察。结果表明,(pNSR16/PCL/CS)/(pNSR16/PCL/Gt)双层血管支架的初始抗弯强度和初始分子量大于(PCL/CS )/(PCL/Gt)支架,并且前者的降解速度、分子量和失重率减少速度快于后者,pNSR16的添加促进了血管支架的降解。在降解过程中,浸泡液 pH 值呈弱酸性和中性,前期迅速降低并在后期呈现稳定的趋势。支架在酶解液中的降解速度快于在水解液中。%The preparation of vascular scaffold becomes a necessary condition for cure for cardiovascular disease. In this paper electrospinning technique was used to prepare the (pNSR1 6/PCL/CS)/(pNSR1 6/PCL/Gt)bilayer vascular scaffold,and its degradation in vitro was studied after being soaked in phosphate buffer solution (pH=7.14)and multiple enzyme solution for different time.The changes of weight loss rate,water absorption rate, pH value of degradation solution,mechanical property and molecular weight were tested after sampling at 2nd, 4th,8ht,12th week respectively,and at the same time the morphology was observed using scanning electron microscope.The results showed that the initial bending strength and molecular weight of (pNSR1 6/PCL/CS)/(pNSR1 6/PCL/Gt)bilayer vascular scaffold were greater than those of (PCL/CS)/(PCL/Gt),the degradation velocity and decreasement velocity of molecular weight and weight loss rate of the former were faster than the latter,and the addition of pNSR1 6 promoted the degradation of vascular scaffold.In the process of degradation, pH value of degradation solution was weakly acidic and neutral,the scaffold decreased rapidly

  1. Improving effects of chitosan nanofiber scaffolds on osteoblast proliferation and maturation.

    Science.gov (United States)

    Ho, Ming-Hua; Liao, Mei-Hsiu; Lin, Yi-Ling; Lai, Chien-Hao; Lin, Pei-I; Chen, Ruei-Ming

    2014-01-01

    Osteoblast maturation plays a key role in regulating osteogenesis. Electrospun nanofibrous products were reported to possess a high surface area and porosity. In this study, we developed chitosan nanofibers and examined the effects of nanofibrous scaffolds on osteoblast maturation and the possible mechanisms. Macro- and micro observations of the chitosan nanofibers revealed that these nanoproducts had a flat surface and well-distributed fibers with nanoscale diameters. Mouse osteoblasts were able to attach onto the chitosan nanofiber scaffolds, and the scaffolds degraded in a time-dependent manner. Analysis by scanning electron microscopy further showed mouse osteoblasts adhered onto the scaffolds along the nanofibers, and cell-cell communication was also detected. Mouse osteoblasts grew much better on chitosan nanofiber scaffolds than on chitosan films. In addition, human osteoblasts were able to adhere and grow on the chitosan nanofiber scaffolds. Interestingly, culturing human osteoblasts on chitosan nanofiber scaffolds time-dependently increased DNA replication and cell proliferation. In parallel, administration of human osteoblasts onto chitosan nanofibers significantly induced osteopontin, osteocalcin, and alkaline phosphatase (ALP) messenger (m)RNA expression. As to the mechanism, chitosan nanofibers triggered runt-related transcription factor 2 mRNA and protein syntheses. Consequently, results of ALP-, alizarin red-, and von Kossa-staining analyses showed that chitosan nanofibers improved osteoblast mineralization. Taken together, results of this study demonstrate that chitosan nanofibers can stimulate osteoblast proliferation and maturation via runt-related transcription factor 2-mediated regulation of osteoblast-associated osteopontin, osteocalcin, and ALP gene expression. PMID:25246786

  2. Fabrication and biocompatibility of poly(l-lactic acid) and chitosan composite scaffolds with hierarchical microstructures.

    Science.gov (United States)

    Lou, Tao; Wang, Xuejun; Yan, Xu; Miao, Yu; Long, Yun-Ze; Yin, Hai-Lei; Sun, Bin; Song, Guojun

    2016-07-01

    The scaffold microstructure is crucial to reconstruct tissue normal functions. In this article, poly(l-lactic acid) and chitosan fiber (PLLA/CTSF) composite scaffolds with hierarchical microstructures both in fiber and pore sizes were successfully fabricated by combining thermal induced phase separation and salt leaching techniques. The composite scaffolds consisted of a nanofibrous PLLA matrix with diameter of 50-500nm, and chitosan fibers with diameter of about 20μm were homogenously distributed in the PLLA matrix as a microsized reinforcer. The composite scaffolds also had high porosity (>94%) and hierarchical pore size, which were consisted of both micropores (50nm-10μm) and macropores (50-300μm). By tailoring the microstructure and chemical composition, the mechanical property, pH buffer and protein adsorption capacity of the composite scaffold were improved significantly compared with those of PLLA scaffold. Cell culture results also revealed that the PLLA/CTSF composite scaffolds supported MG-63 osteoblast proliferation and penetration. PMID:27127062

  3. Selective functionalization of nanofiber scaffolds to regulate salivary gland epithelial cell proliferation and polarity.

    Science.gov (United States)

    Cantara, Shraddha I; Soscia, David A; Sequeira, Sharon J; Jean-Gilles, Riffard P; Castracane, James; Larsen, Melinda

    2012-11-01

    Epithelial cell types typically lose apicobasal polarity when cultured on 2D substrates, but apicobasal polarity is required for directional secretion by secretory cells, such as salivary gland acinar cells. We cultured salivary gland epithelial cells on poly(lactic-co-glycolic acid) (PLGA) nanofiber scaffolds that mimic the basement membrane, a specialized extracellular matrix, and examined cell proliferation and apicobasal polarization. Although cells proliferated on nanofibers, chitosan-coated nanofiber scaffolds stimulated proliferation of salivary gland epithelial cells. Although apicobasal cell polarity was promoted by the nanofiber scaffolds relative to flat surfaces, as determined by the apical localization of ZO-1, it was antagonized by the presence of chitosan. Neither salivary gland acinar nor ductal cells fully polarized on the nanofiber scaffolds, as determined by the homogenous membrane distribution of the mature tight junction marker, occludin. However, nanofiber scaffolds chemically functionalized with the basement membrane protein, laminin-111, promoted more mature tight junctions, as determined by apical localization of occludin, but did not affect cell proliferation. To emulate the multifunctional capabilities of the basement membrane, bifunctional PLGA nanofibers were generated. Both acinar and ductal cell lines responded to signals provided by bifunctional scaffolds coupled to chitosan and laminin-111, demonstrating the applicability of such scaffolds for epithelial cell types.

  4. Enhancement of neurite outgrowth in neuron cancer stem cells by growth on 3-D collagen scaffolds

    International Nuclear Information System (INIS)

    Highlights: ► Neuron cancer stem cells (NCSCs) behave high multiply of growth on collagen scaffold. ► Enhancement of NCSCs neurite outgrowth on porous collagen scaffold. ► 3-D collagen culture of NCSCs shows an advance differentiation than 2-D culture. -- Abstract: Collagen is one component of the extracellular matrix that has been widely used for constructive remodeling to facilitate cell growth and differentiation. The 3-D distribution and growth of cells within the porous scaffold suggest a clinical significance for nerve tissue engineering. In the current study, we investigated proliferation and differentiation of neuron cancer stem cells (NCSCs) on a 3-D porous collagen scaffold that mimics the natural extracellular matrix. We first generated green fluorescence protein (GFP) expressing NCSCs using a lentiviral system to instantly monitor the transitions of morphological changes during growth on the 3-D scaffold. We found that proliferation of GFP-NCSCs increased, and a single cell mass rapidly grew with unrestricted expansion between days 3 and 9 in culture. Moreover, immunostaining with neuronal nuclei (NeuN) revealed that NCSCs grown on the 3-D collagen scaffold significantly enhanced neurite outgrowth. Our findings confirmed that the 80 μm porous collagen scaffold could enhance attachment, viability and differentiation of the cancer neural stem cells. This result could provide a new application for nerve tissue engineering and nerve regeneration.

  5. The CNK2 Scaffold Interacts with Vilse/ARHGAP39 and Modulates Rac Cycling During Morphogenesis of Dendritic Spines

    OpenAIRE

    Lim, Junghwa; Ritt, Daniel A.; Zhou, Ming; Morrison, Deborah K.

    2014-01-01

    Protein scaffolds play an important role in signal transduction, functioning to facilitate protein interactions and localize key pathway components to specific signaling sites. Connector enhancer of KSR-2 (CNK2) is a neuronally-expressed scaffold recently implicated in non-syndromic, X-linked mental retardation (MRX) [1–3]. MRX patients have deficits in cognitive function and their neurons often exhibit dendritic spine abnormalities [4], suggesting a role for CNK2 in synaptic signaling and/or...

  6. Fabrication and evaluation of a sustained-release chitosan-based scaffold embedded with PLGA microspheres

    International Nuclear Information System (INIS)

    Nutrient depletion within three-dimensional (3D) scaffolds is one of the major hurdles in the use of this technology to grow cells for applications in tissue engineering. In order to help in addressing it, we herein propose to use the controlled release of encapsulated nutrients within polymer microspheres into chitosan-based 3D scaffolds, wherein the microspheres are embedded. This method has allowed maintaining a stable concentration of nutrients within the scaffolds over the long term. The polymer microspheres were prepared using multiple emulsions (w/o/w), in which bovine serum albumin (BSA) and poly (lactic-co-glycolic) acid (PLGA) were regarded as the protein pattern and the exoperidium material, respectively. These were then mixed with a chitosan solution in order to form the scaffolds by cryo-desiccation. The release of BSA, entrapped within the embedded microspheres, was monitored with time using a BCA kit. The morphology and structure of the PLGA microspheres containing BSA before and after embedding within the scaffold were observed under a scanning electron microscope (SEM). These had a round shape with diameters in the range of 27–55 μm, whereas the chitosan-based scaffolds had a uniform porous structure with the microspheres uniformly dispersed within their 3D structure and without any morphological change. In addition, the porosity, water absorption and degradation rate at 37 °C in an aqueous environment of 1% chitosan-based scaffolds were (92.99 2.51) %, (89.66 ± 0.66) % and (73.77 ± 3.21) %, respectively. The studies of BSA release from the embedded microspheres have shown a sustained and cumulative tendency with little initial burst, with (20.24 ± 0.83) % of the initial amount released after 168 h (an average rate of 0.12%/h). The protein concentration within the chitosan-based scaffolds after 168 h was found to be (11.44 ± 1.81) × 10−2 mg/mL. This novel chitosan-based scaffold embedded with PLGA microspheres has proven to be a

  7. Fabrication and evaluation of a sustained-release chitosan-based scaffold embedded with PLGA microspheres

    Energy Technology Data Exchange (ETDEWEB)

    Song, Kedong, E-mail: kedongsong@dlut.edu.cn [Dalian R and D Center for Stem Cell and Tissue Engineering, State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024 (China); Liu, Yingchao [Dalian R and D Center for Stem Cell and Tissue Engineering, State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024 (China); Macedo, Hugo M. [Biological Systems Engineering Laboratory, Department of Chemical Engineering, Department of Chemical Engineering, South Kensington Campus, London SW7 2AZ (United Kingdom); Jiang, Lili; Li, Chao; Mei, Guanyu [Dalian R and D Center for Stem Cell and Tissue Engineering, State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024 (China); Liu, Tianqing, E-mail: liutq@dlut.edu.cn [Dalian R and D Center for Stem Cell and Tissue Engineering, State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024 (China)

    2013-04-01

    Nutrient depletion within three-dimensional (3D) scaffolds is one of the major hurdles in the use of this technology to grow cells for applications in tissue engineering. In order to help in addressing it, we herein propose to use the controlled release of encapsulated nutrients within polymer microspheres into chitosan-based 3D scaffolds, wherein the microspheres are embedded. This method has allowed maintaining a stable concentration of nutrients within the scaffolds over the long term. The polymer microspheres were prepared using multiple emulsions (w/o/w), in which bovine serum albumin (BSA) and poly (lactic-co-glycolic) acid (PLGA) were regarded as the protein pattern and the exoperidium material, respectively. These were then mixed with a chitosan solution in order to form the scaffolds by cryo-desiccation. The release of BSA, entrapped within the embedded microspheres, was monitored with time using a BCA kit. The morphology and structure of the PLGA microspheres containing BSA before and after embedding within the scaffold were observed under a scanning electron microscope (SEM). These had a round shape with diameters in the range of 27–55 μm, whereas the chitosan-based scaffolds had a uniform porous structure with the microspheres uniformly dispersed within their 3D structure and without any morphological change. In addition, the porosity, water absorption and degradation rate at 37 °C in an aqueous environment of 1% chitosan-based scaffolds were (92.99 ± 2.51) %, (89.66 ± 0.66) % and (73.77 ± 3.21) %, respectively. The studies of BSA release from the embedded microspheres have shown a sustained and cumulative tendency with little initial burst, with (20.24 ± 0.83) % of the initial amount released after 168 h (an average rate of 0.12%/h). The protein concentration within the chitosan-based scaffolds after 168 h was found to be (11.44 ± 1.81) × 10{sup −2} mg/mL. This novel chitosan-based scaffold embedded with PLGA microspheres has proven to be a

  8. Construction of a 3D rGO-collagen hybrid scaffold for enhancement of the neural differentiation of mesenchymal stem cells

    Science.gov (United States)

    Guo, Weibo; Wang, Shu; Yu, Xin; Qiu, Jichuan; Li, Jianhua; Tang, Wei; Li, Zhou; Mou, Xiaoning; Liu, Hong; Wang, Zhonglin

    2016-01-01

    The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold because the surface of the scaffold can determine the fate of stem cells. A conductive surface is required for a scaffold to direct stem cells toward neural differentiation. However, most conductive polymers are toxic and not amenable to biological degradation, which restricts the design of neural tissue engineering scaffolds. In this study, we used a bioactive three-dimensional (3D) porcine acellular dermal matrix (PADM), which is mainly composed of type I collagen, as a basic material and successfully assembled a layer of reduced graphene oxide (rGO) nanosheets on the surface of the PADM channels to obtain a porous 3D, biodegradable, conductive and biocompatible PADM-rGO hybrid neural tissue engineering scaffold. Compared with the PADM scaffold, assembling the rGO into the scaffold did not induce a significant change in the microstructure but endowed the PADM-rGO hybrid scaffold with good conductivity. A comparison of the neural differentiation of rat bone-marrow-derived mesenchymal stem cells (MSCs) was performed by culturing the MSCs on PADM and PADM-rGO scaffolds in neuronal culture medium, followed by the determination of gene expression and immunofluorescence staining. The results of both the gene expression and protein level assessments suggest that the rGO-assembled PADM scaffold may promote the differentiation of MSCs into neuronal cells with higher protein and gene expression levels after 7 days under neural differentiation conditions. This study demonstrated that the PADM-rGO hybrid scaffold is a promising scaffold for neural tissue engineering; this scaffold can not only support the growth of MSCs at a high proliferation rate but also enhance the differentiation of MSCs into neural cells.The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold

  9. Ceramic Identity Contributes to Mechanical Properties and Osteoblast Behavior on Macroporous Composite Scaffolds

    Directory of Open Access Journals (Sweden)

    J. Kent Leach

    2012-05-01

    Full Text Available Implants formed of metals, bioceramics, or polymers may provide an alternative to autografts for treating large bone defects. However, limitations to each material motivate the examination of composites to capitalize on the beneficial aspects of individual components and to address the need for conferring bioactive behavior to the polymer matrix. We hypothesized that the inclusion of different bioceramics in a ceramic-polymer composite would alter the physical properties of the implant and the cellular osteogenic response. To test this, composite scaffolds formed from poly(lactide-co-glycolide (PLG and either hydroxyapatite (HA, β-tricalcium phosphate (TCP, or bioactive glass (Bioglass 45S®, BG were fabricated, and the physical properties of each scaffold were examined. We quantified cell proliferation by DNA content, osteogenic response of human osteoblasts (NHOsts to composite scaffolds by alkaline phosphatase (ALP activity, and changes in gene expression by qPCR. Compared to BG-PLG scaffolds, HA-PLG and TCP-PLG composite scaffolds possessed greater compressive moduli. NHOsts on BG-PLG substrates exhibited higher ALP activity than those on control, HA-, or TCP-PLG scaffolds after 21 days, and cells on composites exhibited a 3-fold increase in ALP activity between 7 and 21 days versus a minimal increase on control scaffolds. Compared to cells on PLG controls, RUNX2 expression in NHOsts on composite scaffolds was lower at both 7 and 21 days, while expression of genes encoding for bone matrix proteins (COL1A1 and SPARC was higher on BG-PLG scaffolds at both time points. These data demonstrate the importance of selecting a ceramic when fabricating composites applied for bone healing.

  10. Interphotoreceptor matrix-poly(ϵ-caprolactone composite scaffolds for human photoreceptor differentiation

    Directory of Open Access Journals (Sweden)

    Petr Baranov

    2014-10-01

    Full Text Available Tissue engineering has been widely applied in different areas of regenerative medicine, including retinal regeneration. Typically, artificial biopolymers require additional surface modification (e.g. with arginine–glycine–aspartate-containing peptides or adsorption of protein, such as fibronectin, before cell seeding. Here, we describe an alternative approach for scaffold design: the manufacture of hybrid interphotoreceptor matrix-poly (ϵ-caprolactone scaffolds, in which the insoluble extracellular matrix of the retina is incorporated into a biodegradable polymer well suited for transplantation. The incorporation of interphotoreceptor matrix did not change the topography of polycaprolactone film, although it led to a slight increase in hydrophilic properties (water contact angle measurements. This hybrid scaffold provided sufficient stimuli for human retinal progenitor cell adhesion and inhibited proliferation, leading to differentiation toward photoreceptor cells (expression of Crx, Nrl, rhodopsin, ROM1. This scaffold may be used for transplantation of retinal progenitor cells and their progeny to treat retinal degenerative disorders.

  11. Polyacrylamide scaffolds for studying cellular response to substrate stiffness in three dimensions

    Science.gov (United States)

    Lin, Keng-Hui

    2013-03-01

    Recent developments in two-dimensional (2D) culture substrates with tunable stiffness and patterned adhesion ligands have demonstrated that biochemical and mechanical cues regulate the biological functions of living cells. We have extended these cell culture platforms into three dimensions (3D), as in complex biological systems, by producing highly ordered scaffolds of polyacrylamide coated with extracellular matrix proteins. We characterized parameters for the scaffold fabrication. We then grew individual fibroblasts in the identical pores of our scaffolds, examing cellular morphological, cytoskeletal, and adhesion properties. We have observed rich variety of morphologies and anchoring strategies assumed by cells growing on our tunable 3D polyacrylamide scaffolds to demonstrate the richness of cell-mciroenvironment interactions when cell adhesions are not confined to 2D surfaces.

  12. Tropomyosin is localized in the nuclear matrix and chromosome scaffold of physarum polycephalum

    Institute of Scientific and Technical Information of China (English)

    ZENGXIANLU; XIAOGUANGWANG; 等

    1999-01-01

    The nuclei and chromosomes were isolated from plasmodia of Physarum polycephalum.The nuclear matrix and chromosome scaffold were obtained after the DNA and most of the proteins were extracted with DNase I and 2 M NaCl.SD-PAGE analyses revealed that the nuclear matrix and chromosome scaffold contained a 37 kD polypeptide which is equivalent to tropomyosin in molecular weight.Immunofluorescence observations upon slide preparations labeled with anti-tropomyosin antibody showed that the nuclear matrix and chromosome scaffold emanated bright fluorescence,suggesting the presence of the antigen in them.Immunodotting results confirmed the presence of tropomyosin in the nuclear matrix and chromosome scaffold.Immunoelectron microscopic observations further demonstrated that tropomyosin was dispersively distributed in the interphase nuclei and metaphase chromosomes.

  13. Semiotic Scaffolding in Living Systems

    DEFF Research Database (Denmark)

    Hoffmeyer, Jesper

    2008-01-01

    The apparently purposeful nature of living systems is obtained through a sophisticated network of semiotic controls whereby biochemical, physiological and behavioral processes become tuned to the needs of the system. The operation of these semiotic controls takes place and is enabled across...... a diversity of levels. Such semiotic controls may be distinguished from ordinary deterministic control mechanisms through an inbuilt anticipatory capacity based on a distinct kind of causation that I call here "semiotic causation" to denote the bringing about of changes under the guidance of interpretation...... for living systems. Semiotic scaffolding safeguards the optimal performance of organisms through semiotic interaction with cue elements which are characteristically present in dynamic situations. At the cellular level, semiotic scaffolding assures the proper integration of the digital coding system (the...

  14. Biologically improved nanofibrous scaffolds for cardiac tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Bhaarathy, V. [Centre for Nanofibers and Nanotechnology, NUSNNI, Faculty of Engineering, National University of Singapore, 117576 (Singapore); Department of Nanoscience and Technology, School of Physical Sciences, Bharathiar University, Coimbatore 641046 (India); Lee Kong Chian School of Medicine, Nanyang Technological University, 138673 (Singapore); Venugopal, J., E-mail: nnijrv@nus.edu.sg [Centre for Nanofibers and Nanotechnology, NUSNNI, Faculty of Engineering, National University of Singapore, 117576 (Singapore); Gandhimathi, C. [Centre for Nanofibers and Nanotechnology, NUSNNI, Faculty of Engineering, National University of Singapore, 117576 (Singapore); Ponpandian, N.; Mangalaraj, D. [Department of Nanoscience and Technology, School of Physical Sciences, Bharathiar University, Coimbatore 641046 (India); Ramakrishna, S. [Centre for Nanofibers and Nanotechnology, NUSNNI, Faculty of Engineering, National University of Singapore, 117576 (Singapore)

    2014-11-01

    Nanofibrous structure developed by electrospinning technology provides attractive extracellular matrix conditions for the anchorage, migration and differentiation of stem cells, including those responsible for regenerative medicine. Recently, biocomposite nanofibers consisting of two or more polymeric blends are electrospun more tidily in order to obtain scaffolds with desired functional and mechanical properties depending on their applications. The study focuses on one such an attempt of using copolymer Poly(L-lactic acid)-co-poly (ε-caprolactone) (PLACL), silk fibroin (SF) and Aloe Vera (AV) for fabricating biocomposite nanofibrous scaffolds for cardiac tissue engineering. SEM micrographs of fabricated electrospun PLACL, PLACL/SF and PLACL/SF/AV nanofibrous scaffolds are porous, beadless, uniform nanofibers with interconnected pores and obtained fibre diameter in the range of 459 ± 22 nm, 202 ± 12 nm and 188 ± 16 nm respectively. PLACL, PLACL/SF and PLACL/SF/AV electrospun mats obtained at room temperature with an elastic modulus of 14.1 ± 0.7, 9.96 ± 2.5 and 7.0 ± 0.9 MPa respectively. PLACL/SF/AV nanofibers have more desirable properties to act as flexible cell supporting scaffolds compared to PLACL for the repair of myocardial infarction (MI). The PLACL/SF and PLACL/SF/AV nanofibers had a contact angle of 51 ± 12° compared to that of 133 ± 15° of PLACL alone. Cardiac cell proliferation was increased by 21% in PLACL/SF/AV nanofibers compared to PLACL by day 6 and further increased to 42% by day 9. Confocal analysis for cardiac expression proteins myosin and connexin 43 was observed better by day 9 compared to all other nanofibrous scaffolds. The results proved that the fabricated PLACL/SF/AV nanofibrous scaffolds have good potentiality for the regeneration of infarcted myocardium in cardiac tissue engineering. - Highlights: • Fabricated nanofibrous scaffolds are porous, beadless and uniform structures. • PLACL/SF/AV nanofibers improve the

  15. Significant type I and type III collagen production from human periodontal ligament fibroblasts in 3D peptide scaffolds without extra growth factors.

    Directory of Open Access Journals (Sweden)

    Yoshiyuki Kumada

    Full Text Available We here report the development of two peptide scaffolds designed for periodontal ligament fibroblasts. The scaffolds consist of one of the pure self-assembling peptide scaffolds RADA16 through direct coupling to short biologically active motifs. The motifs are 2-unit RGD binding sequence PRG (PRGDSGYRGDS and laminin cell adhesion motif PDS (PDSGR. RGD and laminin have been previously shown to promote specific biological activities including periodontal ligament fibroblasts adhesion, proliferation and protein production. Compared to the pure RADA16 peptide scaffold, we here show that these designer peptide scaffolds significantly promote human periodontal ligament fibroblasts to proliferate and migrate into the scaffolds (for approximately 300 microm/two weeks. Moreover these peptide scaffolds significantly stimulated periodontal ligament fibroblasts to produce extracellular matrix proteins without using extra additional growth factors. Immunofluorescent images clearly demonstrated that the peptide scaffolds were almost completely covered with type I and type III collagens which were main protein components of periodontal ligament. Our results suggest that these designer self-assembling peptide nanofiber scaffolds may be useful for promoting wound healing and especially periodontal ligament tissue regeneration.

  16. XB130: A novel adaptor protein in cancer signal transduction

    Science.gov (United States)

    ZHANG, RUIYAO; ZHANG, JINGYAO; WU, QIFEI; MENG, FANDI; LIU, CHANG

    2016-01-01

    Adaptor proteins are functional proteins that contain two or more protein-binding modules to link signaling proteins together, which affect cell growth and shape and have no enzymatic activity. The actin filament-associated protein (AFAP) family is an important member of the adaptor proteins, including AFAP1, AFAP1L1 and AFAP1L2/XB130. AFAP1 and AFAP1L1 share certain common characteristics and function as an actin-binding protein and a cSrc-activating protein. XB130 exhibits certain unique features in structure and function. The mRNA of XB130 is expressed in human spleen, thyroid, kidney, brain, lung, pancreas, liver, colon and stomach, and the most prominent disease associated with XB130 is cancer. XB130 has a controversial effect on cancer. Studies have shown that XB130 can promote cancer progression and downregulation of XB130-reduced growth of tumors derived from certain cell lines. A higher mRNA level of XB130 was shown to be associated with a better survival in non-small cell lung cancer. Previous studies have shown that XB130 can regulate cell growth, migration and invasion and possibly has the effect through the cAMP-cSrc-phosphoinositide 3-kinase/Akt pathway. Except for cancer, XB130 is also associated with other pathological or physiological procedures, such as airway repair and regeneration. PMID:26998266

  17. Fabrication of multi-biofunctional gelatin-based electrospun fibrous scaffolds for enhancement of osteogenesis of mesenchymal stem cells.

    Science.gov (United States)

    Lin, Wei-Han; Yu, Jiashing; Chen, Guoping; Tsai, Wei-Bor

    2016-02-01

    Biofunctional scaffolds that support the adhesion, proliferation, and osteo-differentiation of mesenchymal stem cells (MSCs) are critical for bone tissue engineering. In this study, a simple in situ UV-crosslinking strategy was utilized to fabricate gelatin electrospun fibrous (GEF) scaffolds with multiple biosignals, including cell adhesive Arg-Gly-Asp (RGD) peptide, osteo-conductive hydroxyapatite (HAp) nanoparticles, and osteo-inductive bone morphogenic protein-2 (BMP-2). The adhesion and proliferation of MSCs on the GEF scaffolds were improved by the incorporation of RGD. Meanwhile, the incorporation of HAp and BMP-2 enhanced osteo-differentiation of MSCs. The three incorporated bio-factors exert a synergistic effect on osteogenesis of MSCs in the GEF scaffolds. This strategy of incorporating multiple biomolecules could be used to fabricate crosslinked electrospun scaffolds of natural polymers for tissue-engineering applications. PMID:26642073

  18. Carbohydrate mimetics and scaffolds: sweet spots in medicinal chemistry.

    Science.gov (United States)

    Cipolla, Laura; La Ferla, Barbara; Airoldi, Cristina; Zona, Cristiano; Orsato, Alexandre; Shaikh, Nasrin; Russo, Laura; Nicotra, Francesco

    2010-04-01

    Several glycoprocessing enzymes and glycoreceptors have been recognized as important targets for therapeutic intervention. This concept has inspired the development of important classes of therapeutics, such as anti-influenza drugs inhibiting influenza virus neuraminidase, anti-inflammatory drugs targeting lectin-sialyl-Lewis X interaction and glycosidase inhibitors against HIV, Gaucher's disease, hepatitis and cancer. These therapeutics are mainly carbohydrate mimics in which proper modifications permit stronger interactions with the target protein, higher stability, better pharmacokinetic properties and easier synthesis. Furthermore, the conformational rigidity and polyfunctionality of carbohydrates stimulate their use as scaffolds for the generation of libraries by combinatorial decoration with different pharmacophores. This mini-review will present examples of how to exploit carbohydrates mimics and scaffolds in drug research. PMID:21426009

  19. Enhanced in vitro osteoblast differentiation on TiO2 scaffold coated with alginate hydrogel containing simvastatin

    Directory of Open Access Journals (Sweden)

    Helen Pullisaar

    2013-11-01

    Full Text Available The aim of this study was to develop a three-dimensional porous bone graft material as vehicle for simvastatin delivery and to investigate its effect on primary human osteoblasts from three donors. Highly porous titanium dioxide (TiO2 scaffolds were submerged into simvastatin containing alginate solution. Microstructure of scaffolds, visualized by scanning electron microscopy and micro-computed tomography, revealed an evenly distributed alginate layer covering the surface of TiO2 scaffold struts. Progressive and sustained simvastatin release was observed for up to 19 days. No cytotoxic effects on osteoblasts were observed by scaffolds with simvastatin when compared to scaffolds without simvastatin. Expression of osteoblast markers (collagen type I alpha 1, alkaline phosphatase, bone morphogenetic protein 2, osteoprotegerin, vascular endothelial growth factor A and osteocalcin was quantified using real-time reverse transcriptase–polymerase chain reaction. Secretion of osteoprotegerin, vascular endothelial growth factor A and osteocalcin was analysed by multiplex immunoassay (Luminex. The relative expression and secretion of osteocalcin was significantly increased by cells cultured on scaffolds with 10 µM simvastatin when compared to scaffolds without simvastatin after 21 days. In addition, secretion of vascular endothelial growth factor A was significantly enhanced from cells cultured on scaffolds with both 10 nM and 10 µM simvastatin when compared to scaffolds without simvastatin at day 21. In conclusion, the results indicate that simvastatin-coated TiO2 scaffolds can support a sustained release of simvastatin and induce osteoblast differentiation. The combination of the physical properties of TiO2 scaffolds with the osteogenic effect of simvastatin may represent a new strategy for bone regeneration in defects where immediate load is wanted or unavailable.

  20. Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model.

    Directory of Open Access Journals (Sweden)

    Xiaoqiang Huang

    Full Text Available Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with

  1. Cell–scaffold interaction within engineered tissue

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Haiping; Liu, Yuanyuan, E-mail: Yuanyuan_liu@shu.edu.cn; Jiang, Zhenglong; Chen, Weihua; Yu, Yongzhe; Hu, Qingxi

    2014-05-01

    The structure of a tissue engineering scaffold plays an important role in modulating tissue growth. A novel gelatin–chitosan (Gel–Cs) scaffold with a unique structure produced by three-dimensional printing (3DP) technology combining with vacuum freeze-drying has been developed for tissue-engineering applications. The scaffold composed of overall construction, micro-pore, surface morphology, and effective mechanical property. Such a structure meets the essential design criteria of an ideal engineered scaffold. The favorable cell–matrix interaction supports the active biocompatibility of the structure. The structure is capable of supporting cell attachment and proliferation. Cells seeded into this structure tend to maintain phenotypic shape and secreted large amounts of extracellular matrix (ECM) and the cell growth decreased the mechanical properties of scaffold. This novel biodegradable scaffold has potential applications for tissue engineering based upon its unique structure, which acts to support cell growth. - Highlights: • The scaffold is not only for providing a surface for cell residence but also for determining cell phenotype and retaining structural integrity. • The mechanical property of scaffold can be affected by activities of cell. • The scaffold provides a microenvironment for cell attachment, growth, and migration.

  2. Preparation of poly(ethylene glycol/polylactide hybrid fibrous scaffolds for bone tissue engineering

    Directory of Open Access Journals (Sweden)

    Ni P

    2011-11-01

    Full Text Available PeiYan Ni, ShaoZhi Fu, Min Fan, Gang Guo, Shuai Shi, JinRong Peng, Feng Luo, ZhiYong QianState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, People's Republic of ChinaAbstract: Polylactide (PLA electrospun fibers have been reported as a scaffold for bone tissue engineering application, however, the great hydrophobicity limits its broad application. In this study, the hybrid amphiphilic poly(ethylene glycol (PEG/hydrophobic PLA fibrous scaffolds exhibited improved morphology with regular and continuous fibers compared to corresponding blank PLA fiber mats. The prepared PEG/PLA fibrous scaffolds favored mesenchymal stem cell (MSC attachment and proliferation by providing an interconnected porous extracellular environment. Meanwhile, MSCs can penetrate into the fibrous scaffold through the interstitial pores and integrate well with the surrounding fibers, which is very important for favorable application in tissue engineering. More importantly, the electrospun hybrid PEG/PLA fibrous scaffolds can enhance MSCs to differentiate into bone-associated cells by comprehensively evaluating the representative markers of the osteogenic procedure with messenger ribonucleic acid quantitation and protein analysis. MSCs on the PEG/PLA fibrous scaffolds presented better differentiation potential with higher messenger ribonucleic acid expression of the earliest osteogenic marker Cbfa-1 and mid-stage osteogenic marker Col I. The significantly higher alkaline phosphatase activity of the PEG/PLA fibrous scaffolds indicated that these can enhance the differentiation of MSCs into osteoblast-like cells. Furthermore, the higher messenger ribonucleic acid level of the late osteogenic differentiation markers OCN (osteocalcin and OPN (osteopontin, accompanied by the positive Alizarin red S staining, showed better maturation of osteogenic induction on the PEG/PLA fibrous scaffolds at the

  3. Mandibular Jaw Bone Regeneration Using Human Dental Cell-Seeded Tyrosine-Derived Polycarbonate Scaffolds.

    Science.gov (United States)

    Zhang, Weibo; Zhang, Zheng; Chen, Shuang; Macri, Lauren; Kohn, Joachim; Yelick, Pamela C

    2016-07-01

    Here we present a new model for alveolar jaw bone regeneration, which uses human dental pulp cells (hDPCs) combined with tyrosine-derived polycarbonate polymer scaffolds [E1001(1k)] containing beta-tricalcium phosphate (β-TCP) [E1001(1k)/β-TCP]. E1001(1k)/β-TCP scaffolds (5 mm diameter × 1 mm thickness) were fabricated to fit a 5 mm rat mandibular ramus critical bone defect. Five experimental groups were examined in this study: (1) E1001(1k)/β-TCP scaffolds seeded with a high density of hDPCs, 5.0 × 10(5) hDPCs/scaffold (CH); (2) E1001(1k)/β-TCP scaffolds seeded with a lower density of hDPCs, 2.5 × 10(5) hDPCs/scaffold (CL); (3) acellular E1001(1k)/β-TCP scaffolds (SA); (4) acellular E1001(1k)/β-TCP scaffolds supplemented with 4 μg recombinant human bone morphogenetic protein-2 (BMP); and (5) empty defects (EDs). Replicate hDPC-seeded and acellular E1001(1k)/β-TCP scaffolds were cultured in vitro in osteogenic media for 1 week before implantation for 3 and 6 weeks. Live microcomputed tomography (μCT) imaging at 3 and 6 weeks postimplantation revealed robust bone regeneration in the BMP implant group. CH and CL groups exhibited similar uniformly distributed mineralized tissue coverage throughout the defects, but less than the BMP implants. In contrast, SA-treated defects exhibited sparse areas of mineralized tissue regeneration. The ED group exhibited slightly reduced defect size. Histological analyses revealed no indication of an immune response. In addition, robust expression of dentin and bone differentiation marker expression was observed in hDPC-seeded scaffolds, whereas, in contrast, BMP and SA implants exhibited only bone and not dentin differentiation marker expression. hDPCs were detected in 3-week but not in 6-week hDPC-seeded scaffold groups, indicating their survival for at least 3 weeks. Together, these results show that hDPC-seeded E1001(1k)/β-TCP scaffolds support the rapid regeneration of osteo

  4. RhBMP-2 microspheres-loaded chitosan/collagen scaffold enhanced osseointegration: an experiment in dog.

    Science.gov (United States)

    Shi, Shanshan; Cheng, Xiangrong; Wang, Jiawei; Zhang, Wei; Peng, Lin; Zhang, Yufeng

    2009-01-01

    The purpose of this study is to develop a novel recombinant human bone morphogenetic protein-2 (rhBMP-2) sustained release scaffold for dental implant osseointegration, and to evaluate the effect of this scaffold on promoting bone formation. RhBMP-2 was encapsulated in the poly-D,L-lactide-co-glycolide (PLGA) biodegradable microspheres, which were subsequently dispersed in a chitosan/collagen composite scaffold. This rhBMP-2 microspheres-loaded scaffold (S-MB) was compared with a chitosan/collagen scaffold without microspheres that directly encapsulated rhBMP-2 (S-B) in vitro and in vivo. The microstructure of the new scaffold was examined with scanning electron microscopy. The release profile of rhBMP-2 in vitro was measured at interval periods. The effect of rhBMP-2 encapsulated scaffolds on enhancing bone formation through implantation in dogs' mandibles was identified by histological examination of the regenerated bone after 4 weeks of implantation. Due to PLGA microspheres being loaded, the S-MB exhibited lower values at porosity and swelling rate, as well as a higher effective release dose than that of the S-B. Bone density, bone-implant contact, and bone-fill values measured from dog experiments demonstrated that the S-MB induced bone regeneration more quickly and was timely substituted by new bone. It was concluded that this sustained carrier scaffold based on microspheres was more effective to induce implant osseointegration. PMID:18667455

  5. In vitro performance of 13-93 bioactive glass fiber and trabecular scaffolds with MLO-A5 osteogenic cells.

    Science.gov (United States)

    Modglin, Vernon C; Brown, Roger F; Fu, Qiang; Rahaman, Mohamed N; Jung, Steven B; Day, Delbert E

    2012-10-01

    This in vitro study was performed to evaluate the ability of two types of porous bioactive glass scaffolds to support the growth and differentiation of an established osteogenic cell line. The two scaffold types tested included 13-93 glass fiber and trabecular-like scaffolds seeded with murine MLO-A5 cells and cultured for intervals of 2 to 12 days. Culture in MTT-containing medium showed metabolically active cells both on the surface and within the interior of the scaffolds. Scanning electron microscopy revealed well-attached cells on both types of scaffolds with a continual increase in cell density over a 6-day period. Protein measurements also showed a linear increase in cell density during the incubation. Activity of alkaline phosphatase, a key indicator of osteoblast differentiation, increased about 10-fold during the 6-day incubation with both scaffold types. The addition of mineralization media to MLO-A5 seeded scaffolds triggered extensive formation of alizarin red-positive mineralized extracellular material, additional evidence of cell differentiation and completion of the final step of bone formation on the constructs. Collectively, the results indicate that the 13-93 glass fiber and trabecular scaffolds promote the attachment, growth, and differentiation of MLO-A5 osteogenic cells and could potentially be used for bone tissue engineering applications. PMID:22528984

  6. Metacognitive scaffolding during collaborative learning: a promising combination

    OpenAIRE

    Molenaar, Inge; Sleegers, Peter; Boxtel, van, M.

    2014-01-01

    This article explores the effect of computerized scaffolding with different scaffolds (structuring vs. problematizing) on intra-group metacognitive interaction. In this study, we investigate 4 types of intra-group social metacognitive activities; namely ignored, accepted, shared and co-constructed metacognitive activities in 18 triads (6 control groups; no scaffolds and 12 experimental groups; 6 structuring scaffolds and 6 problematizing scaffolds).We found that groups receiving scaffolding s...

  7. Preparation and characterization of aligned porous PCL/zein scaffolds as drug delivery systems via improved unidirectional freeze-drying method.

    Science.gov (United States)

    Fereshteh, Zeinab; Fathi, Mohammadhossein; Bagri, Akbar; Boccaccini, Aldo R

    2016-11-01

    A novel type of drug-delivery scaffold based on poly(ε-caprolactone) (PCL) and zein blends was prepared by improved unidirectional freeze-drying. Scaffolds with tube-like pore structure and high porosity, up to 89%, were obtained by adjusting the concentration of the PCL and zein solutions. Characters of the prepared scaffolds, such as microstructural, porosity, and compressive strength, were evaluated. The hydrophilicity and the degradability of the composite films were investigated in contact with phosphate buffer saline (PBS). It was found that the presence of zein accelerates the degradation rate of the scaffolds in the period time of investigation (28days). The results showed an acceptable way for controlling the in vitro degradation behavior of PCL composite scaffolds by adapting the concentration of zein. In vitro protein release and degradation results revealed that the absolute weight loss of the PCL/zein scaffolds exhibited an increasing trend by increasing the amount of zein concentration in the scaffolds. The drug delivery capability of the scaffolds was tested using tetracycline hydrochloride (TCH). Sustained release of the drug was obtained, and it was found that the proportion of zein in the scaffold had a great impact on the drug release kinetics. The results demonstrated the potential of the PCL/zein biocomposite scaffolds as a suitable candidate in tissue engineering strategies for bone defect treatment. PMID:27524061

  8. The effect of the fibre orientation of electrospun scaffolds on the matrix production of rabbit annulus fibrosus-derived stem cells

    Institute of Scientific and Technical Information of China (English)

    Chen Liu; Caihong Zhu; Jun Li; Pinghui Zhou; Min Chen; Huilin Yang; Bin Li

    2015-01-01

    Annulus fibrosus (AF) tissue engineering has recently received increasing attention as a treatment for intervertebral disc (IVD) degeneration;however, such engineering remains challenging because of the remarkable complexity of AF tissue. In order to engineer a functional AF replacement, the fabrication of cell-scaffold constructs that mimic the cellular, biochemical and structural features of native AF tissue is critical. In this study, we fabricated aligned fibrous polyurethane scaffolds using an electrospinning technique and used them for culturing AF-derived stem/progenitor cells (AFSCs). Random fibrous scaffolds, also prepared via electrospinning, were used as a control. We compared the morphology, proliferation, gene expression and matrix production of AFSCs on aligned scaffolds and random scaffolds. There was no apparent difference in the attachment or proliferation of cells cultured on aligned scaffolds and random scaffolds. However, compared to cells on random scaffolds, the AFSCs on aligned scaffolds were more elongated and better aligned, and they exhibited higher gene expression and matrix production of collagen-I and aggrecan. The gene expression and protein production of collagen-II did not appear to differ between the two groups. Together, these findings indicate that aligned fibrous scaffolds may provide a favourable microenvironment for the differentiation of AFSCs into cells similar to outer AF cells, which predominantly produce collagen-I matrix.

  9. A comparative study on in vitro osteogenic priming potential of electron spun scaffold PLLA/HA/Col, PLLA/HA, and PLLA/Col for tissue engineering application.

    Science.gov (United States)

    Balaji Raghavendran, Hanumantha Rao; Puvaneswary, Subramaniam; Talebian, Sepehr; Murali, Malliga R; Raman Murali, Malliga; Naveen, Sangeetha V; Vasudevaraj Naveen, Sangeetha; Krishnamurithy, G; McKean, Robert; Kamarul, Tunku

    2014-01-01

    A comparative study on the in vitro osteogenic potential of electrospun poly-L-lactide/hydroxyapatite/collagen (PLLA/HA/Col, PLLA/HA, and PLLA/Col) scaffolds was conducted. The morphology, chemical composition, and surface roughness of the fibrous scaffolds were examined. Furthermore, cell attachment, distribution, morphology, mineralization, extracellular matrix protein localization, and gene expression of human mesenchymal stromal cells (hMSCs) differentiated on the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA were also analyzed. The electrospun scaffolds with a diameter of 200-950 nm demonstrated well-formed interconnected fibrous network structure, which supported the growth of hMSCs. When compared with PLLA/H%A and PLLA/Col scaffolds, PLLA/Col/HA scaffolds presented a higher density of viable cells and significant upregulation of genes associated with osteogenic lineage, which were achieved without the use of specific medium or growth factors. These results were supported by the elevated levels of calcium, osteocalcin, and mineralization (PCol/HA scaffolds exhibited superior osteoinductivity, when compared with PLLA/Col or PLLA/HA scaffolds. These findings indicated that the fibrous structure and synergistic action of Col and nano-HA with high-molecular-weight PLLA played a vital role in inducing osteogenic differentiation of hMSCs. The data obtained in this study demonstrated that the developed fibrous PLLA/Col/HA biocomposite scaffold may be supportive for stem cell based therapies for bone repair, when compared with the other two scaffolds.

  10. Application of Wnt Pathway Inhibitor Delivering Scaffold for Inhibiting Fibrosis in Urethra Strictures: In Vitro and in Vivo Study

    Directory of Open Access Journals (Sweden)

    Kaile Zhang

    2015-11-01

    Full Text Available Objective: To evaluate the mechanical property and biocompatibility of the Wnt pathway inhibitor (ICG-001 delivering collagen/poly(l-lactide-co-caprolactone (P(LLA-CL scaffold for urethroplasty, and also the feasibility of inhibiting the extracellular matrix (ECM expression in vitro and in vivo. Methods: ICG-001 (1 mg (2 mM was loaded into a (P(LLA-CL scaffold with the co-axial electrospinning technique. The characteristics of the mechanical property and drug release fashion of scaffolds were tested with a mechanical testing machine (Instron and high-performance liquid chromatography (HPLC. Rabbit bladder epithelial cells and the dermal fibroblasts were isolated by enzymatic digestion method. (3-(4,5-Dimethylthiazol-2-yl-2,5-Diphenyltetrazolium Bromide (MTT assay and scanning electron microscopy (SEM were used to evaluate the viability and proliferation of the cells on the scaffolds. Fibrolasts treated with TGF-β1 and ICG-001 released medium from scaffolds were used to evaluate the anti-fibrosis effect through immunofluorescence, real time PCR and western blot. Urethrography and histology were used to evaluate the efficacy of urethral implantation. Results: The scaffold delivering ICG-001 was fabricated, the fiber diameter and mechanical strength of scaffolds with inhibitor were comparable with the non-drug scaffold. The SEM and MTT assay showed no toxic effect of ICG-001 to the proliferation of epithelial cells on the collagen/P(LLA-CL scaffold with ICG-001. After treatment with culture medium released from the drug-delivering scaffold, the expression of Collagen type 1, 3 and fibronectin of fibroblasts could be inhibited significantly at the mRNA and protein levels. In the results of urethrography, urethral strictures and fistulas were found in the rabbits treated with non-ICG-001 delivering scaffolds, but all the rabbits treated with ICG-001-delivering scaffolds showed wide caliber in urethras. Histology results showed less collagen but more

  11. Design of biomimetic and bioactive cold plasma-modified nanostructured scaffolds for enhanced osteogenic differentiation of bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Wang, Mian; Cheng, Xiaoqian; Zhu, Wei; Holmes, Benjamin; Keidar, Michael; Zhang, Lijie Grace

    2014-03-01

    The objective of this study was to design a biomimetic and bioactive tissue-engineered bone construct via a cold atmospheric plasma (CAP) treatment for directed osteogenic differentiation of human bone morrow mesenchymal stem cells (MSCs). Porous nanocrystalline hydroxyapatite/chitosan scaffolds were fabricated via a lyophilization procedure. The nanostructured bone scaffolds were then treated with CAP to create a more favorable surface for cell attachment, proliferation, and differentiation. The CAP-modified scaffolds were characterized via scanning electron microscope, Raman spectrometer, contact angle analyzer, and white light interferometer. In addition, optimal CAP treatment conditions were determined. Our in vitro study shows that MSC adhesion and infiltration were significantly enhanced on CAP modified scaffolds. More importantly, it was demonstrated that CAP-modified nanostructured bone constructs can greatly promote total protein, collagen synthesis, and calcium deposition after 3 weeks of culture, thus making them a promising implantable scaffold for bone regeneration. Moreover, the fibronectin and vitronection adsorption experiments by enzyme-linked immunosorbent assay demonstrated that more adhesion-mediated protein adsorption on the CAP-treated scaffolds. Since the initial specific protein absorption on scaffold surfaces can lead to further recruitment as well as activation of favorable cell functions, it is suggested that our enhanced stem cell growth and osteogenic function may be related to more protein adsorption resulting from surface roughness and wettability modification. The CAP modification method used in this study provides a quick one-step process for cell-favorable tissue-engineered scaffold architecture remodeling and surface property alteration.

  12. Cell alignment induced by anisotropic electrospun fibrous scaffolds alone has limited effect on cardiomyocyte maturation

    Directory of Open Access Journals (Sweden)

    Jingjia Han

    2016-05-01

    Full Text Available Enhancing the maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs will facilitate their applications in disease modeling and drug discovery. Previous studies suggest that cell alignment could enhance hPSC-CM maturation; however, the robustness of this approach has not been well investigated. To this end, we examined if the anisotropic orientation of hPSC-CMs imposed by the underlying aligned fibers within a 3D microenvironment could improve the maturation of hPSC-CMs. Enriched hPSC-CMs were cultured for two weeks on Matrigel-coated anisotropic (aligned and isotropic (random polycaprolactone (PCL fibrous scaffolds, as well as tissue culture polystyrenes (TCPs as a control. As expected, hPSC-CMs grown on the two types of fibrous scaffolds exhibited anisotropic and isotropic orientations, respectively. Similar to cells on TCPs, hPSC-CMs cultured on these scaffolds expressed CM-associated proteins and were pharmacologically responsive to adrenergic receptor agonists, a muscarinic agonist, and a gap junction uncoupler in a dose-dependent manner. Although hPSC-CMs grown on anisotropic fibrous scaffolds displayed the highest expression of genes encoding a number of sarcomere proteins, calcium handling proteins and ion channels, their calcium transient kinetics were slower than cells grown on TCPs. These results suggest that electrospun anisotropic fibrous scaffolds, as a single method, have limited effect on improving the maturation of hPSC-CMs.

  13. A practice scaffolding interactive platform

    DEFF Research Database (Denmark)

    Bundsgaard, Jeppe

    2009-01-01

    , structures the students' activity, and interactively supports subject learning. A PracSIP facilitates students' development of complex competencies, and at the same time it supports the students' development of skills defined in the curriculum. The paper introduces the concept, presents the theoretical......A Practice Scaffolding Interactive Platform (PracSIP) is a social learning platform which supports students in collaborative project based learning by simulating a professional practice. A PracSIP puts the core tools of the simulated practice at the students' disposal, it organizes collaboration...

  14. Information Scaffolding: Application to Technical Animation

    Science.gov (United States)

    Newman, Catherine Claire

    2010-01-01

    Information Scaffolding is a user-centered approach to information design; a method devised to aid "everyday" authors in information composition. Information Scaffolding places a premium on audience-centered documents by emphasizing the information needs and motivations of a multimedia document's intended audience. The aim of this…

  15. Metacognitive scaffolding in an innovative learning arrangement

    NARCIS (Netherlands)

    Molenaar, I.; Boxtel, C.A.M. van; Sleegers, P.J.C.

    2011-01-01

    This study examined the effects of metacognitive scaffolds on learning outcomes of collaborating students in an innovative learning arrangement. The triads were supported by computerized scaffolds, which were dynamically integrated into the learning process and took a structuring or problematizing f

  16. Scaffolding Mathematical Modelling with a Solution Plan

    Science.gov (United States)

    Schukajlow, Stanislaw; Kolter, Jana; Blum, Werner

    2015-01-01

    In the study presented in this paper, we examined the possibility to scaffold mathematical modelling with strategies. The strategies were prompted using an instrument called "solution plan" as a scaffold. The effects of this step by step instrument on mathematical modelling competency and on self-reported strategies were tested using…

  17. Teaching Writing: A Multilayered Participatory Scaffolding Practice

    Science.gov (United States)

    Dix, Stephanie

    2016-01-01

    This article adds to the research on teachers' writing pedagogy. It reviews and challenges the research literature on scaffolding as an instructional practice and presents a more inclusive framework for analysis. As student participation and voice were absent from much of the literature, a participatory scaffolding framework was developed to…

  18. Composite scaffolds for cartilage tissue engineering.

    Science.gov (United States)

    Moutos, Franklin T; Guilak, Farshid

    2008-01-01

    Tissue engineering remains a promising therapeutic strategy for the repair or regeneration of diseased or damaged tissues. Previous approaches have typically focused on combining cells and bioactive molecules (e.g., growth factors, cytokines and DNA fragments) with a biomaterial scaffold that functions as a template to control the geometry of the newly formed tissue, while facilitating the attachment, proliferation, and differentiation of embedded cells. Biomaterial scaffolds also play a crucial role in determining the functional properties of engineered tissues, including biomechanical characteristics such as inhomogeneity, anisotropy, nonlinearity or viscoelasticity. While single-phase, homogeneous materials have been used extensively to create numerous types of tissue constructs, there continue to be significant challenges in the development of scaffolds that can provide the functional properties of load-bearing tissues such as articular cartilage. In an attempt to create more complex scaffolds that promote the regeneration of functional engineered tissues, composite scaffolds comprising two or more distinct materials have been developed. This paper reviews various studies on the development and testing of composite scaffolds for the tissue engineering of articular cartilage, using techniques such as embedded fibers and textiles for reinforcement, embedded solid structures, multi-layered designs, or three-dimensionally woven composite materials. In many cases, the use of composite scaffolds can provide unique biomechanical and biological properties for the development of functional tissue engineering scaffolds.

  19. Evaluation of emulsion electrospun polycaprolactone/hyaluronan/epidermal growth factor nanofibrous scaffolds for wound healing.

    Science.gov (United States)

    Wang, Zhenbei; Qian, Yuna; Li, Linhao; Pan, Lianhong; Njunge, Lucy W; Dong, Lili; Yang, Li

    2016-01-01

    Wound healing scaffolds provide cells with structural integrity and can also deliver biological agents to establish a skin tissue-specific microenvironment to regulate cell functions and to accelerate the healing process. In this study, we fabricated biodegradable nanofibrous scaffolds with an emulsion electrospinning technique. The scaffolds were composed of polycaprolactone, hyaluronan and encapsulating epidermal growth factor. The morphology and core-sheath structure of the nanofibers were characterized by scanning electron microscopy and transmission electron microscopy. The scaffolds were also characterized for chemical composition and hydrophilicity with a Fourier-transform infrared analysis, energy dispersive spectroscopy and the water contact angle. An in vitro model protein bovine serum albumin and epidermal growth factor release study was conducted to evaluate the sustained release potential of the core-sheath structured nanofibers with and without the hyaluronan component. Additionally, an in vitro cultivation of human skin keratinocytes (HaCaT) and fibroblasts on polycaprolactone/hyaluronan and polycaprolactone/hyaluronan-epidermal growth factor scaffolds showed a significant synergistic effect of hyaluronan and epidermal growth factor on cell proliferation and infiltration. Furthermore, there was an up-regulation of the wound-healing-related genes collagen I, collagen III and TGF-β in polycaprolactone/hyaluronan/epidermal growth factor scaffolds compared with control groups. In the full-thickness wound model, the enhanced regeneration of fully functional skin was facilitated by epidermal regeneration in the polycaprolactone/hyaluronan/epidermal growth factor treatment group. Our findings suggest that bioactivity and hemostasis of the hyaluronan-based nanofibrous scaffolds have the capability to encapsulate and control the release of growth factors that can serve as skin tissue engineering scaffolds for wound healing.

  20. The Effect of Sterilization Methods on the Physical Properties of Silk Sericin Scaffolds

    OpenAIRE

    Siritientong, Tippawan; Srichana, Teerapol; Aramwit, Pornanong

    2011-01-01

    Protein-based biomaterials respond differently to sterilization methods. Since protein is a complex structure, heat, or irradiation may result in the loss of its physical or biological properties. Recent investigations have shown that sericin, a degumming silk protein, can be successfully formed into a 3-D scaffolds after mixing with other polymers which can be applied in skin tissue engineering. The objective of this study was to investigate the effectiveness of ethanol, ethylene oxide (EtO)...

  1. Hyaluronan scaffold supports osteogenic differentiation of bone marrow concentrate cells.

    Science.gov (United States)

    Cavallo, C; Desando, G; Ferrari, A; Zini, N; Mariani, E; Grigolo, B

    2016-01-01

    Osteochondral lesions are considered a challenge for orthopedic surgeons. Currently, the treatments available are often unsatisfactory and unable to stimulate tissue regeneration. Tissue engineering offers a new therapeutic strategy, taking into account the role exerted by cells, biomaterial and growth factors in restoring tissue damage. In this light, Mesenchymal Stem Cells (MSCs) have been indicated as a fascinating tool for regenerative medicine thanks to their ability to differentiate into bone, cartilage and adipose tissue. However, in vitro-cultivation of MSCs could be associated with some risks such as de-differentiation/reprogramming, infection and contaminations of the cells. To overcome these shortcomings, a new approach is represented by the use of Bone Marrow Concentrate (BMC), that could allow the delivery of cells surrounded by their microenvironment in injured tissue. For this purpose, cells require a tridimensional scaffold that can support their adhesion, proliferation and differentiation. This study is focused on the potentiality of BMC seeded onto a hyaluronan-based scaffold (Hyaff-11) to differentiate into osteogenic lineage. This process depends on the specific interaction between cells derived from bone marrow (surrounded by their niche) and scaffold, that create an environment able to support the regeneration of damaged tissue. The data obtained from the present study demonstrate that BMC grown onto Hyaff-11 are able to differentiate toward osteogenic sense, producing specific osteogenic genes and matrix proteins.

  2. Proton transfer in organic scaffolds

    Science.gov (United States)

    Basak, Dipankar

    This dissertation focuses on the fundamental understanding of the proton transfer process and translating the knowledge into design/development of new organic materials for efficient non-aqueous proton transport. For example, what controls the shuttling of a proton between two basic sites? a) Distance between two groups? or b) the basicity? c) What is the impact of protonation on molecular conformation when the basic sites are attached to rigid scaffolds? For this purpose, we developed several tunable proton sponges and studied proton transfer in these scaffolds theoretically as well as experimentally. Next we moved our attention to understand long-range proton conduction or proton transport. We introduced liquid crystalline (LC) proton conductor based on triphenylene molecule and established that activation energy barrier for proton transport is lower in the LC phase compared to the crystalline phase. Furthermore, we investigated the impact of several critical factors: the choice of the proton transferring groups, mobility of the charge carriers, intrinsic vs. extrinsic charge carrier concentrations and the molecular architectures on long-range proton transport. The outcome of this research will lead to a deeper understanding of non-aqueous proton transfer process and aid the design of next generation proton exchange membrane (PEM) for fuel cell.

  3. Chitosan scaffolds induce human dental pulp stem cells to neural differentiation: potential roles for spinal cord injury therapy.

    Science.gov (United States)

    Zhang, Jinlong; Lu, Xiaohui; Feng, Guijuan; Gu, Zhifeng; Sun, Yuyu; Bao, Guofeng; Xu, Guanhua; Lu, Yuanzhou; Chen, Jiajia; Xu, Lingfeng; Feng, Xingmei; Cui, Zhiming

    2016-10-01

    Cell-based transplantation strategies hold great potential for spinal cord injury (SCI) repair. Chitosan scaffolds have therapeutic benefits for spinal cord regeneration. Human dental pulp stem cells (DPSCs) are abundant available stem cells with low immunological incompatibility and can be considered for cell replacement therapy. The purpose of this study is to investigate the role of chitosan scaffolds in the neural differentiation of DPSCs in vitro and to assess the supportive effects of chitosan scaffolds in an animal model of SCI. DPSCs were incubated with chitosan scaffolds. Cell viability and the secretion of neurotrophic factors were analyzed. DPSCs incubated with chitosan scaffolds were treated with neural differentiation medium for 14 days and then neural genes and protein markers were analyzed by Western blot and reverse transcription plus the polymerase chain reaction. Our study revealed a higher cell viability and neural differentiation in the DPSC/chitosan-scaffold group. Compared with the control group, the levels of BDNF, GDNF, b-NGF, and NT-3 were significantly increased in the DPSC/chitosan-scaffold group. The Wnt/β-catenin signaling pathway played a key role in the neural differentiation of DPSCs combined with chitosan scaffolds. Transplantation of DPSCs together with chitosan scaffolds into an SCI rat model resulted in the marked recovery of hind limb locomotor functions. Thus, chitosan scaffolds were non-cytotoxic and provided a conducive and favorable microenvironment for the survival and neural differentiation of DPSCs. Transplantation of DPSCs might therefore be a suitable candidate for treating SCI and other neuronal degenerative diseases. PMID:27147262

  4. SYNTHETIC HYDROGELS AS SCAFFOLDS FOR MANIPULATING ENDOTHELIUM CELL BEHAVIORS

    Institute of Scientific and Technical Information of China (English)

    Yong-mei Chen; Jing-jing Yang; Yoshihito Osada; Jian Ping Gong

    2011-01-01

    Synthetic hydrogels can be used as scaffolds that not only favor endothelial cells (ECs) proliferation but also manipulate the behaviors and functions of the ECs. In this review paper, the effect of chemical structure, Young’s modulus (E) and zeta potential (ζ) of synthetic hydrogel scaffolds on static cell behaviors, including cell morphology, proliferation,cytoskeleton structure and focal adhesion, and on dynamic cell behaviors, including migration velocity and morphology oscillation, as well as on EC function such as anti-platelet adhesion, are reported. It was found that negatively charged hydrogels, poly(2-acrylamido-2-methylpropanesulfonie sodium) (PNaAMPS) and poly(sodium p-styrene sulphonate) (PNaSS), can directly promote cell proliferation, with no need of surface modification by any cell-adhesive proteins or peptides at the environment of serum-containing medium. In addition, the Young’s modulus (E) and zeta potential (ζ) of hydrogel scaffolds are quantitatively tuned by copolymer hydrogels, poly(NaAMPS-co-DMAAm) and poly(NaSS-co-DMAAm), in which the two kinds of negatively charged monomers NaAMPS and NaSS are copolymerized with neutral monomer, N,N-dimethylacrylamide (DMAAm). It was found that the critical zeta potential of hydrogels manipulating EC morphology, proliferation, and motility is ζcritical = -20.83 mV and ζcritical = -14.0 mV for poly(NaAMPS-co-DMAAm) and poly(NaSS-co-DMAAm), respectively. The above mentioned EC behaviors well correlate with the adsorption of fibronectin,a kind of cell-adhesive protein, on the hydrogel surfaces. Furthermore, adhered platelets on the EC monolayers cultured on the hydrogel scaffolds obviously decreases with an increase of the Young’s modulus (E) of the hydrogels, especially when E > 60 kPa. Glycocalyx assay and gene expression of ECs demonstrate that the anti-platelet adhesion well correlates with the EC-specific glycocalyx. The above investigation suggests that understanding the relationship

  5. Potential of a PLA-PEO-PLA-based scaffold for skin tissue engineering: in vitro evaluation.

    Science.gov (United States)

    Garric, Xavier; Guillaume, Olivier; Dabboue, Hinda; Vert, Michel; Molès, Jean-Pierre

    2012-01-01

    This study aimed to investigate the in vitro behaviour of porous degradable scaffolds of the PLA-PEO-PLA-type designed prior to in vivo evaluation for skin tissue engineering. Two tri-block co-polymers were synthesized from PEO and DL-lactide and their degradation was studied under conditions that mimic a cutaneous wound environment. 3-D porous scaffolds with interconnected pores were fabricated using the salt leaching method and characterized by ESEM and Hg porosimetry. The degrading action of gamma sterilization was studied on the co-polymers. The less degraded one was selected to make porous scaffolds on which human dermal fibroblasts and human epidermal keratinocytes were cultured. The capacity of such scaffolds to act as a dermal equivalent was also considered. Colonization by human dermal fibroblasts was shown after hematoxylin staining and the production of major proteins normally found in the extracellular matrix was assessed by Western blotting of protein extracts. Finally, a skin substitute was generated by seeding human keratinocytes on the dermal equivalent and a new epidermis was characterized by using immuno-histological staining. Results show that gamma sterilization and that degradation under conditions that mimic skin wound healing were acceptable. The fact that fibroblasts produce extracellular matrix and that keratinocytes generated an epidermal barrier argues in favour of the interest of this type of porous scaffold for skin reconstruction. PMID:21888762

  6. Versatile convergent synthesis of a three peptide loop containing protein mimic of whooping cough pertactin by successive Cu(I)-catalyzed azide alkyne cycloaddition on an orthogonal alkyne functionalized TAC-scaffold

    NARCIS (Netherlands)

    Werkhoven, Paul R; van de Langemheen, Helmus; van der Wal, Steffen; Kruijtzer, John A W; Liskamp, Rob M J

    2014-01-01

    Synthetic mimics of discontinuous epitopes may have a wide range of potential applications, including synthetic vaccines and inhibition of protein-protein interactions. However, synthetic access to these relatively complex peptide molecular constructs is limited. This paper describes a versatile con

  7. Titanate nanotube coatings on biodegradable photopolymer scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Beke, S., E-mail: szabolcs.beke@iit.it [Department of Nanophysics, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genova (Italy); Kőrösi, L. [Department of Biotechnology, Nanophage Therapy Center, Enviroinvest Corporation, Kertváros u. 2, H-7632, Pécs (Hungary); Scarpellini, A. [Department of Nanochemistry, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genova (Italy); Anjum, F.; Brandi, F. [Department of Nanophysics, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genova (Italy)

    2013-05-01

    Rigid, biodegradable photopolymer scaffolds were coated with titanate nanotubes (TNTs) by using a spin-coating method. TNTs were synthesized by a hydrothermal process at 150 °C under 4.7 bar ambient pressure. The biodegradable photopolymer scaffolds were produced by mask-assisted excimer laser photocuring at 308 nm. For scaffold coating, a stable ethanolic TNT sol was prepared by a simple colloid chemical route without the use of any binding compounds or additives. Scanning electron microscopy along with elemental analysis revealed that the scaffolds were homogenously coated by TNTs. The developed TNT coating can further improve the surface geometry of fabricated scaffolds, and therefore it can further increase the cell adhesion. Highlights: ► Biodegradable scaffolds were produced by mask-assisted UV laser photocuring. ► Titanate nanotube deposition was carried out without binding compounds or additives. ► The titanate nanotube coating can further improve the surface geometry of scaffolds. ► These reproducible platforms will be of high importance for biological applications.

  8. Titanate nanotube coatings on biodegradable photopolymer scaffolds

    International Nuclear Information System (INIS)

    Rigid, biodegradable photopolymer scaffolds were coated with titanate nanotubes (TNTs) by using a spin-coating method. TNTs were synthesized by a hydrothermal process at 150 °C under 4.7 bar ambient pressure. The biodegradable photopolymer scaffolds were produced by mask-assisted excimer laser photocuring at 308 nm. For scaffold coating, a stable ethanolic TNT sol was prepared by a simple colloid chemical route without the use of any binding compounds or additives. Scanning electron microscopy along with elemental analysis revealed that the scaffolds were homogenously coated by TNTs. The developed TNT coating can further improve the surface geometry of fabricated scaffolds, and therefore it can further increase the cell adhesion. Highlights: ► Biodegradable scaffolds were produced by mask-assisted UV laser photocuring. ► Titanate nanotube deposition was carried out without binding compounds or additives. ► The titanate nanotube coating can further improve the surface geometry of scaffolds. ► These reproducible platforms will be of high importance for biological applications

  9. Scaffolding With and Through Videos

    DEFF Research Database (Denmark)

    Otrel-Cass, Kathrin; Khoo, Elaine; Cowie, Bronwen

    2012-01-01

    In New Zealand and internationally claims are being made about the potential for information and communication technologies (ICTs) to transform teaching and learning. However, the theoretical underpinnings explaining the complex interplay between the content, pedagogy and technology a teacher needs...... to consider must be expanded. This article explicates theoretical and practical ideas related to teachers’ application of their ICT technology, pedagogy, and content knowledge (TPACK) in science. The article unpacks the social and technological dimensions of teachers’ use of TPACK when they use digital videos...... to scaffold learning. It showcases the intricate interplay between teachers’ knowledge about content, digital video technology, and students’ learning needs based on a qualitative study of two science teachers and their students in a New Zealand primary school....

  10. Electrospinning Auricular Shaped Scaffolds for Tissue Engineering

    OpenAIRE

    Walser, Jochen; Caversaccio, Marco; Ferguson, Stephen J.

    2013-01-01

    Poly(ɛ)caprolactone scaffolds have been electrospun directly into an auricular shaped conductive mould. Bovine chondrocytes were harvested from articular cartilage and seeded onto 16 of the produced scaffolds, which received either an ethanol (group A) or a plasma treatment (group B) for sterilisation before seeding. The seeded scaffolds were cultured for 3 weeks in vitro and analysed with regard to total DNA and GAG content as well as the expression of AGG, COL1, COL2, MMP3 and MMP13. Rap...

  11. Ionic solutes impact collagen scaffold bioactivity.

    Science.gov (United States)

    Pawelec, K M; Husmann, A; Wardale, R J; Best, S M; Cameron, R E

    2015-02-01

    The structure of ice-templated collagen scaffolds is sensitive to many factors. By adding 0.5 wt% of sodium chloride or sucrose to collagen slurries, scaffold structure could be tuned through changes in ice growth kinetics and interactions of the solute and collagen. With ionic solutes (sodium chloride) the entanglements of the collagen molecule decreased, leading to fibrous scaffolds with increased pore size and decreased attachment of chondrocytes. With non-ionic solutes (sucrose) ice growth was slowed, leading to significantly reduced pore size and up-regulated cell attachment. This highlights the large changes in structure and biological function stimulated by solutes in ice-templating systems. PMID:25649518

  12. FERM family proteins and their importance in cellular movements and wound healing (review).

    Science.gov (United States)

    Bosanquet, David C; Ye, Lin; Harding, Keith G; Jiang, Wen G

    2014-07-01

    Motility is a requirement for a number of biological processes, including embryonic development, neuronal development, immune responses, cancer progression and wound healing. Specific to wound healing is the migration of endothelial cells, fibroblasts and other key cellular players into the wound space. Aberrations in wound healing can result in either chronic wounds or abnormally healed wounds. The protein 4.1R, ezrin, radixin, moesin (FERM) superfamily consists of over 40 proteins all containing a three lobed N-terminal FERM domain which binds a variety of cell-membrane associated proteins and lipids. The C-terminal ends of these proteins typically contain an actin-binding domain (ABD). These proteins therefore mediate the linkage between the cell membrane and the actin cytoskeleton, and are involved in cellular movements and migration. Certain FERM proteins have been shown to promote cancer metastasis via this very mechanism. Herein we review the effects of a number of FERM proteins on wound healing and cancer. We show how these proteins typically aid wound healing through their effects on increasing cellular migration and movements, but also typically promote metastasis in cancer. We conclude that FERM proteins play important roles in cellular migration, with markedly different outcomes in the context of cancer and wound healing.

  13. Tolerogenic responses of CD206+, CD83+, FOXP3+, and CTLA-4 to sericin/polyvinyl alcohol/glycerin scaffolds relevant to IL-33 and HSP60 activity.

    Science.gov (United States)

    Ampawong, Sumate; Aramwit, Pornanong

    2016-09-01

    Silk sericin-releasing (sericin/polyvinyl alcohol (PVA)/glycerin) scaffolds have been designed for wound dressing applications using different fabrication techniques that influence scaffold antigenicity. The immunological tolerance of scaffolds depends on the balance of immunogenic and tolerogenic responses modulated by dendritic cells (DCs). An in vivo skin implantation model was used to compare the tolerogenic effect of sericin/PVA/glycerin scaffolds prepared by freeze-drying versus salt-leaching techniques, using an Allevyn® scaffold as a control. Immunohistochemical and histopathological studies were performed to evaluate tolerogenic DCs (CD206+), immunogenic DCs (CD83+), regulatory T-cells (FOXP3+ and CTLA-4), a proinflammatory cytokine (interleukin 33: IL-33), a stress marker (heat shock protein 60; HSP60), histopathological changes and related inflammatory cells. It was found that both sericin/PVA/glycerin scaffolds were tolerogenic and induced early activated Treg functions, while the Allevyn® scaffold was immunogenic. However, the tolerance of the freeze-dried sericin/PVA/glycerin scaffolds was not as consistent as the salt-leached sericin/PVA/glycerin scaffolds, indicated by the low level of CTLA-4 expression. This was probably due to molecular cross-linking and the morphological and mechanical properties of the freeze-drying technique, which would enhance the immune response. Severe inflammatory responses (including mast cell degranulation and foreign body giant cell accumulation) and histopathological changes (including fat infiltration and fibrosis formation) were mainly found with the Allevyn® scaffold, presumably from its architecture and chemical composition, especially polyurethane. The up-regulation of IL-33 and HSP60 with the Allevyn® scaffold was correlated with the inflammatory and pathological levels. Our findings suggested that salt-leached sericin/PVA/glycerin scaffolds were tolerogenic, induced a low inflammatory response and were

  14. Investigating the morphological, mechanical and degradation properties of scaffolds comprising collagen, gelatin and elastin for use in soft tissue engineering.

    Science.gov (United States)

    Grover, Chloe N; Cameron, Ruth E; Best, Serena M

    2012-06-01

    Collagen-based scaffolds can be used to mimic the extracellular matrix (ECM) of soft tissues and provide support during tissue regeneration. To better match the native ECM composition and mechanical properties as well as tailor the degradation resistance and available cell binding motifs, other proteins or different collagen types may be added. The present study has explored the use of components such as gelatin or elastin and investigated their effect on the bulk physical properties of the resulting scaffolds compared to those made from pure collagen type I. The effect of altering the composition and crosslinking was evaluated in terms of the scaffold structure, mechanical properties, swelling, degradation and cell attachment. Results demonstrate that scaffolds based on gelatin had reduced tensile stiffness and degradation time compared with collagen. The addition of elastin reduced the overall strength and stiffness of the scaffolds, with electron microscopy results suggesting that insoluble elastin interacts best with collagen and soluble elastin interacts best with gelatin. Carbodiimide crosslinking was essential for structural stability, strength and degradation resistance for scaffolds of all compositions. In addition, preliminary cell adhesion studies showed these highly porous structures (pore size 130-160 μm) to be able to support HT1080 cell infiltration and growth. Therefore, this study suggests that the use of gelatin in place of collagen, with additions of elastin, can tailor the physical properties of scaffolds and could be a design strategy for reducing the overall material costs. PMID:22520419

  15. S/MARt DB: a database on scaffold/matrix attached regions

    OpenAIRE

    Liebich, Ines; Bode, Jürgen; Frisch, Matthias; Wingender, Edgar

    2002-01-01

    S/MARt DB, the S/MAR transaction database, is a relational database covering scaffold/matrix attached regions (S/MARs) and nuclear matrix proteins that are involved in the chromosomal attachment to the nuclear scaffold. The data are mainly extracted from original publications, but a World Wide Web interface for direct submissions is also available. S/MARt DB is closely linked to the TRANSFAC database on transcription factors and their binding sites. It is freely accessible through the World W...

  16. Analysis of a homologue of the adducin head gene which is a potential target for the Dictyostelium STAT protein Dd-STATa.

    Science.gov (United States)

    Aoshima, Ryota; Hiraoka, Rieko; Shimada, Nao; Kawata, Takefumi

    2006-01-01

    A Dd-STATa-null mutant, which is defective in expression of a Dictyostelium homologue of the metazoan STAT (signal transducers and activators of transcription) proteins, fails to culminate and this phenotype correlates with the loss of expression of various prestalk (pst) genes. An EST clone, SSK395, encodes a close homologue of the adducin amino-terminal head domain and harbors a putative actin-binding domain. We fused promoter fragments of the cognate gene, ahhA (adducin head homologue A), to a lacZ reporter and determined their expression pattern. The proximal promoter region is necessary for the expression of ahhA at an early (pre-aggregative) stage of development and this expression is Dd-STATa independent. The distal promoter region is necessary for expression at later stages of development in pstA cells, of the slug and in upper cup and pstAB cells during culmination. The distal region is partly Dd-STATa-dependent. The ahhA-null mutant develops almost normally until culmination, but it forms slanting culminants that tend to collapse on to the substratum. The mutant also occasionally forms fruiting bodies with swollen papillae and with constrictions in the prestalk region. The AhhA protein localizes to the stalk tube entrance and also to the upper cup cells and in cells at or near to the constricted region where an F-actin ring is localized. These findings suggest that Dd-STATa regulates culmination and may be necessary for straight downward elongation of the stalk, via the putative actin-binding protein AhhA.

  17. Macroporous nanowire nanoelectronic scaffolds for synthetic tissues

    OpenAIRE

    Liu, Jia; Dvir, Tal; Jin, Lihua; Tsui, Jonathan H.; Qing, Quan; Suo, Zhigang; Langer, Robert S.; Kohane, Daniel Solomon; Lieber, Charles M.; Tian, Bozhi

    2012-01-01

    The development of three-dimensional (3D) synthetic biomaterials as structural and bioactive scaffolds is central to fields ranging from cellular biophysics to regenerative medicine. As of yet, these scaffolds cannot electrically probe the physicochemical and biological micro-environments throughout their 3D and macroporous interior, although this capability could have a marked impact in both electronics and biomaterials. Here, we address this challenge using macroporous, flexible and free-st...

  18. Chitosan-collagen porous scaffold and bone marrow mesenchymal stem cell transplantation for ischemic stroke

    Directory of Open Access Journals (Sweden)

    Feng Yan

    2015-01-01

    Full Text Available In this study, we successfully constructed a composite of bone marrow mesenchymal stem cells and a chitosan-collagen scaffold in vitro, transplanted either the composite or bone marrow mesenchymal stem cells alone into the ischemic area in animal models, and compared their effects. At 14 days after co-transplantation of bone marrow mesenchymal stem cells and the hitosan-collagen scaffold, neurological function recovered noticeably. Vascular endothelial growth factor expression and nestin-labeled neural precursor cells were detected in the ischemic area, surrounding tissue, hippocampal dentate gyrus and subventricular zone. Simultaneously, a high level of expression of glial fibrillary acidic protein and a low level of expression of neuron-specific enolase were visible in BrdU-labeled bone marrow mesenchymal stem cells. These findings suggest that transplantation of a composite of bone marrow mesenchymal stem cells and a chitosan-collagen scaffold has a neuroprotective effect following ischemic stroke.

  19. Concave Pit-Containing Scaffold Surfaces Improve Stem Cell-Derived Osteoblast Performance and Lead to Significant Bone Tissue Formation

    Science.gov (United States)

    Cusella-De Angelis, Maria Gabriella; Laino, Gregorio; Piattelli, Adriano; Pacifici, Maurizio; De Rosa, Alfredo; Papaccio, Gianpaolo

    2007-01-01

    Background Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear. Methodology and Findings In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80–120 µm in diameter and 40–100 µm in depth, which we termed primary; and (ii) smaller microcavities of 10–20 µm in diameter and 3–10 µm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct bone-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior bone formation was elicited compared to cells seeded on the smooth scaffold. Conclusion In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and

  20. The influence of biological motifs and dynamic mechanical stimulation in hydrogel scaffold systems on the phenotype of chondrocytes.

    Science.gov (United States)

    Appelman, Taly P; Mizrahi, Joseph; Elisseeff, Jennifer H; Seliktar, Dror

    2011-02-01

    Primary bovine chondrocytes and PEG-based hydrogels were used to investigate the effects of scaffold composition and architecture on the cellular response to large dynamic compressive strain stimulation. Proteins and proteoglycans were conjugated to functionalized poly(ethylene glycol) (PEG) and immobilized in PEG hydrogels to create bio-synthetic scaffolds. Second passage articular chondrocytes were encapsulated into four different scaffold compositions: PEG-Proteoglycan (PP), PEG-Fibrinogen (PF), PEG-Albumin (PA), and PEG only and subjected to 15% dynamic compressive strain at 1-Hz frequency. Cellular response was evaluated in terms of cell number, glycosaminoglycans (GAGs), collagen type II and collagen type I accumulation in the constructs following 24h and 28 days of stimulated and static culture. Stimulation of the constructs resulted in an increase in the cell number in all scaffolds, with no statistical difference measured among them. Dynamic stimulation of PP, PF, PA and PEG constructs resulted in a respective increase in the GAGs by 33%, 53.4%, 240.5%, and 284.5%, compared to their static controls. The permissive PEG and PA scaffolds showed a significantly larger relative increase in the GAGs in comparison to the other scaffolds tested. Collagen type II content in the PF, PA and PEG constructs increased by 78%, 1266% and 896% respectively, compared to their static controls. Permissive constructs showed a significantly larger relative increase and final absolute values of GAGs and type II collagen, compared to the PF constructs. Immunostaining for collagen type I, an indicator for chondrocyte de-differentiation, indicated that stimulation inhibited its production. Correlation maps between scaffold properties highlighted the major differences between permissive and instructive scaffolds. These results support the hypothesis that both compressive strain and scaffold bioactivity have an important effect on the chondrocyte metabolic response to mechanical

  1. Nanocomposite scaffold fabrication by incorporating gold nanoparticles into biodegradable polymer matrix: Synthesis, characterization, and photothermal effect.

    Science.gov (United States)

    Abdelrasoul, Gaser N; Farkas, Balazs; Romano, Ilaria; Diaspro, Alberto; Beke, Szabolcs

    2015-11-01

    Nanoparticle incorporation into scaffold materials is a valuable route to deliver various therapeutic agents, such as drug molecules or large biomolecules, proteins (e.g. DNA or RNA) into their targets. In particular, gold nanoparticles (Au NPs) with their low inherent toxicity, tunable stability and high surface area provide unique attributes facilitating new delivery strategies. A biodegradable, photocurable polymer resin, polypropylene fumarate (PPF) along with Au NPs were utilized to synthesize a hybrid nanocomposite resin, directly exploitable in stereolithography (SL) processes. To increase the particles' colloidal stability, the Au NP nanofillers were coated with polyvinyl pyrrolidone (PVP). The resulting resin was used to fabricate a new type of composite scaffold via mask projection excimer laser stereolithography. The thermal properties of the nanocomposite scaffolds were found to be sensitive to the concentration of NPs. The mechanical properties were augmented by the NPs up to 0.16μM, though further increase in the concentration led to a gradual decrease. Au NP incorporation rendered the biopolymer scaffolds photosensitive, i.e. the presence of Au NPs enhanced the optical absorption of the scaffolds as well, leading to possible localized temperature rise when irradiated with 532nm laser, known as the photothermal effect. PMID:26249594

  2. The differential effect of scaffold composition and architecture on chondrocyte response to mechanical stimulation.

    Science.gov (United States)

    Appelman, Taly P; Mizrahi, Joseph; Elisseeff, Jennifer H; Seliktar, Dror

    2009-02-01

    This study aims to explore the differential effect of scaffold composition and architecture on chondrogenic response to dynamic strain stimulation using encapsulating PEG-based hydrogels and primary bovine chondrocytes. Proteins and proteoglycans were conjugated to functionalized poly(ethylene glycol) (PEG) and immobilized in PEG hydrogels to create bio-synthetic materials to be used as scaffolds. Four different compositions were tested, including: PEG-Proteoglycan (PP), PEG-Fibrinogen (PF), PEG-Albumin (PA), and PEG only. Primary articular chondrocytes were encapsulated in the hydrogel scaffolds and subjected to 15% dynamic compressive strain stimulation at 1-Hz frequency for 28 days. Stimulation of PP, PF, PA and PEG constructs resulted in a respective increase in the unconfined true compressive modulus by 32%, 45.4%, 33.6%, and 28.2%, compared to their static controls. The PF showed a significantly larger relative increase in the modulus in comparison to all other scaffolds tested. These results support the hypothesis that mechanical stimulation and material bioactivity have a significant effect on the reported chondrocyte response. Similar trends were observed with the swelling ratio of the constructs. These findings indicate that while stimulation causes metabolic changes in chondrocytes seeded in PEG hydrogels, the matrix bioactivity has a significant role in enhancing chondrocyte mechanotransduction in encapsulating scaffolds subjected to physical deformations. PMID:19000634

  3. Neocellularization and neovascularization of nanosized bioactive glass-coated decellularized trabecular bone scaffolds

    KAUST Repository

    Gerhardt, Lutz Christian

    2012-09-11

    In this study, the in vivo recellularization and neovascularization of nanosized bioactive glass (n-BG)-coated decellu-larized trabecular bone scaffolds were studied in a rat model and quantified using stereological analyses. Based on the highest amount of vascular endothelial growth factor (VEGF) secreted by human fibroblasts grown on n-BG coatings (0-1.245 mg/cm 2), decellularized trabecular bone samples (porosity: 43-81%) were coated with n-BG particles. Grown on n-BG particles at a coating density of 0.263 mg/cm2, human fibroblasts produced 4.3 times more VEGF than on uncoated controls. After 8 weeks of implantation in Sprague-Dawley rats, both uncoated and n-BG-coated samples were well infiltrated with newly formed tissue (47-48%) and blood vessels (3-4%). No significant differences were found in cellularization and vascularization between uncoated bone scaffolds and n-BG-coated scaffolds. This finding indicates that the decellularized bone itself may exhibit growth-promoting properties induced by the highly interconnected pore microarchitecture and/or proteins left behind on decellularized scaffolds. Even if we did not find proangiogenic effects in n-BG-coated bone scaffolds, a bioactive coating is considered to be beneficial to impart osteoinductive and osteoconductive properties to decellularized bone. n-BG-coated bone grafts have thus high clinical potential for the regeneration of complex tissue defects given their ability for recellularization and neovascularization. © 2012 Wiley Periodicals, Inc.

  4. The KNOTTIN website and database: a new information system dedicated to the knottin scaffold

    OpenAIRE

    Gelly, Jean-Christophe; Gracy, Jérôme; Kaas, Quentin; Le-Nguyen, Dung; Heitz, Annie; Chiche, Laurent

    2004-01-01

    The KNOTTIN website and database organize information about knottins or inhibitor cystine knots, small disulfide-rich proteins with a knotted topology. Thanks to their small size and high stability, knottins provide appealing scaffolds for protein engineering and drug design. Static pages present the main historical and recent results about knottin discoveries, sequences, structures, folding, functions, applications and bibliography. Database searches provide dynamically generated tabular rep...

  5. Bioimprinted Polymer Scaffolds for Selective Recognition of RGD Peptides

    Science.gov (United States)

    Bergmann, Nicole; Peppas, Nicholas A.

    2003-03-01

    Fibronectin and a number of other plasma and extracellular matrix (ECM) adhesion proteins contain the tetrapeptide arginine-glycine-aspartic acid-serine (RGDS), and this sequence can be summarily recognized and bound by integrins present on cell membranes. Upon integrin binding, cells adhere to the substrate, and this adherence encourages ECM deposition and other cellular remodeling events. By targeting specific chemical functional groups on the peptide using non-covalent molecular imprinting, biomimetic polymeric scaffolds can be designed to mimic protein-ECM binding both on the surface and in the bulk during polymer degradation. Methacrylic acid-ethylene glycol dimethacrylate (MAA-g-EGDMA) copolymer films were prepared by free-radical ultraviolet polymerization in the presence of RGDS to create novel imprinted matrices for possible tissue engineering scaffolds. SEM analysis revealed a highly macroporous structure in peptide-imprinted polymers compared to controls. Optimal crosslinking ratios for peptide imprinting were determined using a small molecular weight fluorescent tag, 4-chloro-7-nitrobenzofurazan, and analyzed using fluorescent microscopy. Higher crosslinking ratios yielded better template recognition and gels exhibited specific recognition in aqueous media to RGDS molecules when in the presence of similar tetrapeptides.

  6. Tubular inverse opal scaffolds for biomimetic vessels

    Science.gov (United States)

    Zhao, Ze; Wang, Jie; Lu, Jie; Yu, Yunru; Fu, Fanfan; Wang, Huan; Liu, Yuxiao; Zhao, Yuanjin; Gu, Zhongze

    2016-07-01

    There is a clinical need for tissue-engineered blood vessels that can be used to replace or bypass damaged arteries. The success of such grafts depends strongly on their ability to mimic native arteries; however, currently available artificial vessels are restricted by their complex processing, controversial integrity, or uncontrollable cell location and orientation. Here, we present new tubular scaffolds with specific surface microstructures for structural vessel mimicry. The tubular scaffolds are fabricated by rotationally expanding three-dimensional tubular inverse opals that are replicated from colloidal crystal templates in capillaries. Because of the ordered porous structure of the inverse opals, the expanded tubular scaffolds are imparted with circumferentially oriented elliptical pattern microstructures on their surfaces. It is demonstrated that these tailored tubular scaffolds can effectively make endothelial cells to form an integrated hollow tubular structure on their inner surface and induce smooth muscle cells to form a circumferential orientation on their outer surface. These features of our tubular scaffolds make them highly promising for the construction of biomimetic blood vessels.There is a clinical need for tissue-engineered blood vessels that can be used to replace or bypass damaged arteries. The success of such grafts depends strongly on their ability to mimic native arteries; however, currently available artificial vessels are restricted by their complex processing, controversial integrity, or uncontrollable cell location and orientation. Here, we present new tubular scaffolds with specific surface microstructures for structural vessel mimicry. The tubular scaffolds are fabricated by rotationally expanding three-dimensional tubular inverse opals that are replicated from colloidal crystal templates in capillaries. Because of the ordered porous structure of the inverse opals, the expanded tubular scaffolds are imparted with circumferentially

  7. Combining Scaffolding for Content and Scaffolding for Dialogue to Support Conceptual Breakthroughs in Understanding Probability

    Science.gov (United States)

    Kazak, Sibel; Wegerif, Rupert; Fujita, Taro

    2015-01-01

    In this paper, we explore the relationship between scaffolding, dialogue, and conceptual breakthroughs, using data from a design-based research study that focuses on the development of understanding of probability in 10-12 year old students. The aim of the study is to gain insight into how the combination of scaffolding for content using…

  8. Antioxidant effects of betulin on porcine chondrocyte behavior in gelatin/C6S/C4S/HA modified tricopolymer scaffold

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Wen-Yang; Lin, Feng-Huei [Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan (China); Sadhasivam, S., E-mail: rahulsbio@yahoo.co.in [Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan (China); Savitha, S. [Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan (China)

    2010-05-10

    The antioxidant effects of betulin on porcine chondrocytes cultured in gelatin/C6S/C4S/HA modified tricopolymer scaffold for a period of 4 weeks was investigated. The porous structure of the scaffold and cell attachment was observed by scanning electron microscopy (SEM). Biochemical measures of necrosis, cell proliferation, sulfated glycosaminoglycans (sGAG) content and extracellular matrix related gene expressions were quantitatively evaluated. The cell proliferation data showed good cellular viability in tricopolymer scaffold and increased optical density for total DNA demonstrated that the cells continued to proliferate inside the scaffold. The sGAG production indicated chondrogenic differentiation. Chondrocytes treated with betulin expressed transcripts encoding type II collagen, aggrecan, and decorin. To conclude, the substantiated results supported cell proliferation, production of extracellular matrix proteins and down-regulation of matrix metalloproteases and cytokine, in betulin treated scaffolds.

  9. Scaffolding the "Scaffolding" Metaphor: From Inspiration to a Practical Tool for Kindergarten Teachers

    Science.gov (United States)

    Eshach, Haim; Dor-Ziderman, Yair; Arbel, Yael

    2011-10-01

    The present research aims shifting `scaffolding' from an inspiring metaphor to a practical tool to be used by kindergarten teachers when conducting scientific activities. It identifies scaffolding strategies that three experienced kindergarten teachers, ones acknowledged as excelling in science teaching, implicitly used when conducting science activities. For this end 20 whole-day observations were recorded in each of the three kindergartens and transcribed verbatim. The scaffolding strategies were identified through an inductive analysis performed on the observations and through the relevant literature. The strategies yielded from the analysis were grouped into affective and cognitive domains, each divided into categories and subcategories. The complete set of identified strategies was termed the scaffolding scheme. The scaffolding scheme can assist kindergarten and primary school teachers, as well as researchers, in analyzing scientific activities conducted in the kindergarten and judging how efficient the employed strategies are, what strategies to eliminate, and what other strategies might be needed.

  10. Biodegradable Polymer-Based Scaffolds for Bone Tissue Engineering

    CERN Document Server

    Sultana, Naznin

    2013-01-01

    This book addresses the principles, methods and applications of biodegradable polymer based scaffolds for bone tissue engineering. The general principle of bone tissue engineering is reviewed and the traditional and novel scaffolding materials, their properties and scaffold fabrication techniques are explored. By acting as temporary synthetic extracellular matrices for cell accommodation, proliferation, and differentiation, scaffolds play a pivotal role in tissue engineering. This book does not only provide the comprehensive summary of the current trends in scaffolding design but also presents the new trends and directions for scaffold development for the ever expanding tissue engineering applications.

  11. Elastomeric PGS scaffolds in arterial tissue engineering.

    Science.gov (United States)

    Lee, Kee-Won; Wang, Yadong

    2011-01-01

    Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation. The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS) for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro

  12. Maltodextrin enhances biofilm elimination by electrochemical scaffold

    Science.gov (United States)

    Sultana, Sujala T.; Call, Douglas R.; Beyenal, Haluk

    2016-01-01

    Electrochemical scaffolds (e-scaffolds) continuously generate low concentrations of H2O2 suitable for damaging wound biofilms without damaging host tissue. Nevertheless, retarded diffusion combined with H2O2 degradation can limit the efficacy of this potentially important clinical tool. H2O2 diffusion into biofilms and bacterial cells can be increased by damaging the biofilm structure or by activating membrane transportation channels by exposure to hyperosmotic agents. We hypothesized that e-scaffolds would be more effective against Acinetobacter baumannii and Staphylococcus aureus biofilms in the presence of a hyperosmotic agent. E-scaffolds polarized at −600 mVAg/AgCl were overlaid onto preformed biofilms in media containing various maltodextrin concentrations. E-scaffold alone decreased A. baumannii and S. aureus biofilm cell densities by (3.92 ± 0.15) log and (2.31 ± 0.12) log, respectively. Compared to untreated biofilms, the efficacy of the e-scaffold increased to a maximum (8.27 ± 0.05) log reduction in A. baumannii and (4.71 ± 0.12) log reduction in S. aureus biofilm cell densities upon 10 mM and 30 mM maltodextrin addition, respectively. Overall ~55% decrease in relative biofilm surface coverage was achieved for both species. We conclude that combined treatment with electrochemically generated H2O2 from an e-scaffold and maltodextrin is more effective in decreasing viable biofilm cell density. PMID:27782161

  13. mAKAP – A Master Scaffold for Cardiac Remodeling

    Science.gov (United States)

    Passariello, Catherine L.; Li, Jinliang; Dodge-Kafka, Kimberly; Kapiloff, Michael S.

    2014-01-01

    Cardiac remodeling is regulated by an extensive intracellular signal transduction network. Each of the many signaling pathways in this network contributes uniquely to the control of cellular adaptation. In the last few years, it has become apparent that multimolecular signaling complexes or ‘signalosomes’ are important for fidelity in intracellular signaling and for mediating crosstalk between the different signaling pathways. These complexes integrate upstream signals and control downstream effectors. In the cardiac myocyte, the protein mAKAPβ serves as a scaffold for a large signalosome that is responsive to cAMP, calcium, hypoxia, and mitogen-activated protein kinase signaling. The main function of mAKAPβ signalosomes is to modulate stress-related gene expression regulated by the transcription factors NFATc, MEF2 and HIF-1α and type II histone deacetylases that control pathological cardiac hypertrophy. PMID:25551320

  14. The effect of 3D nanofibrous scaffolds on the chondrogenesis of induced pluripotent stem cells and their application in restoration of cartilage defects.

    Directory of Open Access Journals (Sweden)

    Ji Liu

    Full Text Available The discovery of induced pluripotent stem cells (iPSCs rendered the reprogramming of terminally differentiated cells to primary stem cells with pluripotency possible and provided potential for the regeneration and restoration of cartilage defect. Chondrogenic differentiation of iPSCs is crucial for their application in cartilage tissue engineering. In this study we investigated the effect of 3D nanofibrous scaffolds on the chondrogenesis of iPSCs and articular cartilage defect restoration. Super-hydrophilic and durable mechanic polycaprolactone (PCL/gelatin scaffolds were fabricated using two separate electrospinning processes. The morphological structure and mechanical properties of the scaffolds were characterized. The chondrogenesis of the iPSCs in vitro and the restoration of the cartilage defect was investigated using scanning electron microscopy (SEM, the Cell Counting Kit-8 (CCK-8, histological observation, RT-qPCR, and western blot analysis. iPSCs on the scaffolds expressed higher levels of chondrogenic markers than the control group. In an animal model, cartilage defects implanted with the scaffold-cell complex exhibited an enhanced gross appearance and histological improvements, higher cartilage-specific gene expression and protein levels, as well as subchondral bone regeneration. Therefore, we showed scaffolds with a 3D nanofibrous structure enhanced the chondrogenesis of iPSCs and that iPSC-containing scaffolds improved the restoration of cartilage defects to a greater degree than did scaffolds alone in vivo.

  15. Incorporation of Fucoidan in β-Tricalcium phosphate-Chitosan scaffold prompts the differentiation of human bone marrow stromal cells into osteogenic lineage

    Science.gov (United States)

    Puvaneswary, Subramaniam; Raghavendran, Hanumantharao Balaji; Talebian, Sepehr; Murali, Malliga Raman; A Mahmod, Suhaeb; Singh, Simmrat; Kamarul, Tunku

    2016-01-01

    In our previous study, we reported the fabrication and characterization of a novel tricalcium phosphate-fucoidan-chitosan (TCP-Fu-Ch) biocomposite scaffold. However, the previous report did not show whether the biocomposite scaffold can exhibit osteogenic differentiation of human bone marrow stromal cells in osteogenic media and normal media supplemented with platelet-derived growth factor (PDGF-BB). On day 15, the release of osteocalcin, was significant in the TCP-Fu-Ch scaffold, when compared with that in the TCP-Ch scaffold, and the level of release was approximately 8 and 6 ng/ml in osteogenic and normal media supplemented with PDGF-BB, respectively. Scanning electron microscopy of the TCP-Fu-Ch scaffold demonstrated mineralization and apatite layer formation on day 14, while the addition of PDGF-BB also improved the osteogenic differentiation of the scaffold. An array of gene expression analysis demonstrated that TCP-Fu-Ch scaffold cultured in osteogenic and normal media supplemented with PDGF-BB showed significant improvement in the expression of collagen 1, Runt-related transcription factor 2, osteonectin, bone gamma-carboxyglutamate protein, alkaline phosphatase, and PPA2, but a decline in the expression of integrin. Altogether, the present study demonstrated that fucoidan-incorporated TCP-Ch scaffold could be used in the differentiation of bone marrow stromal cells and can be a potential candidate for the treatment of bone-related ailments through tissue engineering technology. PMID:27068453

  16. TGF-β1-Enhanced TCP-Coated Sensate Scaffolds Can Detect Bone Bonding

    OpenAIRE

    Szivek, J.A.; Margolis, D.S.; Garrison, B.K.; Nelson, E.; Vaidyanathan, R. K.; DeYoung, D. W.

    2005-01-01

    Porous polybutylene terephthalate (PBT) scaffold systems were tested as orthopedic implants to determine whether these scaffolds could be used to detect strain transfer following bone growth into the scaffold. Three types of scaffold systems were tested: porous PBT scaffolds, porous PBT scaffolds with a thin β-tricalcium phosphate coating (LC-PBT), and porous PBT scaffolds with the TCP coating vacuum packed into the scaffold pores (VI-PBT). In addition, the effect of applying TGF-β1 to scaffo...

  17. Anchored design of protein-protein interfaces.

    Directory of Open Access Journals (Sweden)

    Steven M Lewis

    Full Text Available BACKGROUND: Few existing protein-protein interface design methods allow for extensive backbone rearrangements during the design process. There is also a dichotomy between redesign methods, which take advantage of the native interface, and de novo methods, which produce novel binders. METHODOLOGY: Here, we propose a new method for designing novel protein reagents that combines advantages of redesign and de novo methods and allows for extensive backbone motion. This method requires a bound structure of a target and one of its natural binding partners. A key interaction in this interface, the anchor, is computationally grafted out of the partner and into a surface loop on the design scaffold. The design scaffold's surface is then redesigned with backbone flexibility to create a new binding partner for the target. Careful choice of a scaffold will bring experimentally desirable characteristics into the new complex. The use of an anchor both expedites the design process and ensures that binding proceeds against a known location on the target. The use of surface loops on the scaffold allows for flexible-backbone redesign to properly search conformational space. CONCLUSIONS AND SIGNIFICANCE: This protocol was implemented within the Rosetta3 software suite. To demonstrate and evaluate this protocol, we have developed a benchmarking set of structures from the PDB with loop-mediated interfaces. This protocol can recover the correct loop-mediated interface in 15 out of 16 tested structures, using only a single residue as an anchor.

  18. Hydrogel Composite Materials for Tissue Engineering Scaffolds

    Science.gov (United States)

    Shapiro, Jenna M.; Oyen, Michelle L.

    2013-04-01

    Hydrogels are appealing for biomaterials applications due to their compositional similarity with highly hydrated natural biological tissues. However, for structurally demanding tissue engineering applications, hydrogel use is limited by poor mechanical properties. Here, composite materials approaches are considered for improving hydrogel properties while attempting to more closely mimic natural biological tissue structures. A variety of composite material microstructures is explored, based on multiple hydrogel constituents, particle reinforcement, electrospun nanometer to micrometer diameter polymer fibers with single and multiple fiber networks, and combinations of these approaches to form fully three-dimensional fiber-reinforced hydrogels. Natural and synthetic polymers are examined for formation of a range of scaffolds and across a range of engineered tissue applications. Following a discussion of the design and fabrication of composite scaffolds, interactions between living biological cells and composite scaffolds are considered across the full life cycle of tissue engineering from scaffold fabrication to in vivo use. We conclude with a summary of progress in this area to date and make recommendations for continuing research and for advanced hydrogel scaffold development.

  19. SCAFFOLD: TISSUE ENGINEERING AND REGENERATIVE MEDICINE

    Directory of Open Access Journals (Sweden)

    Garg Tarun

    2011-12-01

    Full Text Available Scaffolds are the central components, which are used to deliver the cells, drug and gene into the body. Polymeric scaffolds may be prepared as typical 3-D porous matrix, nanofibrous matrix, thermo sensitive sol-gel transition hydrogel or porous microsphere, which provide suitable substrate for cell attachment, cell proliferation, differentiated function, and cell migration. Scaffold matrices have specific advantage over other novel drug delivery systems by achieving high drug loading. This study has been conducted to illustrate the various fabrication techniques of scaffold like Particulate leaching, freeze-drying, Supercritical fluid technology, thermally induced phase separation, Rapid prototyping, powder compaction, sol-gel, melt moulding etc. These techniques allow the preparation of porous structures with regular porosity. The main conclusion of this study is Scaffold provides adequate signals (e.g., through the use of adhesion peptides and growth factors to the cells, to induce and maintain them in their desired differentiation stage and for their survival and growth and their successful utilisation in various fields like bone formation, joint pain inflammation, tumor, periodontal regeneration, In-vivo generation of dental pulp, diabetes, osteochondrogenesis, wound dressing, inhibit bacterial growth, heart disease, repair of nasal and auricular malformation, cartilage development, regulated non-viral gene delivery, as artificial corneas, as heart valve, antiepileptic effect, tendon repair, ligament replacement, plasmid delivery, etc.

  20. In vitro osteoclastogenesis on textile chitosan scaffold

    Directory of Open Access Journals (Sweden)

    C Heinemann

    2010-02-01

    Full Text Available Textile chitosan fibre scaffolds were evaluated in terms of interaction with osteoclast-like cells, derived from human primary monocytes. Part of the scaffolds was further modified by coating with fibrillar collagen type I in order to make the surface biocompatible. Monocytes were cultured directly on the scaffolds in the presence of macrophage colony stimulating factor (M-CSF and receptor activator of nuclear factor kappaB ligand (RANKL for up to 18 days. Confocal laser scanning microscopy (CLSM as well as scanning electron microscopy (SEM revealed the formation of multinuclear osteoclast-like cells on both the raw chitosan fibres and the collagen-coated scaffolds. The modified surface supported the osteoclastogenesis. Differentiation towards the osteoclastic lineage was confirmed by the microscopic detection of cathepsin K, tartrate resistant acid phosphatase (TRAP, acidic compartments using 3-(2,4-dinitroanillino-3’-amino-N-methyldipropylamine (DAMP, immunological detection of TRAP isoform 5b, and analysis of gene expression of the osteoclastic markers TRAP, cathepsin K, vitronectin receptor, and calcitonin receptor using reverse transcription-polymerase chain reaction (RT-PCR. The feature of the collagen-coated but also of the raw chitosan fibre scaffolds to support attachment and differentiation of human monocytes facilitates cell-induced material resorption – one main requirement for successful bone tissue engineering.

  1. Heterogeneity of Scaffold Biomaterials in Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Lauren Edgar

    2016-05-01

    Full Text Available Tissue engineering (TE offers a potential solution for the shortage of transplantable organs and the need for novel methods of tissue repair. Methods of TE have advanced significantly in recent years, but there are challenges to using engineered tissues and organs including but not limited to: biocompatibility, immunogenicity, biodegradation, and toxicity. Analysis of biomaterials used as scaffolds may, however, elucidate how TE can be enhanced. Ideally, biomaterials should closely mimic the characteristics of desired organ, their function and their in vivo environments. A review of biomaterials used in TE highlighted natural polymers, synthetic polymers, and decellularized organs as sources of scaffolding. Studies of discarded organs supported that decellularization offers a remedy to reducing waste of donor organs, but does not yet provide an effective solution to organ demand because it has shown varied success in vivo depending on organ complexity and physiological requirements. Review of polymer-based scaffolds revealed that a composite scaffold formed by copolymerization is more effective than single polymer scaffolds because it allows copolymers to offset disadvantages a single polymer may possess. Selection of biomaterials for use in TE is essential for transplant success. There is not, however, a singular biomaterial that is universally optimal.

  2. Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications.

    Science.gov (United States)

    Tiede, Christian; Tang, Anna A S; Deacon, Sarah E; Mandal, Upasana; Nettleship, Joanne E; Owen, Robin L; George, Suja E; Harrison, David J; Owens, Raymond J; Tomlinson, Darren C; McPherson, Michael J

    2014-05-01

    We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (Tm ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Å resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality and complexity comprising 1.3 × 10(10) clones. To demonstrate library efficacy, we screened against the yeast Small Ubiquitin-like Modifier (SUMO). In selected clones, variable region 1 often contained sequences homologous to the known SUMO interactive motif (V/I-X-V/I-V/I). Four Adhirons were further characterised and displayed low nanomolar affinities and high specificity for yeast SUMO with essentially no cross-reactivity to human SUMO protein isoforms. We have identified binders against >100 target molecules to date including as examples, a fibroblast growth factor (FGF1), platelet endothelial cell adhesion molecule (PECAM-1; CD31), the SH2 domain Grb2 and a 12-aa peptide. Adhirons are highly stable and well expressed allowing highly specific binding reagents to be selected for use in molecular recognition applications. PMID:24668773

  3. Sustained Release of Cx43 Antisense Oligodeoxynucleotides from Coated Collagen Scaffolds Promotes Wound Healing.

    Science.gov (United States)

    Gilmartin, Daniel J; Soon, Allyson; Thrasivoulou, Christopher; Phillips, Anthony R J; Jayasinghe, Suwan N; Becker, David L

    2016-07-01

    Antisense oligodeoxynucleotides targeting the mRNA of the gap junction protein Cx43 promote tissue repair in a variety of different wounds. Delivery of the antisense drug has most often been achieved by a thermoreversible hydrogel, Pluronic F-127, which is very effective in the short term but does not allow for sustained delivery over several days. For chronic wounds that take a long time to heal, repeated dosing with the drug may be desirable but is not always compatible with conventional treatments such as the weekly changing of compression bandages on venous leg ulcers. Here the coating of collagen scaffolds with antisense oligonucleotides is investigated and a way to provide protection of the oligodeoxynucleotide drug is found in conjunction with sustained release over a 7 d period. This approach significantly reduces the normal foreign body reaction to the scaffold, which induces an increase of Cx43 protein and an inhibition of healing. As a result of the antisense integration into the scaffold, inflammation is reduced with the rate of wound healing and contracture is significantly improved. This coated scaffold approach may be very useful for treating venous leg ulcers and also for providing a sustained release of any other types of oligonucleotide drugs that are being developed. PMID:27253638

  4. Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Baeyens, Nicolas; Latrache, Iman; Yerna, Xavier [Laboratory of Cell Physiology, IoNS, Université Catholique de Louvain (Belgium); Noppe, Gauthier; Horman, Sandrine [Pôle de Recherche Cardiovasculaire, IREC, Université Catholique de Louvain (Belgium); Morel, Nicole, E-mail: nicole.morel@uclouvain.be [Laboratory of Cell Physiology, IoNS, Université Catholique de Louvain (Belgium)

    2013-11-22

    Highlights: •The three ERM proteins are expressed in vascular smooth muscle cell. •ERM depletion inhibited PDGF-evoked migration redundantly. •ERM depletion increased cell adhesion redundantly. •ERM depletion did not affect PDGF-evoked Ca signal, Rac1 activation, proliferation. •ERM proteins control PDGF-induced migration by regulating adhesion. -- Abstract: Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca{sup 2+} signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.

  5. Peptide functionalized polyhydroxyalkanoate nanofibrous scaffolds enhance Schwann cells activity

    NARCIS (Netherlands)

    Masaeli, E.; Wieringa, P.A.; Morshed, M.; Nasr-Esfahani, M.H.; Sadri, S.; Blitterswijk, van C.A.; Moroni, L.

    2014-01-01

    Interactions between Schwann cells (SCs) and scaffolds are important for tissue development during nerve regeneration, because SCs physiologically assist in directing the growth of regenerating axons. In this study, we prepared electrospun scaffolds combining poly (3-hydroxybutyrate) (PHB) and poly

  6. Development of a nanostructured DNA delivery scaffold via electrospinning of PLGA and PLA-PEG block copolymers

    Science.gov (United States)

    Luu, Y. K.; Kim, K.; Hsiao, B. S.; Chu, B.; Hadjiargyrou, M.; Hadjiargyou, M. (Principal Investigator)

    2003-01-01

    The present work utilizes electrospinning to fabricate synthetic polymer/DNA composite scaffolds for therapeutic application in gene delivery for tissue engineering. The scaffolds are non-woven, nano-fibered, membranous structures composed predominantly of poly(lactide-co-glycolide) (PLGA) random copolymer and a poly(D,L-lactide)-poly(ethylene glycol) (PLA-PEG) block copolymer. Release of plasmid DNA from the scaffolds was sustained over a 20-day study period, with maximum release occurring at approximately 2 h. Cumulative release profiles indicated amounts released were approximately 68-80% of the initially loaded DNA. Variations in the PLGA to PLA-PEG block copolymer ratio vastly affected the overall structural morphology, as well as both the rate and efficiency of DNA release. Results indicated that DNA released directly from these electrospun scaffolds was indeed intact, capable of cellular transfection, and successfully encoded the protein beta-galactosidase. When tested under tensile loads, the electrospun polymer/DNA composite scaffolds exhibited tensile moduli of approximately 35 MPa, with approximately 45% strain initially. These values approximate those of skin and cartilage. Taken together, this work represents the first successful demonstration of plasmid DNA incorporation into a polymer scaffold using electrospinning.

  7. Three-Dimensional Polydopamine Functionalized Coiled Microfibrous Scaffolds Enhance Human Mesenchymal Stem Cells Colonization and Mild Myofibroblastic Differentiation.

    Science.gov (United States)

    Taskin, Mehmet Berat; Xu, Ruodan; Gregersen, Hans; Nygaard, Jens Vinge; Besenbacher, Flemming; Chen, Menglin

    2016-06-29

    Electrospinning has been widely applied for tissue engineering due to its versatility of fabricating extracellular matrix (ECM) mimicking fibrillar scaffolds. Yet there are still challenges such as that these two-dimensional (2D) tightly packed, hydrophobic fibers often hinder cell infiltration and cell-scaffold integration. In this study, polycaprolactone (PCL) was electrospun into a grounded coagulation bath collector, resulting in 3D coiled microfibers with in situ surface functionalization with hydrophilic, catecholic polydopamine (pDA). The 3D scaffolds showed biocompatibility and were well-integrated with human bone marrow derived human mesenchymal stem cells (hMSCs), with significantly higher cell penetration depth compared to that of the 2D PCL microfibers from traditional electrospinning. Further differentiation of human mesenchymal stem cells (hMSCs) into fibroblast phenotype in vitro indicates that, compared to the stiff, tightly packed, 2D scaffolds which aggravated myofibroblasts related activities, such as upregulated gene and protein expression of α-smooth muscle actin (α-SMA), 3D scaffolds induced milder myofibroblastic differentiation. The flexible 3D fibers further allowed contraction with the well-integrated, mechanically active myofibroblasts, monitored under live-cell imaging, whereas the stiff 2D scaffolds restricted that. PMID:27265317

  8. Scaffold-mediated BMP-2 minicircle DNA delivery accelerated bone repair in a mouse critical-size calvarial defect model.

    Science.gov (United States)

    Keeney, Michael; Chung, Michael T; Zielins, Elizabeth R; Paik, Kevin J; McArdle, Adrian; Morrison, Shane D; Ransom, Ryan C; Barbhaiya, Namrata; Atashroo, David; Jacobson, Gunilla; Zare, Richard N; Longaker, Michael T; Wan, Derrick C; Yang, Fan

    2016-08-01

    Scaffold-mediated gene delivery holds great promise for tissue regeneration. However, previous attempts to induce bone regeneration using scaffold-mediated non-viral gene delivery rarely resulted in satisfactory healing. We report a novel platform with sustained release of minicircle DNA (MC) from PLGA scaffolds to accelerate bone repair. MC was encapsulated inside PLGA scaffolds using supercritical CO2 , which showed prolonged release of MC. Skull-derived osteoblasts transfected with BMP-2 MC in vitro result in higher osteocalcin gene expression and mineralized bone formation. When implanted in a critical-size mouse calvarial defect, scaffolds containing luciferase MC lead to robust in situ protein production up to at least 60 days. Scaffold-mediated BMP-2 MC delivery leads to substantially accelerated bone repair as early as two weeks, which continues to progress over 12 weeks. This platform represents an efficient, long-term nonviral gene delivery system, and may be applicable for enhancing repair of a broad range of tissues types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2099-2107, 2016. PMID:27059085

  9. Mechanical and in vitro performance of 13-93 bioactive glass scaffolds prepared by a polymer foam replication technique.

    Science.gov (United States)

    Fu, Qiang; Rahaman, Mohamed N; Bal, B Sonny; Brown, Roger F; Day, Delbert E

    2008-11-01

    A polymer foam replication technique was used to prepare porous scaffolds of 13-93 bioactive glass with a microstructure similar to that of human trabecular bone. The scaffolds, with a porosity of 85+/-2% and pore size of 100-500 microm, had a compressive strength of 11+/-1 MPa, and an elastic modulus of 3.0+/-0.5 GPa, approximately equal to the highest values reported for human trabecular bone. The strength was also considerably higher than the values reported for polymeric, bioactive glass-ceramic and hydroxyapatite constructs prepared by the same technique and with the equivalent level of porosity. The in vitro bioactivity of the scaffolds was observed by the conversion of the glass surface to a nanostructured hydroxyapatite layer within 7 days in simulated body fluid at 37 degrees C. Protein and MTT assays of in vitro cell cultures showed an excellent ability of the scaffolds to support the proliferation of MC3T3-E1 preosteoblastic cells, both on the surface and in the interior of the porous constructs. Scanning electron microscopy showed cells with a closely adhering, well-spread morphology and a continuous increase in cell density on the scaffolds during 6 days of culture. The results indicate that the 13-93 bioactive glass scaffolds could be applied to bone repair and regeneration. PMID:18519173

  10. Scaffolding for Three-Dimensional Embryonic Vasculogenesis

    Science.gov (United States)

    Kraehenbuehl, Thomas P.; Aday, Sezin; Ferreira, Lino S.

    Biomaterial scaffolds have great potential to support efficient vascular differentiation of embryonic stem cells. Vascular cell fate-specific biochemical and biophysical cues have been identified and incorporated into three-dimensional (3D) biomaterials to efficiently direct embryonic vasculogenesis. The resulting vascular-like tissue can be used for regenerative medicine applications, further elucidation of biophysical and biochemical cues governing vasculogenesis, and drug discovery. In this chapter, we give an overview on the following: (1) developmental cues for directed differentiation of human embryonic stem cells (hESCs) into vascular cells, (2) 3D vascular differentiation in embryoid bodies (EBs), (3) preparation of 3D scaffolds for the vascular differentiation of hESCs, and (4) the most significant studies combining scaffolding and hESCs for development of vascular-like tissue.

  11. Scaffolds for central nervous system tissue engineering

    Science.gov (United States)

    He, Jin; Wang, Xiu-Mei; Spector, Myron; Cui, Fu-Zhai

    2012-03-01

    Traumatic injuries to the brain and spinal cord of the central nervous system (CNS) lead to severe and permanent neurological deficits and to date there is no universally accepted treatment. Owing to the profound impact, extensive studies have been carried out aiming at reducing inflammatory responses and overcoming the inhibitory environment in the CNS after injury so as to enhance regeneration. Artificial scaffolds may provide a suitable environment for axonal regeneration and functional recovery, and are of particular importance in cases in which the injury has resulted in a cavitary defect. In this review we discuss development of scaffolds for CNS tissue engineering, focusing on mechanism of CNS injuries, various biomaterials that have been used in studies, and current strategies for designing and fabricating scaffolds.

  12. Nano/macro porous bioactive glass scaffold

    Science.gov (United States)

    Wang, Shaojie

    Bioactive glass (BG) and ceramics have been widely studied and developed as implants to replace hard tissues of the musculo-skeletal system, such as bones and teeth. Recently, instead of using bulk materials, which usually do not degrade rapidly enough and may remain in the human body for a long time, the idea of bioscaffold for tissue regeneration has generated much interest. An ideal bioscaffold is a porous material that would not only provide a three-dimensional structure for the regeneration of natural tissue, but also degrade gradually and, eventually be replaced by the natural tissue completely. Among various material choices the nano-macro dual porous BG appears as the most promising candidate for bioscaffold applications. Here macropores facilitate tissue growth while nanopores control degradation and enhance cell response. The surface area, which controls the degradation of scaffold can also be tuned by changing the nanopore size. However, fabrication of such 3D structure with desirable nano and macro pores has remained challenging. In this dissertation, sol-gel process combined with spinodal decomposition or polymer sponge replication method has been developed to fabricate the nano-macro porous BG scaffolds. Macropores up to 100microm are created by freezing polymer induced spinodal structure through sol-gel transition, while larger macropores (>200um) of predetermined size are obtained by the polymer sponge replication technique. The size of nanopores, which are inherent to the sol-gel method of glass fabrication, has been tailored using several approaches: Before gel point, small nanopores are generated using acid catalyst that leads to weakly-branched polymer-like network. On the other hand, larger nanopores are created with the base-catalyzed gel with highly-branched cluster-like structure. After the gel point, the nanostructure can be further modified by manipulating the sintering temperature and/or the ammonia concentration used in the solvent

  13. Knowledge scaffolding visualizations: A guiding framework

    Directory of Open Access Journals (Sweden)

    Elitsa Alexander

    2015-06-01

    Full Text Available In this paper we provide a guiding framework for understanding and selecting visual representations in the knowledge management (KM practice. We build on an interdisciplinary analogy between two connotations of the notion of “scaffolding”: physical scaffolding from an architectural-engineering perspective and scaffolding of the “everyday knowing in practice” from a KM perspective. We classify visual structures for knowledge communication in teams into four types of scaffolds: grounded (corresponding e.g., to perspectives diagrams or dynamic facilitation diagrams, suspended (e.g., negotiation sketches, argument maps, panel (e.g., roadmaps or timelines and reinforcing (e.g., concept diagrams. The article concludes with a set of recommendations in the form of questions to ask whenever practitioners are choosing visualizations for specific KM needs. Our recommendations aim at providing a framework at a broad-brush level to aid choosing a suitable visualization template depending on the type of KM endeavour.

  14. Research Diary: A Tool for Scaffolding

    Directory of Open Access Journals (Sweden)

    Marion Engin Ed.D

    2011-09-01

    Full Text Available Diaries have long been seen as tools for reflection in learning languages, and learning about teaching. Despite this recognition of the importance of narratives in diary writing, little attention has been paid to the role of research diaries in the process of learning about research, and learning how to be a researcher. During the author's own research into the construction of teaching knowledge by pre-service trainees, she became aware that her research diary was scaffolding her own construction of research knowledge. In this article the author discusses the role of a research diary based on a socio-cultural theory of learning. The diary acts as the expert other in the scaffolding of research knowledge by the novice researcher. The discussion of the nature of the scaffolding and the role of diary writing draws on examples from the author's research diary written during her doctoral studies.

  15. Effect of different calcium phosphate scaffold ratios on odontogenic differentiation of human dental pulp cells

    Energy Technology Data Exchange (ETDEWEB)

    AbdulQader, Sarah Talib [School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Department of Pedodontic and Preventive Dentistry, College of Dentistry, University of Baghdad, Baghdad (Iraq); Kannan, Thirumulu Ponnuraj, E-mail: kannan@usm.my [School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Rahman, Ismail Ab [School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia); Ismail, Hanafi [School of Materials and Minerals Resource Engineering, Universiti Sains Malaysia, 14300 Penang (Malaysia); Mahmood, Zuliani [School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia)

    2015-04-01

    Calcium phosphate (CaP) scaffolds have been widely and successfully used with osteoblast cells for bone tissue regeneration. However, it is necessary to investigate the effects of these scaffolds on odontoblast cells' proliferation and differentiation for dentin tissue regeneration. In this study, three different hydroxyapatite (HA) to beta tricalcium phosphate (β-TCP) ratios of biphasic calcium phosphate (BCP) scaffolds, BCP20, BCP50, and BCP80, with a mean pore size of 300 μm and 65% porosity were prepared from phosphoric acid (H{sub 2}PO{sub 4}) and calcium carbonate (CaCO{sub 3}) sintered at 1000 °C for 2 h. The extracts of these scaffolds were assessed with regard to cell viability and differentiation of odontoblasts. The high alkalinity, more calcium, and phosphate ions released that were exhibited by BCP20 decreased the viability of human dental pulp cells (HDPCs) as compared to BCP50 and BCP80. However, the cells cultured with BCP20 extract expressed high alkaline phosphatase activity and high expression level of bone sialoprotein (BSP), dental matrix protein-1 (DMP-1), and dentin sialophosphoprotein (DSPP) genes as compared to that cultured with BCP50 and BCP80 extracts. The results highlighted the effect of different scaffold ratios on the cell microenvironment and demonstrated that BCP20 scaffold can support HDPC differentiation for dentin tissue regeneration. - Highlights: • BCPs of different HA/β-TCP ratios influence cell microenvironment. • BCP20 decreases cell viability of HDPCs as compared to BCP50 and BCP80. • HDPCs cultured with BCP20 express highest ALP activity. • HDPCs cultured with BCP20 up-regulate BSP, DMP-1 and DSPP gene expressions. • BCP20 can support HDPC differentiation for dentin tissue regeneration.

  16. Nanocomposite scaffold fabrication by incorporating gold nanoparticles into biodegradable polymer matrix: Synthesis, characterization, and photothermal effect

    Energy Technology Data Exchange (ETDEWEB)

    Abdelrasoul, Gaser N.; Farkas, Balazs; Romano, Ilaria; Diaspro, Alberto; Beke, Szabolcs, E-mail: szabolcs.beke@iit.it

    2015-11-01

    Nanoparticle incorporation into scaffold materials is a valuable route to deliver various therapeutic agents, such as drug molecules or large biomolecules, proteins (e.g. DNA or RNA) into their targets. In particular, gold nanoparticles (Au NPs) with their low inherent toxicity, tunable stability and high surface area provide unique attributes facilitating new delivery strategies. A biodegradable, photocurable polymer resin, polypropylene fumarate (PPF) along with Au NPs were utilized to synthesize a hybrid nanocomposite resin, directly exploitable in stereolithography (SL) processes. To increase the particles' colloidal stability, the Au NP nanofillers were coated with polyvinyl pyrrolidone (PVP). The resulting resin was used to fabricate a new type of composite scaffold via mask projection excimer laser stereolithography. The thermal properties of the nanocomposite scaffolds were found to be sensitive to the concentration of NPs. The mechanical properties were augmented by the NPs up to 0.16 μM, though further increase in the concentration led to a gradual decrease. Au NP incorporation rendered the biopolymer scaffolds photosensitive, i.e. the presence of Au NPs enhanced the optical absorption of the scaffolds as well, leading to possible localized temperature rise when irradiated with 532 nm laser, known as the photothermal effect. - Highlights: • Gold nanoparticle incorporation into biopolymer resin was realized. • Gold incorporation into biopolymer resin is a big step in tissue engineering. • Composite scaffolds were synthesized and thoroughly characterized. • Gold nanoparticles are remarkable candidates to be utilized as “transport vehicles”. • The photothermal effect was demonstrated using a 532-nm laser.

  17. Microsphere-based scaffolds encapsulating tricalcium phosphate and hydroxyapatite for bone regeneration.

    Science.gov (United States)

    Gupta, Vineet; Lyne, Dina V; Barragan, Marilyn; Berkland, Cory J; Detamore, Michael S

    2016-07-01

    Bioceramic mixtures of tricalcium phosphate (TCP) and hydroxyapatite (HAp) are widely used for bone regeneration because of their excellent cytocompatibility, osteoconduction, and osteoinduction. Therefore, we hypothesized that incorporation of a mixture of TCP and HAp in microsphere-based scaffolds would enhance osteogenesis of rat bone marrow stromal cells (rBMSCs) compared to a positive control of scaffolds with encapsulated bone-morphogenic protein-2 (BMP-2). Poly(D,L-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds encapsulating TCP and HAp mixtures in two different ratios (7:3 and 1:1) were fabricated with the same net ceramic content (30 wt%) to evaluate how incorporation of these ceramic mixtures would affect the osteogenesis in rBMSCs. Encapsulation of TCP/HAp mixtures impacted microsphere morphologies and the compressive moduli of the scaffolds. Additionally, TCP/HAp mixtures enhanced the end-point secretion of extracellular matrix components relevant to bone tissue compared to the "blank" (PLGA-only) microsphere-based scaffolds as evidenced by the biochemical, gene expression, histology, and immunohistochemical characterization. Moreover, the TCP/HAp mixture groups even surpassed the BMP-2 positive control group in some instances in terms of matrix synthesis and gene expression. Lastly, gene expression data suggested that the rBMSCs responded differently to different TCP/HAp ratios presented to them. Altogether, it can be concluded that TCP/HAp mixtures stimulated the differentiation of rBMSCs toward an osteoblastic phenotype, and therefore may be beneficial in gradient microsphere-based scaffolds for osteochondral regeneration. PMID:27272903

  18. Nanocomposite scaffold fabrication by incorporating gold nanoparticles into biodegradable polymer matrix: Synthesis, characterization, and photothermal effect

    International Nuclear Information System (INIS)

    Nanoparticle incorporation into scaffold materials is a valuable route to deliver various therapeutic agents, such as drug molecules or large biomolecules, proteins (e.g. DNA or RNA) into their targets. In particular, gold nanoparticles (Au NPs) with their low inherent toxicity, tunable stability and high surface area provide unique attributes facilitating new delivery strategies. A biodegradable, photocurable polymer resin, polypropylene fumarate (PPF) along with Au NPs were utilized to synthesize a hybrid nanocomposite resin, directly exploitable in stereolithography (SL) processes. To increase the particles' colloidal stability, the Au NP nanofillers were coated with polyvinyl pyrrolidone (PVP). The resulting resin was used to fabricate a new type of composite scaffold via mask projection excimer laser stereolithography. The thermal properties of the nanocomposite scaffolds were found to be sensitive to the concentration of NPs. The mechanical properties were augmented by the NPs up to 0.16 μM, though further increase in the concentration led to a gradual decrease. Au NP incorporation rendered the biopolymer scaffolds photosensitive, i.e. the presence of Au NPs enhanced the optical absorption of the scaffolds as well, leading to possible localized temperature rise when irradiated with 532 nm laser, known as the photothermal effect. - Highlights: • Gold nanoparticle incorporation into biopolymer resin was realized. • Gold incorporation into biopolymer resin is a big step in tissue engineering. • Composite scaffolds were synthesized and thoroughly characterized. • Gold nanoparticles are remarkable candidates to be utilized as “transport vehicles”. • The photothermal effect was demonstrated using a 532-nm laser

  19. Effect of different calcium phosphate scaffold ratios on odontogenic differentiation of human dental pulp cells

    International Nuclear Information System (INIS)

    Calcium phosphate (CaP) scaffolds have been widely and successfully used with osteoblast cells for bone tissue regeneration. However, it is necessary to investigate the effects of these scaffolds on odontoblast cells' proliferation and differentiation for dentin tissue regeneration. In this study, three different hydroxyapatite (HA) to beta tricalcium phosphate (β-TCP) ratios of biphasic calcium phosphate (BCP) scaffolds, BCP20, BCP50, and BCP80, with a mean pore size of 300 μm and 65% porosity were prepared from phosphoric acid (H2PO4) and calcium carbonate (CaCO3) sintered at 1000 °C for 2 h. The extracts of these scaffolds were assessed with regard to cell viability and differentiation of odontoblasts. The high alkalinity, more calcium, and phosphate ions released that were exhibited by BCP20 decreased the viability of human dental pulp cells (HDPCs) as compared to BCP50 and BCP80. However, the cells cultured with BCP20 extract expressed high alkaline phosphatase activity and high expression level of bone sialoprotein (BSP), dental matrix protein-1 (DMP-1), and dentin sialophosphoprotein (DSPP) genes as compared to that cultured with BCP50 and BCP80 extracts. The results highlighted the effect of different scaffold ratios on the cell microenvironment and demonstrated that BCP20 scaffold can support HDPC differentiation for dentin tissue regeneration. - Highlights: • BCPs of different HA/β-TCP ratios influence cell microenvironment. • BCP20 decreases cell viability of HDPCs as compared to BCP50 and BCP80. • HDPCs cultured with BCP20 express highest ALP activity. • HDPCs cultured with BCP20 up-regulate BSP, DMP-1 and DSPP gene expressions. • BCP20 can support HDPC differentiation for dentin tissue regeneration

  20. Hemocompatible surface of electrospun nanofibrous scaffolds by ATRP modification

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Wenjie [School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Feng, Yakai, E-mail: yakaifeng@hotmail.com [School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin 300072 (China); Tianjin University-Helmholtz-Zentrum Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, Weijin Road 92, 300072 Tianjin (China); Wang, Heyun [School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); School of Chemistry and Chemical Engineering, Shihezi University, Shihezi 832002 (China); Yang, Dazhi [School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); An, Bo [Department of Orthopedics, Affiliated Hospital of Logistics University of Chinese People' s Armed Police Force, Tianjin 300162 (China); Zhang, Wencheng [Department of Physiology and Pathophysiology, Logistics University of Chinese People' s Armed Police Force, Tianjin 300162 (China); Khan, Musammir [School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Guo, Jintang [School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Tianjin University-Helmholtz-Zentrum Geesthacht, Joint Laboratory for Biomaterials and Regenerative Medicine, Weijin Road 92, 300072 Tianjin (China)

    2013-10-15

    The electrospun scaffolds are potential application in vascular tissue engineering since they can mimic the nano-sized dimension of natural extracellular matrix (ECM). We prepared a fibrous scaffold from polycarbonateurethane (PCU) by electrospinning technology. In order to improve the hydrophilicity and hemocompatibility of the fibrous scaffold, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto the fiber surface by surface-initiated atom transfer radical polymerization (SI-ATRP) method. Although SI-ATRP has been developed and used for surface modification for many years, there are only few studies about the modification of electrospun fiber by this method. The modified fibrous scaffolds were characterized by SEM, Fourier transform infrared (FTIR), and X-ray photoelectron spectroscopy (XPS). The scaffold morphology showed no significant difference when PEGMA was grafted onto the scaffold surface. Based on the water contact angle measurement, the surface hydrophilicity of the scaffold surface was improved significantly after grafting hydrophilic PEGMA (P = 0.0012). The modified surface showed effective resistance for platelet adhesion compared with the unmodified surface. Activated partial thromboplastin time (APTT) of the PCU-g-PEGMA scaffold was much longer than that of the unmodified PCU scaffold. The cyto-compatibility of electrospun nanofibrous scaffolds was tested by human umbilical vein endothelial cells (HUVECs). The images of 7-day cultured cells on the scaffold surface were observed by SEM. The modified scaffolds showed high tendency to induce cell adhesion. Moreover, the cells reached out pseudopodia along the fibrous direction and formed a continuous monolayer. Hemolysis test showed that the grafted chains of PEGMA reduced blood coagulation. These results indicated that the modified electrospun nanofibrous scaffolds were potential application as artificial blood vessels. Highlights: • Electrospun nanofibrous scaffolds were successfully

  1. Fabrication and characterization of multiscale electrospun scaffolds for cartilage regeneration

    OpenAIRE

    Levorson, Erica J.; Sreerekha, Perumcherry Raman; Chennazhi, Krishna Prasad; Kasper, F. Kurtis; Nair, Shantikumar V.; Mikos, Antonios G.

    2013-01-01

    Recently, scaffolds for tissue regeneration purposes have looked to utilize nanoscale features in an effort to reap the cellular benefits of scaffold features resembling extracellular matrix (ECM) components. However, one complication surrounding electrospun nanofibers is limited cellular infiltration. One method to ameliorate this negative effect is by incorporating nanofibers into microfibrous scaffolds. This study shows that it is feasible to fabricate electrospun scaffolds containing two ...

  2. Hemocompatible surface of electrospun nanofibrous scaffolds by ATRP modification

    International Nuclear Information System (INIS)

    The electrospun scaffolds are potential application in vascular tissue engineering since they can mimic the nano-sized dimension of natural extracellular matrix (ECM). We prepared a fibrous scaffold from polycarbonateurethane (PCU) by electrospinning technology. In order to improve the hydrophilicity and hemocompatibility of the fibrous scaffold, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto the fiber surface by surface-initiated atom transfer radical polymerization (SI-ATRP) method. Although SI-ATRP has been developed and used for surface modification for many years, there are only few studies about the modification of electrospun fiber by this method. The modified fibrous scaffolds were characterized by SEM, Fourier transform infrared (FTIR), and X-ray photoelectron spectroscopy (XPS). The scaffold morphology showed no significant difference when PEGMA was grafted onto the scaffold surface. Based on the water contact angle measurement, the surface hydrophilicity of the scaffold surface was improved significantly after grafting hydrophilic PEGMA (P = 0.0012). The modified surface showed effective resistance for platelet adhesion compared with the unmodified surface. Activated partial thromboplastin time (APTT) of the PCU-g-PEGMA scaffold was much longer than that of the unmodified PCU scaffold. The cyto-compatibility of electrospun nanofibrous scaffolds was tested by human umbilical vein endothelial cells (HUVECs). The images of 7-day cultured cells on the scaffold surface were observed by SEM. The modified scaffolds showed high tendency to induce cell adhesion. Moreover, the cells reached out pseudopodia along the fibrous direction and formed a continuous monolayer. Hemolysis test showed that the grafted chains of PEGMA reduced blood coagulation. These results indicated that the modified electrospun nanofibrous scaffolds were potential application as artificial blood vessels. Highlights: • Electrospun nanofibrous scaffolds were successfully

  3. A novel technique for the production of electrospun scaffolds with tailored three-dimensional micro-patterns employing additive manufacturing

    International Nuclear Information System (INIS)

    Electrospinning is a common technique used to fabricate fibrous scaffolds for tissue engineering applications. There is now growing interest in assessing the ability of collector plate design to influence the patterning of the fibres during the electrospinning process. In this study, we investigate a novel method to generate hybrid electrospun scaffolds consisting of both random fibres and a defined three-dimensional (3D) micro-topography at the surface, using patterned resin formers produced by rapid prototyping (RP). Poly(D,L-lactide-co-glycolide) was electrospun onto the engineered RP surfaces and the ability of these formers to influence microfibre patterning in the resulting scaffolds visualized by scanning electron microscopy. Electrospun scaffolds with patterns mirroring the microstructures of the formers were successfully fabricated. The effect of the resulting fibre patterns and 3D geometries on mammalian cell adhesion and proliferation was investigated by seeding enhanced green fluorescent protein labelled 3T3 fibroblasts onto the scaffolds. Following 24 h and four days of culture, the seeded scaffolds were visually assessed by confocal macro- and microscopy. The patterning of the fibres guided initial cell adhesion to the scaffold with subsequent proliferation over the geometry resulting in the cells being held in a 3D micro-topography. Such patterning could be designed to replicate a specific in vivo structure; we use the dermal papillae as an exemplar here. In conclusion, a novel, versatile and scalable method to produce hybrid electrospun scaffolds has been developed. The 3D directional cues of the patterned fibres have been shown to influence cell behaviour and could be used to culture cells within a similar 3D micro-topography as experienced in vivo. (paper)

  4. SCAFFOLDING IN CONNECTIVIST MOBILE LEARNING ENVIRONMENT

    Directory of Open Access Journals (Sweden)

    Ozlem OZAN

    2013-04-01

    Full Text Available Social networks and mobile technologies are transforming learning ecology. In this changing learning environment, we find a variety of new learner needs. The aim of this study is to investigate how to provide scaffolding to the learners in connectivist mobile learning environment: Ø to learn in a networked environment, Ø to manage their networked learning process, Ø to interact in a networked society, and Ø to use the tools belonging to the network society. The researcher described how Vygotsky's “scaffolding” concept, Berge’s “learner support” strategies, and Siemens’ “connectivism” approach can be used together to satisfy mobile learners’ needs. A connectivist mobile learning environment was designed for the research, and the research was executed as a mixed-method study. Data collection tools were Facebook wall entries, personal messages, chat records; Twitter, Diigo, blog entries; emails, mobile learning management system statistics, perceived learning survey and demographic information survey. Results showed that there were four major aspects of scaffolding in connectivist mobile learning environment as type of it, provider of it, and timing of it and strategies of it. Participants preferred mostly social scaffolding, and then preferred respectively, managerial, instructional and technical scaffolding. Social scaffolding was mostly provided by peers, and managerial scaffolding was mostly provided by instructor. Use of mobile devices increased the learner motivation and interest. Some participants stated that learning was more permanent by using mobile technologies. Social networks and mobile technologies made it easier to manage the learning process and expressed a positive impact on perceived learning.

  5. Electrospun aliphatic polycarbonates as tailored tissue scaffold materials.

    Science.gov (United States)

    Welle, Alexander; Kröger, Mario; Döring, Manfred; Niederer, Kerstin; Pindel, Elvira; Chronakis, Ioannis S

    2007-04-01

    Two different aliphatic polycarbonates were synthesised from CO(2) and the respective epoxides. Poly(propyl carbonate) (PPC) was prepared by heterogeneous catalysis with zinc glutarate. Poly(cyclohexyl carbonate) (PCHC) was prepared via living copolymerisation homogeneously catalysed by a 3-amino-2-cyanoimidoacrylate zinc acetate complex and subjected to electrospinning. The obtained nanofibres had a well-defined morphology free of beads along the fibres and with slightly porous structures on their surface. Subsequently, low-power deep UV irradiations, previously applied for photochemical surface modifications of two-dimensional and three-dimensional scaffolds from biostable polymers, were performed. Here, an effect on surface and bulk properties of PPC nanofibres was observed. Surface modifications of both polymers affected plasma protein adsorption. Photochemical bulk modifications observed for the first time on PPC nanofibres are indicating the possibility of spatial control of biodegradation rates, hence allow for control of the progression of host/implant interactions in vivo. In particular PPC was used for cell culture of L929 fibroblasts and primary rat hepatocytes. Even delicate primary cells showed good adhesion to the scaffolds and high viability.

  6. Scaffolds of polycaprolactone with hydroxyapatite fibers

    International Nuclear Information System (INIS)

    Scaffolds of poly (ε-caprolactone) has been studied in many researches in tissue engineering. The used of hydroxyapatite fibers, allowed increase its resistance mechanical, beside the character bioactive and osteoconductive. Improving, its role in tissue engineering. The aim in this study was developed polycaprolactone matrix with dispersed hydroxyapatite fibers. The characterizations were by scanning electron microscopy (SEM), X- Ray Diffractometer (XRD), X-Ray Fluorescence (XRF) and Energy dispersive X-Ray Detector (EDX). Was able reviewed its composition, morphology and possible contaminations. The results were scaffolds with porosity and distribution of the fibers in all its area. (author)

  7. Porous polymer/hydroxyapatite scaffolds: characterization and biocompatibility investigations.

    NARCIS (Netherlands)

    Douglas, T.E.L.; Pamula, E.; Hauk, D.; Wiltfang, J.; Sivananthan, S.; Sherry, E.; Warnke, P.H.

    2009-01-01

    Poly-lactic-glycolic acid (PLGA) has been widely used as a scaffold material for bone tissue engineering applications. 3D sponge-like porous scaffolds have previously been generated through a solvent casting and salt leaching technique. In this study, polymer-ceramic composite scaffolds were created

  8. Design, fabrication and application of tissue engineering used cells scaffold

    Institute of Scientific and Technical Information of China (English)

    WANG Shenguo; BEI Jianzhong

    2001-01-01

    @@ FUNCTIONS OF CELLS SCAFFOLD IN THE TISSUE ENGINEERINGCell, cells scaffold and the construction of tissue and organ are three main factors for the Tissue Engineering. A main function of cells scaffold in tissue engineering is to provide an environment for cells propagation.

  9. SHOP: scaffold hopping by GRID-based similarity searches

    DEFF Research Database (Denmark)

    Bergmann, Rikke; Linusson, Anna; Zamora, Ismael

    2007-01-01

    A new GRID-based method for scaffold hopping (SHOP) is presented. In a fully automatic manner, scaffolds were identified in a database based on three types of 3D-descriptors. SHOP's ability to recover scaffolds was assessed and validated by searching a database spiked with fragments of known...

  10. Synthetic scaffold morphology controls human dermal connective tissue formation

    NARCIS (Netherlands)

    Wang, H.; Pieper, J.; Peters, F.; Blitterswijk, C.A. van; Lamme, E.N.

    2005-01-01

    Engineering tissues in bioreactors is often hampered by disproportionate tissue formation at the surface of scaffolds. This hinders nutrient flow and retards cell proliferation and tissue formation inside the scaffold. The objective of this study was to optimize scaffold morphology to prevent this f

  11. Mesoporous bioactive glass surface modified poly(lactic-co-glycolic acid electrospun fibrous scaffold for bone regeneration

    Directory of Open Access Journals (Sweden)

    Chen SJ

    2015-06-01

    after a 7-day immersion in simulated body fluid. In vitro studies showed that the incorporation of MBG favored cell proliferation and osteogenic differentiation of human mesenchymal stem cells on the PLGA scaffolds. Moreover, the MBG surface-modified PLGA (PLGA-MBG scaffolds were shown to be capable of providing the improved adsorption/release behaviors of bone morphogenetic protein-2 (BMP-2. It is very significant that PLGA-MBG scaffolds could be effective for BMP-2 delivery and bone regeneration. Keywords: mesoporous, scaffolds, bone regeneration, stem cells, BMP-2

  12. Role of chondroitin sulphate tethered silk scaffold in cartilaginous disc tissue regeneration.

    Science.gov (United States)

    Bhattacharjee, Maumita; Chawla, Shikha; Chameettachal, Shibu; Murab, Sumit; Bhavesh, Neel Sarovar; Ghosh, Sourabh

    2016-04-01

    Strategies for tissue engineering focus on scaffolds with tunable structure and morphology as well as optimum surface chemistry to simulate the anatomy and functionality of the target tissue. Silk fibroin has demonstrated its potential in supporting cartilaginous tissue formation both in vitro and in vivo. In this study, we investigate the role of controlled lamellar organization and chemical composition of biofunctionalized silk scaffolds in replicating the structural properties of the annulus region of an intervertebral disc using articular chondrocytes. Covalent attachment of chondroitin sulfate (CS) to silk is characterized. CS-conjugated silk constructs demonstrate enhanced cellular metabolic activity and chondrogenic redifferentiation potential with significantly improved mechanical properties over silk-only constructs. A matrix-assisted laser desorption ionization-time of flight analysis and protein-protein interaction studies help to generate insights into how CS conjugation can facilitate the production of disc associated matrix proteins, compared to a silk-only based construct. An in-depth understanding of the interplay between such extra cellular matrix associated proteins should help in designing more rational scaffolds for cartilaginous disc regeneration needs. PMID:27068621

  13. S-SCAM/MAGI-2 is an essential synaptic scaffolding molecule for the GluA2-containing maintenance pool of AMPA receptors

    OpenAIRE

    Danielson, Eric; Zhang, Nanyan; Metallo, Jacob; Kaleka, Kanwardeep; Shin, Seung Min; Gerges, Nashaat; Lee, Sang H.

    2012-01-01

    Synaptic plasticity, the cellular basis of learning and memory, involves the dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses. One of the remaining key unanswered aspects of AMPAR trafficking is the mechanism by which synaptic strength is preserved in spite of protein turnover. In particular, the identity of AMPAR scaffolding molecule(s) involved in the maintenance of GluA2-containing AMPARs is completely unknown. Here we report that Synaptic scaffolding molecule (S-SCA...

  14. ACTIBIND, an actin-binding fungal T2-RNase with antiangiogenic and anticarcinogenic characteristics.

    OpenAIRE

    Roiz, Levava; Smirnoff, Patricia; Bar-Eli, Menashe; Schwartz, Betty; Shoseyov, Oded

    2006-01-01

    KEYWORDS CLASSIFICATION: Agriculture;Angiogenesis Inhibitors;Animals;Anticarcinogenic Agents;Aspergillus niger;blood;Biopsy,Needle;Breast Neoplasms;cytology;Colorectal Neoplasms;dietary modulation of cancer & cancer biomarkers;drug effects;drug therapy;Disease Models,Animal;Endoribonucleases;Female;Food;Humans;Immunohistochemistry;Israel;lifestyle modulation of cancer & cancer biomarkers;Male;Mice;Mice,Nude;Neoplasm Transplantation;Neovascularization,Pathologic;pathology;pharmacology;preventi...

  15. Stem Cells Grown in Osteogenic Medium on PLGA, PLGA/HA, and Titanium Scaffolds for Surgical Applications

    Directory of Open Access Journals (Sweden)

    Annalia Asti

    2010-01-01

    Full Text Available Pluripotent adipose tissue-derived stem cells (hASCs can differentiate into various mesodermal cell types such as osteoblasts, chondroblasts, and myoblasts. We isolated hASCs from subcutaneous adipose tissue during orthopaedic surgery and induced the osteogenic differentiation for 28 days on three different synthetic scaffolds such as polylactide-co-glycolide (PLGA, polylactide-co-glycolide/hydroxyapatite (PLGA/HA, and trabecular titanium scaffolds (Ti6Al4V. Pore size can influence certain criteria such as cell attachment, infiltration, and vascularization. The aim of this study was to investigate the performance of PLGA and PLGA/HA scaffolds with a higher porosity, ranging between 75% and 84%, with respect to Ti scaffolds but with smaller pore size, seeded with hASCs to develop a model that could be used in the treatment of bone defects and fractures. Osteogenesis was assessed by ELISA quantitation of extracellular matrix protein expression, von Kossa staining, X-ray microanalysis, and scanning electron microscopy. The higher amount of protein matrix on the Ti scaffold with respect to PLGA and PLGA/HA leads to the conclusion that not only the type of material but the structure significantly affects cell proliferation.

  16. Differential input by Ste5 scaffold and Msg5 phosphatase route a MAPK cascade to multiple outcomes.

    Science.gov (United States)

    Andersson, Jessica; Simpson, David M; Qi, Maosong; Wang, Yunmei; Elion, Elaine A

    2004-07-01

    Pathway specificity is poorly understood for mitogen-activated protein kinase (MAPK) cascades that control different outputs in response to different stimuli. In yeast, it is not known how the same MAPK cascade activates Kss1 MAPK to promote invasive growth (IG) and proliferation, and both Fus3 and Kss1 MAPKs to promote mating. Previous work has suggested that the Kss1 MAPK cascade is activated independently of the mating G protein (Ste4)-scaffold (Ste5) system during IG. Here we demonstrate that Ste4 and Ste5 activate Kss1 during IG and in response to multiple stimuli including butanol. Ste5 activates Kss1 by generating a pool of active MAPKKK (Ste11), whereas additional scaffolding is needed to activate Fus3. Scaffold-independent activation of Kss1 can occur at multiple steps in the pathway, whereas Fus3 is strictly dependent on the scaffold. Pathway specificity is linked to Kss1 immunity to a MAPK phosphatase that constitutively inhibits basal activation of Fus3 and blocks activation of the mating pathway. These findings reveal the versatility of scaffolds and how a single MAPK cascade mediates different outputs. PMID:15192700

  17. A poly(glycerol sebacate)-coated mesoporous bioactive glass scaffold with adjustable mechanical strength, degradation rate, controlled-release and cell behavior for bone tissue engineering.

    Science.gov (United States)

    Lin, Dan; Yang, Kai; Tang, Wei; Liu, Yutong; Yuan, Yuan; Liu, Changsheng

    2015-07-01

    Various requirements in the field of tissue engineering have motivated the development of three-dimensional scaffold with adjustable physicochemical properties and biological functions. A series of multiparameter-adjustable mesoporous bioactive glass (MBG) scaffolds with uncrosslinked poly(glycerol sebacate) (PGS) coating was prepared in this article. MBG scaffold was prepared by a modified F127/PU co-templating process and then PGS was coated by a simple adsorption and lyophilization process. Through controlling macropore parameters and PGS coating amount, the mechanical strength, degradation rate, controlled-release and cell behavior of the composite scaffold could be modulated in a wide range. PGS coating successfully endowed MBG scaffold with improved toughness and adjustable mechanical strength covering the bearing range of trabecular bone (2-12MPa). Multilevel degradation rate of the scaffold and controlled-release rate of protein from mesopore could be achieved, with little impact on the protein activity owing to an "ultralow-solvent" coating and "nano-cavity entrapment" immobilization method. In vitro studies indicated that PGS coating promoted cell attachment and proliferation in a dose-dependent manner, without affecting the osteogenic induction capacity of MBG substrate. These results first provide strong evidence that uncrosslinked PGS might also yield extraordinary achievements in traditional MBG scaffold. With the multiparameter adjustability, the composite MBG/PGS scaffolds would have a hopeful prospect in bone tissue engineering. The design considerations and coating method of this study can also be extended to other ceramic-based artificial scaffolds and are expected to provide new thoughts on development of future tissue engineering materials.

  18. Fluorescent composite scaffolds made of nanodiamonds/polycaprolactone

    Science.gov (United States)

    Cao, Li; Hou, Yanwen; Lafdi, Khalid; Urmey, Kirk

    2015-11-01

    Polycaprolactone (PCL) has been widely studied for biological applications. Biodegradable PCL fibrous scaffold can work as an appropriate substrate for tissue regeneration. In this letter, fluorescent nanodiamonds (FNDs) were prepared after surface passivation with octadecylamine. The FNDs were then mixed with PCL polymer and subsequently electrospun into FNDs/PCL fibrous scaffolds. The obtained scaffolds not only exhibited photoluminescence, but also showed reinforced mechanical strength. Toxicity study indicated FNDs/PCL scaffolds were nontoxic. This biocompatible fluorescent composite fibrous scaffold can support in vitro cell growth and also has the potential to act as an optical probe for tissue engineering application in vitro and in vivo.

  19. Comparison of TALEN scaffolds in Xenopus tropicalis

    Directory of Open Access Journals (Sweden)

    Keisuke Nakajima

    2013-11-01

    Transcription activator-like effector nucleases (TALENs are facile and potent tools used to modify a gene of interest for targeted gene knockout. TALENs consist of an N-terminal domain, a DNA-binding domain, and a C-terminal domain, which are derived from a transcription activator-like effector, and the non-specific nuclease domain of FokI. Using Xenopus tropicalis (X. tropicalis, we compared the toxicities and somatic mutation activities of four TALEN architectures in a side-by-side manner: a basic TALEN, a scaffold with the same truncated N- and C-terminal domains as GoldyTALEN, a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain, and a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric Sharkey nuclease domain. The strongest phenotype and targeted somatic gene mutation were induced by the injection of TALEN mRNAs containing the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain. The obligate heterodimeric TALENs exhibited reduced toxicity compared to the homodimeric TALENs, and the homodimeric GoldyTALEN-type scaffold showed both a high activity of somatic gene modification and high toxicity. The Sharkey mutation in the heterodimeric nuclease domain reduced the TALEN-mediated somatic mutagenesis.

  20. Scaffolding in Group-Oriented Japanese Preschools

    Science.gov (United States)

    Izumi-Taylor, Satomi

    2013-01-01

    Akira, a 4-year-old boy, wanders into a classroom of 3-year-olds, observing the younger children. He goes over to Masaharu who is having a problem making a piece fit on a puzzle and offers him help. Masaharu succeeds and his mentor applauds his efforts. This interaction shows an older child supporting a younger child through scaffolding. Using…

  1. A conceptualisation of whole-class scaffolding

    NARCIS (Netherlands)

    Smit, J.; van Eerde, H.A.A.; Bakker, A.

    2013-01-01

    The concept of scaffolding refers to temporary and adaptive support, originally in dyadic adult– child interaction. It has become widely used, also in whole-class settings, but often in loose ways. The aim of this paper is to theoretically and empirically ground a conceptualisation of whole-class sc

  2. Projection printing of 3-dimensional tissue scaffolds.

    Science.gov (United States)

    Lu, Yi; Chen, Shaochen

    2012-01-01

    Our ability to create precise, predesigned, spatially patterned biochemical and physical microenvironments inside polymer scaffolds could provide a powerful tool in studying progenitor cell behavior and differentiation under biomimetic, three-dimensional (3D) culture conditions. The development of freeform fabrication technology has become a promising tool for the manufacturing of biological scaffolds for tissue regeneration and stem cell engineering. Freeform fabrication is a very promising technology due to the efficient and simple process for creating bona fide 3D microstructures, such as closed channels and cavities. It is also capable of encapsulating biomolecules and even living cells. This chapter describes direct projection printing of 3D tissue engineering scaffolds by using a digital micromirror-array device (DMD) in a layer-by-layer process. This simple and fast microstereolithography system consists of an ultraviolet (UV) light source, a digital micromirror masking device, imaging optics, and controlling devices. Images of UV light are projected onto the photocurable resin by creating the "dynamic photomask" design with graphic software. Multilayered scaffolds are microfabricated through a photopolymerization process. PMID:22692617

  3. Scaffolding in Connectivist Mobile Learning Environment

    Science.gov (United States)

    Ozan, Ozlem

    2013-01-01

    Social networks and mobile technologies are transforming learning ecology. In this changing learning environment, we find a variety of new learner needs. The aim of this study is to investigate how to provide scaffolding to the learners in connectivist mobile learning environment: (1) to learn in a networked environment; (2) to manage their…

  4. Engineered Biopolymeric Scaffolds for Chronic Wound Healing.

    Science.gov (United States)

    Dickinson, Laura E; Gerecht, Sharon

    2016-01-01

    Skin regeneration requires the coordinated integration of concomitant biological and molecular events in the extracellular wound environment during overlapping phases of inflammation, proliferation, and matrix remodeling. This process is highly efficient during normal wound healing. However, chronic wounds fail to progress through the ordered and reparative wound healing process and are unable to heal, requiring long-term treatment at high costs. There are many advanced skin substitutes, which mostly comprise bioactive dressings containing mammalian derived matrix components, and/or human cells, in clinical use. However, it is presently hypothesized that no treatment significantly outperforms the others. To address this unmet challenge, recent research has focused on developing innovative acellular biopolymeric scaffolds as more efficacious wound healing therapies. These biomaterial-based skin substitutes are precisely engineered and fine-tuned to recapitulate aspects of the wound healing milieu and target specific events in the wound healing cascade to facilitate complete skin repair with restored function and tissue integrity. This mini-review will provide a brief overview of chronic wound healing and current skin substitute treatment strategies while focusing on recent engineering approaches that regenerate skin using synthetic, biopolymeric scaffolds. We discuss key polymeric scaffold design criteria, including degradation, biocompatibility, and microstructure, and how they translate to inductive microenvironments that stimulate cell infiltration and vascularization to enhance chronic wound healing. As healthcare moves toward precision medicine-based strategies, the potential and therapeutic implications of synthetic, biopolymeric scaffolds as tunable treatment modalities for chronic wounds will be considered. PMID:27547189

  5. Acellular organ scaffolds for tumor tissue engineering

    Science.gov (United States)

    Guller, Anna; Trusova, Inna; Petersen, Elena; Shekhter, Anatoly; Kurkov, Alexander; Qian, Yi; Zvyagin, Andrei

    2015-12-01

    Rationale: Tissue engineering (TE) is an emerging alternative approach to create models of human malignant tumors for experimental oncology, personalized medicine and drug discovery studies. Being the bottom-up strategy, TE provides an opportunity to control and explore the role of every component of the model system, including cellular populations, supportive scaffolds and signalling molecules. Objectives: As an initial step to create a new ex vivo TE model of cancer, we optimized protocols to obtain organ-specific acellular matrices and evaluated their potential as TE scaffolds for culture of normal and tumor cells. Methods and results: Effective decellularization of animals' kidneys, ureter, lungs, heart, and liver has been achieved by detergent-based processing. The obtained scaffolds demonstrated biocompatibility and growthsupporting potential in combination with normal (Vero, MDCK) and tumor cell lines (C26, B16). Acellular scaffolds and TE constructs have been characterized and compared with morphological methods. Conclusions: The proposed methodology allows creation of sustainable 3D tumor TE constructs to explore the role of organ-specific cell-matrix interaction in tumorigenesis.

  6. Designing Appropriate Scaffolding for Student Science Projects

    Science.gov (United States)

    Johnson, Marie; Smith, Mark

    2008-01-01

    The authors have developed a successful approach to teaching and inspiring undergraduate science and nonscience majors to complete creditable, semester-long, hands-on science research projects. This approach utilizes a carefully developed scaffolding consisting of in-class exercises and discussions, preparatory homework and lab events, and three…

  7. Scaffolding English Language Learners' Reading Performance

    Science.gov (United States)

    McKenzie, Lolita D.

    2011-01-01

    English language learners (ELLs) spend a majority of their instructional time in mainstream classrooms with mainstream teachers. Reading is an area with which many ELLs are challenged when placed within mainstream classrooms. Scaffolding has been identified as one of the best teaching practices for helping students read. ELL students in a local…

  8. Biodegradable elastomeric scaffolds for soft tissue engineering

    NARCIS (Netherlands)

    Pego, Ana Paula; Poot, André A.; Grijpma, Dirk W.; Feijen, Jan

    2003-01-01

    Elastomeric copolymers of 1,3-trimethylene carbonate (TMC) and ε-caprolactone (CL) and copolymers of TMC and D,L-lactide (DLLA) have been evaluated as candidate materials for the preparation of biodegradable scaffolds for soft tissue engineering. TMC-DLLA copolymers are amorphous and degrade more r

  9. Composite tissue engineering on polycaprolactone nanofiber scaffolds.

    Science.gov (United States)

    Reed, Courtney R; Han, Li; Andrady, Anthony; Caballero, Montserrat; Jack, Megan C; Collins, James B; Saba, Salim C; Loboa, Elizabeth G; Cairns, Bruce A; van Aalst, John A

    2009-05-01

    Tissue engineering has largely focused on single tissue-type reconstruction (such as bone); however, the basic unit of healing in any clinically relevant scenario is a compound tissue type (such as bone, periosteum, and skin). Nanofibers are submicron fibrils that mimic the extracellular matrix, promoting cellular adhesion, proliferation, and migration. Stem cell manipulation on nanofiber scaffolds holds significant promise for future tissue engineering. This work represents our initial efforts to create the building blocks for composite tissue reflecting the basic unit of healing. Polycaprolactone (PCL) nanofibers were electrospun using standard techniques. Human foreskin fibroblasts, murine keratinocytes, and periosteal cells (4-mm punch biopsy) harvested from children undergoing palate repair were grown in appropriate media on PCL nanofibers. Human fat-derived mesenchymal stem cells were osteoinduced on PCL nanofibers. Cell growth was assessed with fluorescent viability staining; cocultured cells were differentiated using antibodies to fibroblast- and keratinocyte-specific surface markers. Osteoinduction was assessed with Alizarin red S. PCL nanofiber scaffolds supported robust growth of fibroblasts, keratinocytes, and periosteal cells. Cocultured periosteal cells (with fibroblasts) and keratinocytes showed improved longevity of the keratinocytes, though growth of these cell types was randomly distributed throughout the scaffold. Robust osteoinduction was noted on PCL nanofibers. Composite tissue engineering using PCL nanofiber scaffolds is possible, though the major obstacles to the trilaminar construct are maintaining an appropriate interface between the tissue types and neovascularization of the composite structure. PMID:19387150

  10. Development of a scaffold displaying exoloops of RXFP1.

    Science.gov (United States)

    Diepenhorst, Natalie A; Gooley, Paul R; Stone, Martin J; Bathgate, Ross A D

    2013-01-01

    Relaxin family peptide receptor 1 (RXFP1), the cognate receptor for relaxin, is a G-protein coupled receptor (GPCR) possessing a unique extracellular region consisting of a domain of 10 leucine rich repeats (LRRs) linked to an N-terminal low density lipoprotein Class A module. Relaxin binds to its receptor primarily by a high affinity interaction with the LRRs. An additional low-affinity interaction has been proposed to occur between relaxin and the the exoloops (ELs) of the transmembrane domain, however the molecular detail of this interaction remains undefined. While site directed mutagenesis and subsequent functional characterisation of these mutants traditionally allows identification of residues contributing to receptor function, in this case results are complicated by the presence of the high affinity binding site in the LRRs. To create a tool to investigate the low-affinity interaction, a protein scaffold system displaying exoloops 1 and 2 from RXFP1 was designed. This was achieved by inserting RXFP1 exoloops 1 and 2 into the native loops of a thermostabilised 6 kDa GB1 protein creating EL1/EL2-GB1. This protein has been expressed and purified in milligram quantities and used in conjunction with biophysical techniques such as NMR to explore relaxin binding to the exoloops of RXFP1. PMID:24640555

  11. Fabrication and Mechanical Characterization of Hydrogel Infused Network Silk Scaffolds

    Science.gov (United States)

    Kundanati, Lakshminath; Singh, Saket K.; Mandal, Biman B.; Murthy, Tejas G.; Gundiah, Namrata; Pugno, Nicola M.

    2016-01-01

    Development and characterization of porous scaffolds for tissue engineering and regenerative medicine is of great importance. In recent times, silk scaffolds were developed and successfully tested in tissue engineering and drug release applications. We developed a novel composite scaffold by mechanical infusion of silk hydrogel matrix into a highly porous network silk scaffold. The mechanical behaviour of these scaffolds was thoroughly examined for their possible use in load bearing applications. Firstly, unconfined compression experiments show that the denser composite scaffolds displayed significant enhancement in the elastic modulus as compared to either of the components. This effect was examined and further explained with the help of foam mechanics principles. Secondly, results from confined compression experiments that resemble loading of cartilage in confinement, showed nonlinear material responses for all scaffolds. Finally, the confined creep experiments were performed to calculate the hydraulic permeability of the scaffolds using soil mechanics principles. Our results show that composite scaffolds with some modifications can be a potential candidate for use of cartilage like applications. We hope such approaches help in developing novel scaffolds for tissue engineering by providing an understanding of the mechanics and can further be used to develop graded scaffolds by targeted infusion in specific regions. PMID:27681725

  12. Astrocytes Grown in Alvetex(®) Three Dimensional Scaffolds Retain a Non-reactive Phenotype.

    Science.gov (United States)

    Ugbode, Christopher I; Hirst, Warren D; Rattray, Marcus

    2016-08-01

    Protocols which permit the extraction of primary astrocytes from either embryonic or postnatal mice are well established however astrocytes in culture are different to those in the mature CNS. Three dimensional (3D) cultures, using a variety of scaffolds may enable better phenotypic properties to be developed in culture. We present data from embryonic (E15) and postnatal (P4) murine primary cortical astrocytes grown on coated coverslips or a 3D polystyrene scaffold, Alvetex. Growth of both embryonic and postnatal primary astrocytes in the 3D scaffold changed astrocyte morphology to a mature, protoplasmic phenotype. Embryonic-derived astrocytes in 3D expressed markers of mature astrocytes, namely the glutamate transporter GLT-1 with low levels of the chondroitin sulphate proteoglycans, NG2 and SMC3. Embryonic astrocytes derived in 3D show lower levels of markers of reactive astrocytes, namely GFAP and mRNA levels of LCN2, PTX3, Serpina3n and Cx43. Postnatal-derived astrocytes show few protein changes between 2D and 3D conditions. Our data shows that Alvetex is a suitable scaffold for growth of astrocytes, and with appropriate choice of cells allows the maintenance of astrocytes with the properties of mature cells and a non-reactive phenotype. PMID:27099962

  13. Design and testing of biological scaffolds for delivering reparative cells to target sites in the lung.

    Science.gov (United States)

    Ingenito, Edward P; Sen, Elif; Tsai, Larry W; Murthy, Shankar; Hoffman, Andrew

    2010-06-01

    This study summarizes the development and testing of a scaffold to promote engraftment of cells in the distal lung. A fibrinogen-fibronectin-vitronectin hydrogel (FFVH) was developed and optimized with respect to its mechanical and biological properties for this application. In vitro, FFVH scaffolds promoted attachment, histiotypic growth and expression of basement membrane proteins by primary ovine lung mesenchymal cells derived from lung biopsies. In vivo testing was then performed to assess the ability of FFVHs to promote cell engraftment in the sheep lung. Treatment with autologous cells delivered using FFVH was clinically well tolerated. Cells labelled with a fluorescent dye (PKH-26) were detected at treatment sites after 1 month. Tissue mass (assessed by CT imaging) and lung perfusion (assessed by nuclear scintigraphy) were increased at emphysema test sites. Post-treatment histology demonstrated cell proliferation and increased elastin expression without scarring or collapse. No treatment-related pathology was observed at healthy control sites. FFVH scaffolds promote cell attachment, spreading and extracellular matrix expression in vitro and apparent engraftment in vivo, with evidence of trophic effects on the surrounding tissue. Scaffolds of this type may contribute to the development of cell-based therapies for patients with end-stage pulmonary diseases. PMID:20020503

  14. Polyelectrolyte-complex nanostructured fibrous scaffolds for tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Verma, Devendra [Department of Civil Engineering, North Dakota State University, Fargo, ND 58105 (United States); Katti, Kalpana S., E-mail: Kalpana.Katti@ndsu.edu [Department of Civil Engineering, North Dakota State University, Fargo, ND 58105 (United States); Katti, Dinesh R. [Department of Civil Engineering, North Dakota State University, Fargo, ND 58105 (United States)

    2009-08-31

    In the current work, polyelectrolyte complex (PEC) fibrous scaffolds for tissue engineering have been synthesized and a mechanism of their formation has been investigated. The scaffolds are synthesized using polygalacturonic acid and chitosan using the freeze drying methodology. Highly interconnected pores of sizes in the range of 5-20 {mu}m are observed in the scaffolds. The thickness of the fibers was found to be in the range of 1-2 {mu}m. Individual fibers have a nanogranular structure as observed using AFM imaging. In these scaffolds, PEC nanoparticles assemble together at the interface of ice crystals during freeze drying process. Further investigation shows that the freezing temperature and concentration have a remarkable effect on structure of scaffolds. Biocompatibility studies show that scaffold containing chitosan, polygalacturonic acid and hydroxyapatite promotes cell adhesion and proliferation. On the other hand, cells on scaffolds fabricated without hydroxyapatite nanoparticles showed poor adhesion.

  15. The host response to allogeneic and xenogeneic biological scaffold materials.

    Science.gov (United States)

    Keane, Timothy J; Badylak, Stephen F

    2015-05-01

    The clinical use of biological scaffold materials has become commonplace. Such scaffolds are composed of extracellular matrix (ECM), or components of ECM, derived from allogeneic or xenogeneic tissues. Such scaffold materials vary widely in their source tissue, processing methods and sterilization methods. The success or failure of an ECM scaffold for a given application is dependent on the host response following implantation; a response that is largely mediated by the innate immune system and which is influenced by a numerous factors, including the processing methods used in the preparation of biological scaffolds. The present paper reviews various aspects of the host response to biological scaffolds and factors that affect this response. In addition, some of the logistical, regulatory and reconstructive implications associated with the use of biological scaffolds are discussed. PMID:24668694

  16. Nature's lessons in design: nanomachines to scaffold, remodel and shape membrane compartments.

    OpenAIRE

    Beales, PA; Ciani, B; Cleasby, AJ

    2015-01-01

    Compartmentalisation of cellular processes is fundamental to regulation of metabolism in Eukaryotic organisms and is primarily provided by membrane-bound organelles. These organelles are dynamic structures whose membrane barriers are continually shaped, remodelled and scaffolded by a rich variety of highly sophisticated protein complexes. Towards the goal of bottom-up assembly of compartmentalised protocells in synthetic biology, we believe it will be important to harness and reconstitute the...

  17. Biomechanical properties of Achilles tendon repair augmented with a bioadhesive-coated scaffold

    OpenAIRE

    Brodie, Michael; Vollenweider, Laura; John L. Murphy; Xu, Fangmin; Lyman, Arinne; Lew, William D; Lee, Bruce P.

    2011-01-01

    The Achilles tendon is the most frequently ruptured tendon. Both acute and chronic (neglected) tendon ruptures can dramatically affect a patient’s quality of life, and require a prolonged period of recovery before return to pre-injury activity levels. This paper describes the use of an adhesive-coated biologic scaffold to augment primary suture repair of transected Achilles tendons. The adhesive portion consisted of a synthetic mimic of mussel adhesive proteins that can adhere to various surf...

  18. The cytoskeletal protein α-catenin unfurls upon binding to vinculin.

    Science.gov (United States)

    Rangarajan, Erumbi S; Izard, Tina

    2012-05-25

    Adherens junctions (AJs) are essential for cell-cell contacts, morphogenesis, and the development of all higher eukaryotes. AJs are formed by calcium-dependent homotypic interactions of the ectodomains of single membrane-pass cadherin family receptors. These homotypic interactions in turn promote binding of the intracellular cytoplasmic tail domains of cadherin receptors with β-catenin, a multifunctional protein that plays roles in both transcription and AJs. The cadherin receptor-β-catenin complex binds to the cytoskeletal protein α-catenin, which is essential for both the formation and the stabilization of these junctions. Precisely how α-catenin contributes to the formation and stabilization of AJs is hotly debated, although the latter is thought to involve its interactions with the cytoskeletal protein vinculin. Here we report the crystal structure of the vinculin binding domain (VBD) of α-catenin in complex with the vinculin head domain (Vh1). This structure reveals that α-catenin is in a unique unfurled mode allowing dimer formation when bound to vinculin. Finally, binding studies suggest that vinculin must be in an activated state to bind to α-catenin and that this interaction is stabilized by the formation of a ternary α-catenin-vinculin-F-actin complex, which can be formed via the F-actin binding domain of either protein. We propose a feed-forward model whereby α-catenin-vinculin interactions promote their binding to the actin cytoskeleton to stabilize AJs. PMID:22493458

  19. Capping protein beta is required for actin cytoskeleton organisation and cell migration during Drosophila oogenesis.

    Science.gov (United States)

    Ogienko, Anna A; Karagodin, Dmitry A; Lashina, Valentina V; Baiborodin, Sergey I; Omelina, Eugeniya S; Baricheva, Elina M

    2013-02-01

    Capping protein (CP) is a well-characterised actin-binding protein important for regulation of actin filament (AF) assembly. CP caps the barbed end of AFs, inhibiting the addition and loss of actin monomers. In Drosophila melanogaster, the gene encoding CP β-subunit is named capping protein beta (cpb; see Hopmann et al. [1996] J Cell Biol 133: 1293-305). The cpb level is reduced in the Drosophila bristle actin cytoskeleton and becomes disorganised with abnormal morphology. A reduced level of the CP protein in ovary results in disruption of oocyte determination, and disturbance of nurse cell (NC) cortical integrity and dumping. We describe novel defects appearing in cpb mutants during oogenesis, in which cpb plays an important role in border and centripetal follicle cell migration, ring canal development and cytoplasmic AF formation. The number of long cytoplasmic AFs was dramatically reduced in cpb hypomorphs and abnormal actin aggregates was seen on the inner side of NC membranes. A hypothesis to explain the formation of abnormal short-cut cytoplasmic AFs and actin aggregates in the cpb mutant NCs was proffered, along with a discussion of the reasons for 'dumpless' phenotype formation in the mutants.

  20. SCAFFOLDING DALAM MICROTEACHING KIMIA BERBASIS PEMBELAJARAN LANGSUNG DAN SIKLUS BELAJAR

    Directory of Open Access Journals (Sweden)

    Abdullatif Nusu

    2014-09-01

    Full Text Available Abstract: Scaffolding in Chemistry Microteaching Utilizing  Direct Instruction and Learning Cycle. This study concerns developing students’ competence in conducting microteaching in chemistry, especially in preparing lesson plans using direct instruction and learning cycle and in implementing the lesson plans in peer teaching. The microteaching skills of 26 students are enhanced using scaffolding, implemented gradually and integratedly. The scaffolding comprises three stages: orientation of the task, revising the lesson plan, and carrying out peer teaching. Scaffolding is found to enable the students to develop lesson plans and to realize the lesson plans in peer teaching, as can be seen from their scores on the two aspects. In addi­tion, the students respond positively to the use of scaffolding in microteaching. Keywords: scaffolding, lesson plan writing, peer teaching, chemistry microteaching Abstrak: Scaffolding dalam Microteaching Kimia Berbasis Pembelajaran Langsung dan Siklus Be­lajar. Penelitian tentang kemampuan mahasiswa dalam melaksanakan microteaching kimia, khususnya dalam menulis rencana pelaksanaan pembelajaran berbasis pembelajaran langsung dan siklus belajar serta menerapkannya dalam peer teaching, telah dilakukan terhadap 26 mahasiswa Program Studi Pendidikan Kimia Universitas Haluoleo di Kendari, Sulawesi Tenggara. Kemampuan melaksanakan microteaching mahasiswa ditingkatkan dengan menggunakan scaffolding yang dilakukan secara bertahap dan terpadu. Scaffolding tersebut terdiri dari tiga tahap yaitu orientasi tugas dan memodelkan cara menggunakan sum­ber scaffolding, revisi Rencana Pelaksanaan Pembelajaran (RPP melalui artikulasi dan refleksi untuk menghasilkan RPP kelompok, dan melaksanakan peer teaching. Keberhasilan scaffolding dalam micro­teaching kimia ditunjukkan dengan tercapainya skor penulisan RPP dan skor pelaksanaan peer teaching yang memenuhi kriteria ketuntasan minimal. Hasil penelitian menunjukkan bahwa

  1. Computational design of a PAK1 binding protein

    OpenAIRE

    Jha, Ramesh K; Leaver-Fay, Andrew; Yin, Shuangye; Wu, YiBing; Butterfoss, Glenn L.; Szyperski, Thomas; Dokholyan, Nikolay V.; Kuhlman, Brian

    2010-01-01

    We describe a computational protocol, called DDMI, for redesigning scaffold proteins to bind to a specified region on a target protein. The DDMI protocol is implemented within the Rosetta molecular modeling program and uses rigid-body docking, sequence design, and gradient-based minimization of backbone and side chain torsion angles to design low energy interfaces between the scaffold and target protein. Iterative rounds of sequence design and conformational optimization were needed to produc...

  2. Identification of the nuclear matrix and chromosome scaffold in dinoflagellate Crypthecodinium cohnii

    Institute of Scientific and Technical Information of China (English)

    CAISHUTAO; CONGMEIZENG; 等

    1992-01-01

    Dinolflagellate is one of the primitive eukaryotes,whose nucleus may represent one of the transition stages from prokaryotic nucleoid to typical eukaryotic nucleus,Using selective extraction together with embeddment-free section and whole mount electron microscopy,a delicate nuclear matrix filament network was shown,for the first time,in dinoflagellate Crypthecodinium cohnii nucleus,Chromosome residues are connected with nuclear matrix filaments to form a complete network spreading over the nucleus,Moreover,we demonstrated that the dinoflagellate chromosome retains a protein scafflod after the depletion of DNA and soluble proteins.This scaffold preserves the characterstic morphology of the chromosome.Two dimensional electrophoreses indicated that the nuclear matrix and chromosome scaffold are mainly composed of acidic proteins.Our results demonstrated that a framework similar th the nuclear matrix and chromosome scaffold in mammalian cells appears in this primitive eukaryote,suggesting that these structures may have been originated from the early stages of eukaryote evolution.

  3. Immobilized Lentivirus Vector on Chondroitin Sulfate-Hyaluronate Acid-Silk Fibroin Hybrid Scaffold for Tissue-Engineered Ligament-Bone Junction

    Directory of Open Access Journals (Sweden)

    Liguo Sun

    2014-01-01

    Full Text Available The lack of a fibrocartilage layer between graft and bone remains the leading cause of graft failure after anterior cruciate ligament (ACL reconstruction. The objective of this study was to develop a gene-modified silk cable-reinforced chondroitin sulfate-hyaluronate acid-silk fibroin (CHS hybrid scaffold for reconstructing the fibrocartilage layer. The scaffold was fabricated by lyophilizing the CHS mixture with braided silk cables. The scanning electronic microscopy (SEM showed that microporous CHS sponges were formed around silk cables. Each end of scaffold was modified with lentiviral-mediated transforming growth factor-β3 (TGF-β3 gene. The cells on scaffold were transfected by bonded lentivirus. In vitro culture demonstrated that mesenchymal stem cells (MSCs on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of cartilage-specific genes also increased with culture time. After 2 weeks, the MSCs were distributed uniformly throughout scaffold. Deposited collagen was also found to increase. The chondral differentiation of MSCs was verified by expressions of collagen II and TGF-β3 genes in mRNA and protein level. Histology also confirmed the production of cartilage extracellular matrix (ECM components. The results demonstrated that gene-modified silk cable-reinforced CHS scaffold was capable of supporting cell proliferation and differentiation to reconstruct the cartilage layer of interface.

  4. Poly(lactic-co-glycolic Acid/Nanohydroxyapatite Scaffold Containing Chitosan Microspheres with Adrenomedullin Delivery for Modulation Activity of Osteoblasts and Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Lin Wang

    2013-01-01

    Full Text Available Adrenomedullin (ADM is a bioactive regulatory peptide that affects migration and proliferation of diverse cell types, including endothelial cells, smooth muscle cells, and osteoblast-like cells. This study investigated the effects of sustained release of ADM on the modulation activity of osteoblasts and vascular endothelial cells in vitro. Chitosan microspheres (CMs were developed for ADM delivery. Poly(lactic-co-glycolic acid and nano-hydroxyapatite were used to prepare scaffolds containing microspheres with ADM. The CMs showed rough surface morphology and high porosity, and they were well-distributed. The scaffolds exhibited relatively uniform pore sizes with interconnected pores. The addition of CMs improved the mechanical properties of the scaffolds without affecting their high porosity. In vitro degradation tests indicated that the addition of CMs increased the water absorption of the scaffolds and inhibited pH decline of phosphate-buffered saline medium. The expression levels of osteogenic-related and angiogenic-related genes were determined in MG63 cells and in human umbilical vein endothelial cells cultured on the scaffolds, respectively. The expression levels of osteogenic-related and angiogenic-related proteins were also detected by western blot analysis. Their expression levels in cells were improved on the ADM delivery scaffolds at a certain time point. The in vitro evaluation suggests that the microsphere-scaffold system is suitable as a model for bone tissue engineering.

  5. Effect of hydroxyapatite-containing microspheres embedded into three-dimensional magnesium phosphate scaffolds on the controlled release of lysozyme and in vitro biodegradation

    Directory of Open Access Journals (Sweden)

    Lee JM

    2014-09-01

    Full Text Available Jongman Lee, Hui-suk YunPowder and Ceramics Division, Korea Institute of Materials Science, Changwon, Republic of KoreaAbstract: The functionality of porous three-dimensional (3D magnesium phosphate (MgP scaffold was investigated for the development of a novel protein delivery system and biomimetic bone tissue engineering scaffold. This enhancement can be achieved by incorporation of hydroxyapatite (HA-containing polymeric microspheres (MSs into a bulk MgP matrix, and a paste-extruding deposition (PED system. In this work, the amount of MS and HA was precisely controlled when manufacturing MS-embedded MgP (MS/MgP composite scaffolds. The main influence was researched in terms of in vitro lysozyme-release, in vitro biodegradation, mechanical properties, and in vitro calcification. The controlled release of lysozyme was indicated, while showing graded release patterns according to HA content. The composite scaffolds degraded gradually with MS content and degradation time. Due to the effect of HA inclusion, the higher HA-containing MS/MgP scaffolds could, not only delay the biodegradation process but also, compensate for the possible loss of mechanical properties. In this regard, it is reasonable to confirm the inverse relationship between biodegradation and corresponding compressive properties. In order to encourage bioactivity and osteoconductivity, the MS/MgP composite scaffolds were subjected to simulated body fluid treatment. Calcium deposition was, in turn, improved with increasing MS and HA content over time. This quantitative result was also proved using morphological and elemental analysis. In summary, a significant transformation of a monolithic MgP scaffold was directed toward a multifunctional bone tissue engineering scaffold equipped with controlled protein delivery, biodegradability, and bioactivity.Keywords: protein delivery, bone tissue engineering

  6. Biomimetic component coating on 3D scaffolds using high bioactivity of mesoporous bioactive ceramics

    Directory of Open Access Journals (Sweden)

    Yun HS

    2011-10-01

    Full Text Available Hui-suk Yun1, Sang-Hyun Kim2, Dongwoo Khang3, Jungil Choi4, Hui-hoon Kim2, Minji Kang31Functional Materials Division, Korea Institute of Materials Science, Gyeongnam, Korea; 2Department of Pharmacology, School of Medicine, Kyungpook National University, Jung-Gu, Daegu, Korea; 3School of Nano and Advanced Materials Science and Engineering and Center for NMBE, Gyeongsang National University, Jinju, Korea; 4Department of Anatomy, Institute of Health Science and School of Medicine, Gyeongsang National University, Jinju, Gyeongnam, KoreaBackground: Mesoporous bioactive glasses (MBGs are very attractive materials for use in bone tissue regeneration because of their extraordinarily high bone-forming bioactivity in vitro. That is, MBGs may induce the rapid formation of hydroxy apatite (HA in simulated body fluid (SBF, which is a major inorganic component of bone extracellular matrix (ECM and comes with both good osteoconductivity and high affinity to adsorb proteins. Meanwhile, the high bioactivity of MBGs may lead to an abrupt initial local pH variation during the initial Ca ion-leaching from MBGs at the initial transplant stage, which may induce unexpected negative effects on using them in in vivo application. In this study we suggest a new way of using MBGs in bone tissue regeneration that can improve the strength and make up for the weakness of MBGs. We applied the outstanding bone-forming bioactivity of MBG to coat the main ECM components HA and collagen on the MBG-polycarplolactone (PCL composite scaffolds for improving their function as bone scaffolds in tissue regeneration. This precoating process can also expect to reduce initial local pH variation of MBGs.Methods and materials: The MBG-PCL scaffolds were immersed in the mixed solution of the collagen and SBF at 37°C for 24 hours. The coating of ECM components on the MBG-PCL scaffolds and the effect of ECM coating on in vitro cell behaviors were confirmed.Results: The ECM components were fully

  7. The Unique-5 and -6 Motifs of ZO-1 Regulate Tight Junction Strand Localization and Scaffolding Properties

    OpenAIRE

    Fanning, Alan S.; Little, Brent P.; Rahner, Christoph; Utepbergenov, Darkhan; Walther, Zenta; James M Anderson

    2007-01-01

    The proper cellular location and sealing of tight junctions is assumed to depend on scaffolding properties of ZO-1, a member of the MAGUK protein family. ZO-1 contains a conserved SH3-GUK module that is separated by a variable region (unique-5), which in other MAGUKs has proven regulatory functions. To identify motifs in ZO-1 critical for its putative scaffolding functions, we focused on the SH3-GUK module including unique-5 (U5) and unique-6 (U6), a motif immediately C-terminal of the GUK do...

  8. Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: an improved approach in cartilage tissue engineering.

    Science.gov (United States)

    Moroz, Andrei; Bittencourt, Renata Aparecida Camargo; Almeida, Renan Padron; Felisbino, Sérgio Luis; Deffune, Elenice

    2013-01-01

    Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n = 5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1 × 10(5)) were than encapsulated inside 60 µl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI.

  9. Cdk2 silencing via a DNA/PCL electrospun scaffold suppresses proliferation and increases death of breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Clément Achille

    Full Text Available RNA interference (RNAi is a promising approach for cancer treatment. Site specific and controlled delivery of RNAi could be beneficial to the patient, while at the same time reducing undesirable off-target side effects. We utilized electrospinning to generate a biodegradable scaffold capable of incorporating and delivering a bioactive plasmid encoding for short hairpin (sh RNA against the cell cycle specific protein, Cdk2. Three electrospun scaffolds were constructed, one using polycaprolactone (PCL alone (Control and PCL with plasmid DNA encoding for either Cdk2 (Cdk2i and EGFP (EGFPi, also served as a control shRNA. Scaffold fiber diameters ranged from 1 to 20 µm (DNA containing and 0.2-3 µm (Control. While the electrospun fibers remained intact for more than two weeks in physiological buffer, degradation was visible during the third week of incubation. Approximately 20-60 ng/ml (~2.5% cumulative release of intact and bioactive plasmid DNA was released over 21 days. Further, Cdk2 mRNA expression in cells plated on the Cdk2i scaffold was decreased by ~51% and 30%, in comparison with that of cells plated on Control or EGFPi scaffold, respectively. This decrease in Cdk2 mRNA by the Cdk2i scaffold translated to a ~40% decrease in the proliferation of the breast cancer cell line, MCF-7, as well as the presence of increased number of dead cells. Taken together, these results represent the first successful demonstration of the delivery of bioactive RNAi-based plasmid DNA from an electrospun polymer scaffold, specifically, in disrupting cell cycle regulation and suppressing proliferation of cancer cells.

  10. Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

    Science.gov (United States)

    Prabhakaran, Molamma P.; Venugopal, J.; Chan, Casey K.; Ramakrishna, S.

    2008-11-01

    The development of biodegradable polymeric scaffolds with surface properties that dominate interactions between the material and biological environment is of great interest in biomedical applications. In this regard, poly-ɛ-caprolactone (PCL) nanofibrous scaffolds were fabricated by an electrospinning process and surface modified by a simple plasma treatment process for enhancing the Schwann cell adhesion, proliferation and interactions with nanofibers necessary for nerve tissue formation. The hydrophilicity of surface modified PCL nanofibrous scaffolds (p-PCL) was evaluated by contact angle and x-ray photoelectron spectroscopy studies. Naturally derived polymers such as collagen are frequently used for the fabrication of biocomposite PCL/collagen scaffolds, though the feasibility of procuring large amounts of natural materials for clinical applications remains a concern, along with their cost and mechanical stability. The proliferation of Schwann cells on p-PCL nanofibrous scaffolds showed a 17% increase in cell proliferation compared to those on PCL/collagen nanofibrous scaffolds after 8 days of cell culture. Schwann cells were found to attach and proliferate on surface modified PCL nanofibrous scaffolds expressing bipolar elongations, retaining their normal morphology. The results of our study showed that plasma treated PCL nanofibrous scaffolds are a cost-effective material compared to PCL/collagen scaffolds, and can potentially serve as an ideal tissue engineered scaffold, especially for peripheral nerve regeneration.

  11. Fabrication and characterization of multiscale electrospun scaffolds for cartilage regeneration

    International Nuclear Information System (INIS)

    Recently, scaffolds for tissue regeneration purposes have been observed to utilize nanoscale features in an effort to reap the cellular benefits of scaffold features resembling extracellular matrix (ECM) components. However, one complication surrounding electrospun nanofibers is limited cellular infiltration. One method to ameliorate this negative effect is by incorporating nanofibers into microfibrous scaffolds. This study shows that it is feasible to fabricate electrospun scaffolds containing two differently scaled fibers interspersed evenly throughout the entire construct as well as scaffolds containing fibers composed of two discrete materials, specifically fibrin and poly(ε-caprolactone). In order to accomplish this, multiscale fibrous scaffolds of different compositions were generated using a dual extrusion electrospinning setup with a rotating mandrel. These scaffolds were then characterized for fiber diameter, porosity and pore size and seeded with human mesenchymal stem cells to assess the influence of scaffold architecture and composition on cellular responses as determined by cellularity, histology and glycosaminoglycan (GAG) content. Analysis revealed that nanofibers within a microfiber mesh function to maintain scaffold cellularity under serum-free conditions as well as aid the deposition of GAGs. This supports the hypothesis that scaffolds with constituents more closely resembling native ECM components may be beneficial for cartilage regeneration. (paper)

  12. Hydroxyapatite reinforced collagen scaffolds with improved architecture and mechanical properties.

    Science.gov (United States)

    Kane, Robert J; Weiss-Bilka, Holly E; Meagher, Matthew J; Liu, Yongxing; Gargac, Joshua A; Niebur, Glen L; Wagner, Diane R; Roeder, Ryan K

    2015-04-01

    Hydroxyapatite (HA) reinforced collagen scaffolds have shown promise for synthetic bone graft substitutes and tissue engineering scaffolds. Freeze-dried HA-collagen scaffolds are readily fabricated and have exhibited osteogenicity in vivo, but are limited by an inherent scaffold architecture that results in a relatively small pore size and weak mechanical properties. In order to overcome these limitations, HA-collagen scaffolds were prepared by compression molding HA reinforcements and paraffin microspheres within a suspension of concentrated collagen fibrils (∼ 180 mg/mL), cross-linking the collagen matrix, and leaching the paraffin porogen. HA-collagen scaffolds exhibited an architecture with high porosity (85-90%), interconnected pores ∼ 300-400 μm in size, and struts ∼ 3-100 μm in thickness containing 0-80 vol% HA whisker or powder reinforcements. HA reinforcement enabled a compressive modulus of up to ∼ 1 MPa, which was an order of magnitude greater than unreinforced collagen scaffolds. The compressive modulus was also at least one order of magnitude greater than comparable freeze-dried HA-collagen scaffolds and two orders of magnitude greater than absorbable collagen sponges used clinically. Moreover, scaffolds reinforced with up to 60 vol% HA exhibited fully recoverable elastic deformation upon loading to 50% compressive strain for at least 100,000 cycles. Thus, the scaffold mechanical properties were well-suited for surgical handling, fixation, and bearing osteogenic loads during bone regeneration. The scaffold architecture, permeability, and composition were shown to be conducive to the infiltration and differentiation of adipose-derive stromal cells in vitro. Acellular scaffolds were demonstrated to induce angiogenesis and osteogenesis after subcutaneous ectopic implantation by recruiting endogenous cell populations, suggesting that the scaffolds were osteoinductive.

  13. Modeling of porous scaffold deformation induced by medium perfusion.

    Science.gov (United States)

    Podichetty, Jagdeep T; Madihally, Sundararajan V

    2014-05-01

    In this study, we tested the possibility of calculating permeability of porous scaffolds utilized in soft tissue engineering using pore size and shape. We validated the results using experimental measured pressure drop and simulations with the inclusion of structural deformation. We prepared Polycaprolactone (PCL) and Chitosan-Gelatin (CG) scaffolds by salt leaching and freeze drying technique, respectively. Micrographs were assessed for pore characteristics and mechanical properties. Porosity for both scaffolds was nearly same but the permeability varied 10-fold. Elastic moduli were 600 and 9 kPa for PCL and CG scaffolds, respectively, while Poisson's ratio was 0.3 for PCL scaffolds and ∼1.0 for CG scaffolds. A flow-through bioreactor accommodating a 10 cm diameter and 0.2 cm thick scaffold was used to determine the pressure-drop at various flow rates. Additionally, computational fluid dynamic (CFD) simulations were performed by coupling fluid flow, described by Brinkman equation, with structural mechanics using a dynamic mesh. The experimentally obtained pressure drop matched the simulation results of PCL scaffolds. Simulations were extended to a broad range of permeabilities (10(-10) m(2) to 10(-14) m(2) ), elastic moduli (10-100,000 kPa) and Poisson's ratio (0.1-0.49). The results showed significant deviation in pressure drop due to scaffold deformation compared to rigid scaffold at permeabilities near healthy tissues. Also, considering the scaffold as a nonrigid structure altered the shear stress profile. In summary, scaffold permeability can be calculated using scaffold pore characteristics and deformation could be predicted using CFD simulation. These relationships could potentially be used in monitoring tissue regeneration noninvasively via pressure drop. PMID:24259467

  14. Ectopic osteogenic tissue formation by MC3T3-E1 cell-laden chitosan/hydroxyapatite composite scaffold.

    Science.gov (United States)

    Koç, Aysel; Elçin, Ayşe Eser; Elçin, Yaşar Murat

    2016-09-01

    This study evaluates the suitability of a macroporous three-dimensional chitosan/hydroxyapatite (CS/HA) composite as a bone tissue engineering scaffold using MC3T3-E1 cells. The CS/HA scaffold was produced by freeze-drying, and characterized by means of SEM and FTIR. In vitro findings demonstrated that CS/HA supported attachment and proliferation of cells, and stimulated extracellular matrix (ECM) production. Tissue biocompatibility and osteogenic capacity of the cell-laden constructs were evaluated in an ectopic Wistar rat model. In vivo results showed that the MC3T3-E1 cell-laden CS/HA was essentially histocompatible, promoted neovascularization and calcified matrix formation, and secreted osteoblast-specific protein. We conclude that the composite scaffold evaluated has potential for applications in bone regeneration. PMID:25968048

  15. Bioprinted Scaffolds for Cartilage Tissue Engineering.

    Science.gov (United States)

    Kang, Hyun-Wook; Yoo, James J; Atala, Anthony

    2015-01-01

    Researchers are focusing on bioprinting technology as a viable option to overcome current difficulties in cartilage tissue engineering. Bioprinting enables a three-dimensional (3-D), free-form, computer-designed structure using biomaterials, biomolecules, and/or cells. The inner and outer shape of a scaffold can be controlled by this technology with great precision. Here, we introduce a hybrid bioprinting technology that is a co-printing process of multiple materials including high-strength synthetic polymer and cell-laden hydrogel. The synthetic polymer provides mechanical support for shape maintenance and load bearing, while the hydrogel provides the biological environment for artificial cartilage regeneration. This chapter introduces the procedures for printing of a 3-D scaffold using our hybrid bioprinting technology and includes the source materials for preparation of 3-D printing. PMID:26445837