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Sample records for actin-binding scaffold protein

  1. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    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.

    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.

    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.

    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

    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

    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

    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. Mechanosensitive kinetic preference of actin-binding protein to actin filament

    Inoue, Yasuhiro; Adachi, Taiji

    2016-04-01

    The kinetic preference of actin-binding proteins to actin filaments is altered by external forces on the filament. Such an altered kinetic preference is largely responsible for remodeling the actin cytoskeletal structure in response to intracellular forces. During remodeling, actin-binding proteins and actin filaments interact under isothermal conditions, because the cells are homeostatic. In such a temperature homeostatic state, we can rigorously and thermodynamically link the chemical potential of actin-binding proteins to stresses on the actin filaments. From this relationship, we can construct a physical model that explains the force-dependent kinetic preference of actin-binding proteins to actin filaments. To confirm the model, we have analyzed the mechanosensitive alternation of the kinetic preference of Arp2/3 and cofilin to actin filaments. We show that this model captures the qualitative responses of these actin-binding proteins to the forces, as observed experimentally. Moreover, our theoretical results demonstrate that, depending on the structural parameters of the binding region, actin-binding proteins can show different kinetic responses even to the same mechanical signal tension, in which the double-helix nature of the actin filament also plays a critical role in a stretch-twist coupling of the filament.

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

    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.

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

    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

    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.

    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.

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

    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

  15. Regulation of blood-testis barrier by actin binding proteins and protein kinases.

    Li, Nan; Tang, Elizabeth I; Cheng, C Yan

    2016-03-01

    The blood-testis barrier (BTB) is an important ultrastructure in the testis, since the onset of meiosis and spermiogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular, at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin-binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases. PMID:26628556

  16. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression.

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the "status" of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

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

    Leite, Sérgio Carvalho; Sampaio, Paula; Sousa, Vera Filipe; Nogueira-Rodrigues, Joana; Pinto-Costa, Rita; Peters, Luanne Laurel; Brites, Pedro; Sousa, Mónica Mendes

    2016-04-19

    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. PMID:27068466

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

    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

  19. Increased expression of secretory actin-binding protein (SABP) on human spermatozoa is associated with poor semen quality

    Čapková, Jana; Elzeinová, Fatima; Novák, Petr

    2007-01-01

    Roč. 22, č. 5 (2007), s. 1396-1404. ISSN 0268-1161 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50520701; CEZ:AV0Z50200510 Keywords : monoclonal antibody * secretory actin-binding protein * human spermatozoa Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.543, year: 2007

  20. The actin-binding ERM protein Moesin directly regulates spindle assembly and function during mitosis.

    Vilmos, Péter; Kristó, Ildikó; Szikora, Szilárd; Jankovics, Ferenc; Lukácsovich, Tamás; Kari, Beáta; Erdélyi, Miklós

    2016-06-01

    Ezrin-Radixin-Moesin proteins are highly conserved, actin-binding cytoskeletal proteins that play an essential role in microvilli formation, T-cell activation, and tumor metastasis by linking actin filaments to the plasma membrane. Recent studies demonstrated that the only Ezrin-Radixin-Moesin protein of Drosophila melanogaster, Moesin, is involved in mitotic spindle function through stabilizing cell shape and microtubules at the cell cortex. We previously observed that Moesin localizes to the mitotic spindle; hence, we tested for the biological significance of this surprising localization and investigated whether it plays a direct role in spindle function. To separate the cortical and spindle functions of Moesin during mitosis we combined cell biological and genetic methods. We used early Drosophila embryos, in which mitosis occurs in the absence of a cell cortex, and found in vivo evidence for the direct requirement of Moesin in mitotic spindle assembly and function. We also found that the accumulation of Moesin precedes the construction of the microtubule spindle, and the fusiform structure formed by Moesin persists even after the microtubules have disassembled. PMID:27006187

  1. Studies on the actin-binding protein HS1 in platelets

    Auger Jocelyn M

    2007-11-01

    Full Text Available Abstract Background The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex plays a vital role in these processes, providing the protrusive force for lamellipodia formation. The Arp2/3 complex is highly regulated by a number of actin-binding proteins including the haematopoietic-specific protein HS1 and its homologue cortactin. The present study investigates the role of HS1 in platelets using HS1-/- mice. Results The present results demonstrate that HS1 is not required for platelet activation, shape change or aggregation. Platelets from HS1-/- mice spread normally on a variety of adhesion proteins and have normal F-actin and Arp2/3 complex distributions. Clot retraction, an actin-dependent process, is also normal in these mice. Platelet aggregation and secretion is indistinguishable between knock out and littermates and there is no increase in bleeding using the tail bleeding assay. Conclusion This study concludes that HS1 does not play a major role in platelet function. It is possible that a role for HS1 is masked by the presence of cortactin.

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

    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.

  3. Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

    Humphries, Christine L.; Balcer, Heath I.; D'Agostino, Jessica L.; Winsor, Barbara; Drubin, David G.; Barnes, Georjana; Andrews, Brenda J.; Goode, Bruce L.

    2002-01-01

    Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 compl...

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

    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

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

    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

    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

    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. The actin-binding proteins eps8 and gelsolin have complementary roles in regulating the growth and stability of mechanosensory hair bundles of mammalian cochlear outer hair cells.

    Jennifer Olt

    Full Text Available Sound transduction depends upon mechanosensitive channels localized on the hair-like bundles that project from the apical surface of cochlear hair cells. Hair bundles show a stair-case structure composed of rows of stereocilia, and each stereocilium contains a core of tightly-packed and uniformly-polarized actin filaments. The growth and maintenance of the stereociliary actin core are dynamically regulated. Recently, it was shown that the actin-binding protein gelsolin is expressed in the stereocilia of outer hair cells (OHCs and in its absence they become long and straggly. Gelsolin is part of a whirlin scaffolding protein complex at the stereocilia tip, which has been shown to interact with other actin regulatory molecules such as Eps8. Here we investigated the physiological effects associated with the absence of gelsolin and its possible overlapping role with Eps8. We found that, in contrast to Eps8, gelsolin does not affect mechanoelectrical transduction during immature stages of development. Moreover, OHCs from gelsolin knockout mice were able to mature into fully functional sensory receptors as judged by the normal resting membrane potential and basolateral membrane currents. Mechanoelectrical transducer current in gelsolin-Eps8 double knockout mice showed a profile similar to that observed in the single mutants for Eps8. We propose that gelsolin has a non-overlapping role with Eps8. While Eps8 is mainly involved in the initial growth of stereocilia in both inner hair cells (IHCs and OHCs, gelsolin is required for the maintenance of mature hair bundles of low-frequency OHCs after the onset of hearing.

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

    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.

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

    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...

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

    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....

  12. Characterization of the myosin light chain kinase from smooth muscle as an actin-binding protein that assembles actin filaments in vitro.

    Hayakawa, K; Okagaki, T; Ye, L H; Samizo, K; Higashi-Fujime, S; Takagi, T; Kohama, K

    1999-05-01

    In addition to its kinase activity, myosin light chain kinase has an actin-binding activity, which results in bundling of actin filaments [Hayakawa et al., Biochem. Biophys. Res. Commun. 199, 786-791, 1994]. There are two actin-binding sites on the kinase: calcium- and calmodulin-sensitive and insensitive sites [Ye et al., J. Biol. Chem. 272, 32182-32189, 1997]. The calcium/calmodulin-sensitive, actin-binding site is located at Asp2-Pro41 and the insensitive site is at Ser138-Met213. The cyanogen bromide fragment, consisting of Asp2-Met213, is furnished with both sites and is the actin-binding core of myosin light chain kinase. Cross-linking between the two sites assembles actin filaments into bundles. Breaking of actin-binding at the calcium/calmodulin-sensitive site by calcium/calmodulin disassembles the bundles. PMID:10231551

  13. Interaptin, an Actin-binding Protein of the α-Actinin Superfamily in Dictyostelium discoideum, Is Developmentally and cAMP-regulated and Associates with Intracellular Membrane Compartments

    Rivero, Francisco; Kuspa, Adam; Brokamp, Regine; Matzner, Monika; Noegel, Angelika A.

    1998-01-01

    In a search for novel members of the α-actinin superfamily, a Dictyostelium discoideum genomic library in yeast artificial chromosomes (YAC) was screened under low stringency conditions using the acting-binding domain of the gelation factor as probe. A new locus was identified and 8.6 kb of genomic DNA were sequenced that encompassed the whole abpD gene. The DNA sequence predicts a protein, interaptin, with a calculated molecular mass of 204,300 D that is constituted by an actin-binding domain, a central coiled-coil rod domain and a membrane-associated domain. In Northern blot analyses a cAMP-stimulated transcript of 5.8 kb is expressed at the stage when cell differentiation occurs. Monoclonal antibodies raised against bacterially expressed interaptin polypeptides recognized a 200-kD developmentally and cAMP-regulated protein and a 160-kD constitutively expressed protein in Western blots. In multicellular structures, interaptin appears to be enriched in anterior-like cells which sort to the upper and lower cups during culmination. The protein is located at the nuclear envelope and ER. In mutants deficient in interaptin development is delayed, but the morphology of the mature fruiting bodies appears normal. When starved in suspension abpD− cells form EDTA-stable aggregates, which, in contrast to wild type, dissociate. Based on its domains and location, interaptin constitutes a potential link between intracellular membrane compartments and the actin cytoskeleton. PMID:9700162

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

    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

  15. Actin-binding protein alpha-actinin 4 (ACTN4) is a transcriptional co-activator of RelA/p65 sub-unit of NF-kB

    Aksenova, Vasilisa; Turoverova, Lidia; Khotin, Mikhail; Magnusson, Karl-Eric; Tulchinsky, Eugene; Melino, Gerry; Pinaev, George P.; Barlev, Nickolai; Tentler, Dmitri

    2013-01-01

    ACTN4 is an actin-binding protein that participates in cytoskeleton organisation. It resides both in the cytoplasm and nucleus and physically associates with various transcription factors. Here, we describe an effect of ACTN4 expression on transcriptional activity of the RelA/p65 subunit of NF-kB. We demonstrate that ACTN4 enhances RelA/p65-dependant expression of c-fos, MMP-3 and MMP-1 genes, but it does not affect TNC, ICAM1 and FN1 expression. Importantly, actin-binding domains of ACTN4 are not critical for the nuclear translocation and co-activation of RelA/p65-dependent transcription. Collectively, our data suggest that in the nucleus, ACTN4 functions as a selective transcriptional co-activator of RelA/p65. PMID:23482348

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

    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.

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

    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...

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

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

    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

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

    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

  1. Concentration profiles of actin-binding molecules in lamellipodia

    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.

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

    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

  3. Protein Scaffolding for Small Molecule Catalysts

    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.

  4. 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

    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...

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

    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)

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

    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.

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

    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

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

    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

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

    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.

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

    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

  11. Polycaprolactone coated porous tricalcium phosphate scaffolds for controlled release of protein for tissue engineering

    Xue, Weichang; Bandyopadhyay, Amit; Bose, Susmita

    2009-01-01

    Polycaprolactone (PCL) was coated on porous tricalcium phosphate (TCP) scaffolds to achieve controlled protein delivery. Porous TCP scaffolds were fabricated using reticulated polyurethane foam as sacrificial scaffold with a porosity of 70–90 vol %. PCL was coated on sintered porous TCP scaffolds by dipping-drying process. The compressive strength of TCP scaffolds increased significantly after PCL coating. The highest strength of 2.41 MPa at a porosity of 70% was obtained for the TCP scaffold...

  12. Programmable DNA scaffolds for spatially-ordered protein assembly

    Chandrasekaran, Arun Richard

    2016-02-01

    Ever since the notion of using DNA as a material was realized, it has been employed in the construction of complex structures that facilitate the assembly of nanoparticles or macromolecules with nanometer-scale precision. Specifically, tiles fashioned from DNA strands and DNA origami sheets have been shown to be suitable as scaffolds for immobilizing proteins with excellent control over their spatial positioning. Supramolecular assembly of proteins into periodic arrays in one or more dimensions is one of the most challenging aspects in the design of scaffolds for biomolecular investigations and macromolecular crystallization. This review provides a brief overview of how various biomolecular interactions with high degree of specificity such as streptavidin-biotin, antigen-antibody, and aptamer-protein interactions have been used to fabricate linear and multidimensional assemblies of structurally intact and functional proteins. The use of DNA-binding proteins as adaptors, polyamide recognition on DNA scaffolds and oligonucleotide linkers for protein assembly are also discussed.Ever since the notion of using DNA as a material was realized, it has been employed in the construction of complex structures that facilitate the assembly of nanoparticles or macromolecules with nanometer-scale precision. Specifically, tiles fashioned from DNA strands and DNA origami sheets have been shown to be suitable as scaffolds for immobilizing proteins with excellent control over their spatial positioning. Supramolecular assembly of proteins into periodic arrays in one or more dimensions is one of the most challenging aspects in the design of scaffolds for biomolecular investigations and macromolecular crystallization. This review provides a brief overview of how various biomolecular interactions with high degree of specificity such as streptavidin-biotin, antigen-antibody, and aptamer-protein interactions have been used to fabricate linear and multidimensional assemblies of structurally

  13. Gold nanoparticle capture within protein crystal scaffolds.

    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

  14. Gold nanoparticle capture within protein crystal scaffolds

    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

  15. Twigged streptavidin polymer as a scaffold for protein assembly.

    Matsumoto, Takuya; Isogawa, Yuki; Minamihata, Kosuke; Tanaka, Tsutomu; Kondo, Akihiko

    2016-05-10

    Protein assemblies are an emerging tool that is finding many biological and bioengineering applications. We here propose a method for the site-specific assembly of proteins on a twigged streptavidin (SA) polymer using streptavidin as a functional scaffold. SA was genetically appended with a G tag (sortase A recognition sequence) and a Y tag (HRP recognition sequence) on its N- and C-termini, respectively, to provide G-SA-Y. G-SA-Y was polymerized using HPR-mediated tyrosine coupling, then fluorescent proteins were immobilized on the polymer by biotin-SA affinity and sortase A-mediated ligation. Fluorescence measurements showed that the proteins were immobilized in close proximity to each other. Hydrolyzing enzymes were also functionally assembled on the G-SA-Y polymer. The site-specific assembly of proteins on twigged SA polymer may find new applications in various biological and bioengineering fields. PMID:27002233

  16. Protein: MPB4 [TP Atlas

    Full Text Available MPB4 Sema3A signaling molecules FLNA FLN, FLN1 FLNA Filamin-A Actin-binding protein 280, Alpha-f ... Endothelial actin-binding protein, Filamin-1, Non-muscle ... filamin 9606 Homo sapiens P21333 2316 3CNK, 2AAV, ...

  17. Assembly of designed protein scaffolds into monolayers for nanoparticle patterning.

    Mejias, Sara H; Couleaud, Pierre; Casado, Santiago; Granados, Daniel; Garcia, Miguel Angel; Abad, Jose M; Cortajarena, Aitziber L

    2016-05-01

    The controlled assembly of building blocks to achieve new nanostructured materials with defined properties at different length scales through rational design is the basis and future of bottom-up nanofabrication. This work describes the assembly of the idealized protein building block, the consensus tetratricopeptide repeat (CTPR), into monolayers by oriented immobilization of the blocks. The selectivity of thiol-gold interaction for an oriented immobilization has been verified by comparing a non-thiolated protein building block. The physical properties of the CTPR protein thin biomolecular films including topography, thickness, and viscoelasticity, are characterized. Finally, the ability of these scaffolds to act as templates for inorganic nanostructures has been demonstrated by the formation of well-packed gold nanoparticles (GNPs) monolayer patterned by the CTPR monolayer. PMID:26844645

  18. Role of Scaffolding Proteins in the Regulation of TRPC-Dependent Calcium Entry.

    Constantin, Bruno

    2016-01-01

    Plasma membrane ion channels, and in particular TRPC channels need a specific membrane environment and association with scaffolding, signaling, and cytoskeleton proteins in order to play their important functional role. The molecular composition of TRPC channels is an important factor in determining channel activation mechanisms. TRPC proteins are incorporated in macromolecular complexes including several key Ca(2 +) signaling proteins as well as proteins involved in vesicle trafficking, cytoskeletal interactions, and scaffolding. Evidence has been provided for association of TRPC with calmodulin (CaM), IP3R, PMCA, Gq/11, RhoA, and a variety of scaffolding proteins. The interaction between TRPC channels with adaptor proteins, determines their mode of regulation as well as their cellular localization and function. Adaptor proteins do not display any enzymatic activity but act as scaffold for the building of signaling complexes. The scaffolding proteins are involved in the assembling of these Ca(2+) signaling complexes, the correct sub-cellular localization of protein partners, and the regulation of the TRPC channelosome. In particular, these proteins, via their multiple protein-protein interaction motifs, can interact with various ion channels involved in the transmembrane potential, and membrane excitability. Scaffolding proteins are key components for the functional organization of TRPC channelosomes that serves as a platform regulating slow Ca(2+) entry, spatially and temporally controlled [Ca(2+)]i signals and Ca(2+) -dependent cellular functions. PMID:27161237

  19. Role of the scaffolding protein Homer 1a in cardiac hypertrophy

    Chiarello, Carmelina

    2013-01-01

    Homer proteins are a family of scaffolding proteins involved in many intracellular signaling pathways, in both excitable and non-excitable cells. These proteins participate in the assembly and regulation of functional signaling complexes, facilitating the cross-talk between surface membrane receptors and channels in the membranes of intracellular compartments (Worley PF. et al., 2007). Homer proteins are constitutively expressed in the brain, where their scaffolding function is important for ...

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

    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...

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

    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.

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

    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...

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

    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

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

    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.

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

    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

  6. Role of scaffolding protein CipC of Clostridium cellulolyticum in cellulose degradation.

    Pagès, S.; Gal, L; Bélaïch, A; Gaudin, C; Tardif, C; Bélaïch, J P

    1997-01-01

    The role of a miniscaffolding protein, miniCipC1, forming part of Clostridium cellulolyticum scaffolding protein CipC in insoluble cellulose degradation was investigated. The parameters of the binding of miniCipC1, which contains a family III cellulose-binding domain (CBD), a hydrophilic domain, and a cohesin domain, to four insoluble celluloses were determined. At saturating concentrations, about 8.2 micromol of protein was bound per g of bacterial microcrystalline cellulose, while Avicel, c...

  7. Altered sensitivities to morphine and cocaine in scaffold protein tamalin knockout mice

    Ogawa, Masaaki; Miyakawa, Tsuyoshi; Nakamura, Kenji; Kitano, Jun; Furushima, Kenryo; Kiyonari, Hiroshi; Nakayama, Rika; Nakao, Kazuki; Moriyoshi, Koki; Nakanishi, Shigetada

    2007-01-01

    Tamalin is a scaffold protein that interacts with metabotropic glutamate receptors and the kinase-deficient neurotrophin TrkCT1 receptor and forms a protein complex with multiple protein-trafficking and intracellular signaling molecules. In culture, tamalin promotes intracellular trafficking of group 1 metabotropic glutamate receptors through its interaction with guanine nucleotide exchange factor cytohesins and causes actin reorganization and membrane ruffling via the TrkCT1/cytohesin-2 sign...

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

    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

  9. A one-pot functionalization strategy for immobilizing proteins onto linear dsDNA scaffolds

    Functional DNA scaffolds can be defined as DNA-based structures comprising chemical moieties facilitating and guiding the immobilization of additional nanocomponents. Due to the limited reactivity of DNA there is currently a need to develop rapid routes to expand its chemical repertoire and increase its versatility as a nanostructuring scaffold. We report a simple synthetic strategy for generating linear and stable double-stranded DNA scaffolds functionalized with multiple sites reactive towards free thiols, and the utility of this approach is demonstrated by immobilizing a model protein containing an accessible free thiol. This procedure is very versatile and could be easily expanded to other types of chemistries. This approach could also potentially be employed for the specific, oriented immobilization of various biomolecules and nanoparticles on predefined DNA architectures.

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

    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.

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

    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

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

    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

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

    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.

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

    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

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

    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.

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

    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.

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

    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.

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

    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.

  19. Axl Phosphorylates Elmo Scaffold Proteins To Promote Rac Activation and Cell Invasion

    Abu-Thuraia, Afnan; Gauthier, Rosemarie; Chidiac, Rony; Fukui, Yoshinori; Screaton, Robert A; Gratton, Jean-Philippe; Côté, Jean-François

    2014-01-01

    The receptor tyrosine kinase Axl contributes to cell migration and invasion. Expression of Axl correlates with metastatic progression in cancer patients, yet the specific signaling events promoting invasion downstream of Axl are poorly defined. Herein, we report Elmo scaffolds to be direct substrates and binding partners of Axl. Elmo proteins are established to interact with Dock family guanine nucleotide exchange factors to control Rac-mediated cytoskeletal dynamics. Proteomics and mutagenes...

  20. Alpha-class glutathione transferases as steroid isomerases and scaffolds for protein redesign

    Pettersson, Pär L.

    2002-01-01

    The present work focuses on the glutathione transferase (GST) Alpha-class enzymes, their characteristics as steroid isomerases and structural plasticity as malleable scaffolds for protein design. The GSTs are a family of detoxication enzymes that appears to have a wider variety of additional functions. Kinetic steady-state parameters for human GST A1-1 with the steroid isomerase substrate Δ5-androstene-3,17-dione (AD), an intermediate in steroid hormone biosynthesis, were determined. It was e...

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

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

    2016-01-01

    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......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......-tail 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....

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

    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

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

    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.

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

    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.

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

    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 O2 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. Preparation of scaffolds from human hair proteins for tissue-engineering applications

    Human hair proteins were isolated and purified for the fabrication of tissue-engineering scaffolds. Their cellular compatibility was studied using NIH3T3 mice fibroblast cells. The proteins were characterized using FTIR spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis for molecular weights and two-dimensional polyacrylamide gel electrophoresis for their isoelectric points (pIs). The molecular weights of keratins were in the range of 40-60 kilo-Daltons (kDa) and of matrix proteins were in the range of 15-30 kDa. The pIs of keratins were found to be in the range of 4.5-5.3. Sponges of the proteins were formed by lyophilization. Scanning electron microscopy was performed to examine the surface. Swelling studies were carried out in phosphate buffer saline at physiological pH 7.4. The hydrophilic character of the protein surface was studied by determining an average contact angle, which came to be 370. The wells of tissue culture plates were coated with these proteins for studying the attachment and morphology of the cells. The protein detachment study was done to ensure the adsorption of proteins on the wells until the completion of the experiments. The cellular growth on a protein-coated surface showed three-dimensional 'bulged' morphology due to cell-cell and cell-matrix contacts. The sponges of human hair proteins supported more cells for a longer period than control. The morphology and cell proliferation studies exhibited by NIH3T3 cells on these proteins have shown their potential to be used as tissue-engineering scaffolds with better cell-cell contacts and leucine-aspartic acid-valine (LDV)-mediated cell-matrix interactions

  7. Naphthalimide Scaffold Provides Versatile Platform for Selective Thiol Sensing and Protein Labeling.

    Zhou, Pengcheng; Yao, Juan; Hu, Guodong; Fang, Jianguo

    2016-04-15

    Reversible thiol modifications are fundamental of cellular redox regulation. Specific thiol detection, including thiol sensing and protein thiols labeling, is critical to study such modifications. We reported the discovery of 4-methylsulfonyl-N-n-butyl-1,8-naphthalimide (MSBN), a highly selective fluorogenic probe for thiols based on the 1,8-naphthalimide scaffold. Thiols react with MSBN nearly quantitatively via nucleophilic aromatic substitution to replace the methylsulfonyl group and restore the quenched fluorescence (>100-fold increase). MSBN was employed to selectively image thiols in live cells and specifically label protein thiols with a turn-on signal to determine diverse reversible protein thiol modifications. In addition, we introduced a bulky group into the MSBN as a mass tag to create a probe MSBN-TPP, which readily discriminates the reduced thioredoxin from the oxidized one. The specific reaction of MSBN with thiols and the easy manipulation of the naphthalimide unit enable MSBN a versatile scaffold in developing novel probes for thiol-based protein bioconjugation and studying various thiol modifications. PMID:26813105

  8. Increased expression of secretory actin-binding protein (SABP) on human spermatozoa is assiciated with poor semen quality

    Čapková, Jana; Elzeinová, Fatima; Novák, P.

    Rio de Janeiro: ImmunoRIO, 2007. -----. [Immuno RIO 2007, International Congrass of Immunology /13./. 21.08.2007-25.08.2007, Rio de Janeiro] Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50520701 Keywords : SABP * human spermatozoa * pathological spermiogram Subject RIV: EB - Genetics ; Molecular Biology

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

    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 ...

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

    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

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

    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

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

    Parmeggiani, Fabio; Pellarin, Riccardo; Larsen, Anders Peter; Varadamsetty, Gautham; Stumpp, Michael T; Zerbe, Oliver; Caflisch, Amedeo; Plückthun, Andreas

    2007-01-01

    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...... constitute the starting point for the generation of designed armadillo repeat protein libraries for the selection of peptide binders, exploiting their modular structure and their conserved binding mode....

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

    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

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

    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

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

    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.

  16. Structural and functional characterization of an iron-sulfur cluster assembly scaffold protein-SufA from Plasmodium vivax.

    Pala, Zarna Rajeshkumar; Saxena, Vishal; Saggu, Gagandeep Singh; Yadav, Sushil Kumar; Pareek, R P; Kochar, Sanjay Kumar; Kochar, Dhanpat Kumar; Garg, Shilpi

    2016-07-01

    Iron-sulfur (Fe-S) clusters are utilized as prosthetic groups in all living organisms for diverse range of cellular processes including electron transport in respiration and photosynthesis, sensing of ambient conditions, regulation of gene expression and catalysis. In Plasmodium, two Fe-S cluster biogenesis pathways are reported, of which the Suf pathway in the apicoplast has been shown essential for the erythrocytic stages of the parasite. While the initial components of this pathway detailing the sulfur mobilization have been elucidated, the components required for the assembly and transfer of Fe-S clusters are not reported from the parasite. In Escherichia coli, SufB acts as a scaffold protein and SufA traffics the assembled Fe-S cluster from SufB to target apo-proteins. However, in Plasmodium, the homologs of these proteins are yet to be characterized for their function. Here, we report a putative SufA protein from Plasmodium vivax with signature motifs of A-type scaffold proteins, which is evolutionarily conserved. The presence of the [Fe4S4](3+) cluster under reduced conditions was confirmed by UV-visible and EPR spectroscopy and the interaction of these clusters with the conserved cysteine residues of chains A and B of PvSufA, validates its existence as a dimer, similar to that in E. coli. The H-bond interactions at the PvSufA-SufB interface demonstrate SufA as a scaffold protein in conjunction with SufB for the pre-assembly of Fe-S clusters and their transfer to the target proteins. Co-localization of the protein to the apicoplast further provides an experimental evidence of a functional scaffold protein SufA for the biogenesis of Fe-S clusters in apicoplast of Plasmodium. PMID:27033210

  17. 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

    Jayo Asier

    2012-08-01

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

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

    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

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

    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.

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

    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

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

    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.

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

    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

  3. Pushing the limits of automatic computational protein design: Design, expression, and characterization of a large synthetic protein based on a fungal laccase scaffold

    Glykys D.J.; Szilvay G.R.; Tortosa P.; Suarez Diez M.; Jaramillo A.; Banta S.

    2011-01-01

    The de novo engineering of new proteins will allow the design of complex systems in synthetic biology. But the design of large proteins is very challenging due to the large combinatorial sequence space to be explored and the lack of a suitable selection system to guide the evolution and optimization. One way to approach this challenge is to use computational design methods based on the current crystallographic data and on molecular mechanics. We have used a laccase protein fold as a scaffold ...

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

    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

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

    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

  6. Axl phosphorylates Elmo scaffold proteins to promote Rac activation and cell invasion.

    Abu-Thuraia, Afnan; Gauthier, Rosemarie; Chidiac, Rony; Fukui, Yoshinori; Screaton, Robert A; Gratton, Jean-Philippe; Côté, Jean-François

    2015-01-01

    The receptor tyrosine kinase Axl contributes to cell migration and invasion. Expression of Axl correlates with metastatic progression in cancer patients, yet the specific signaling events promoting invasion downstream of Axl are poorly defined. Herein, we report Elmo scaffolds to be direct substrates and binding partners of Axl. Elmo proteins are established to interact with Dock family guanine nucleotide exchange factors to control Rac-mediated cytoskeletal dynamics. Proteomics and mutagenesis studies reveal that Axl phosphorylates Elmo1/2 on a conserved carboxyl-terminal tyrosine residue. Upon Gas6-dependent activation of Axl, endogenous Elmo2 becomes phosphorylated on Tyr-713 and enters into a physical complex with Axl in breast cancer cells. Interfering with Elmo2 expression prevented Gas6-induced Rac1 activation in breast cancer cells. Similarly to blocking of Axl, Elmo2 knockdown or pharmacological inhibition of Dock1 abolishes breast cancer cell invasion. Interestingly, Axl or Elmo2 knockdown diminishes breast cancer cell proliferation. Rescue of Elmo2 knockdown cells with the wild-type protein but not with Elmo2 harboring Tyr-713-Phe mutations restores cell invasion and cell proliferation. These results define a new mechanism by which Axl promotes cell proliferation and invasion and identifies inhibition of the Elmo-Dock pathway as a potential therapeutic target to stop Axl-induced metastases. PMID:25332238

  7. Carbohydrate particles as protein carriers and scaffolds: physico-chemical characterization and collagen stability

    Peres, Ivone; Rocha, Sandra; Loureiro, Joana A.; Carmo Pereira, Maria do [University of Porto, LEPAE, Chemical Engineering Department, Faculty of Engineering (Portugal); Ivanova, Galya [Universidade do Porto, REQUIMTE, Departamento de Quimica, Faculdade de Ciencias (Portugal); Coelho, Manuel, E-mail: mcoelho@fe.up.pt [University of Porto, LEPAE, Chemical Engineering Department, Faculty of Engineering (Portugal)

    2012-09-15

    The preservation of protein properties after entrapping into polymeric matrices and the effects of drying the emulsions still remains uncertain and controversial. Carbohydrate particles were designed and prepared by homogenization of gum arabic and maltodextrin mixture, with collagen hydrolysate (CH) followed by spray-drying. The encapsulation of CH in the carbohydrate matrix was achieved with an efficiency of 85 {+-} 2 %. The morphology and the size of the particles, before (40-400 nm) and after spray-drying (<20 {mu}m), were characterized by scanning electron microscopy and dynamic light scattering. Measurements of the nuclear relaxation times and application of diffusion ordered spectroscopy, obtained through pulsed field gradient NMR experiments, have been performed to determine the structure of the CH-polysaccharide conjugates and to clarify the mechanism of CH immobilization in the polysaccharide matrix. In vitro release profiles in ultrapure water and in cellular medium reveal that the diffusion rate of CH from the polymeric matrix to the dialysis solution decreases in average 30-50 % over time, compared to free CH molecules. In cellular medium at 37 Degree-Sign C, the complete release of CH from the particles is achieved only after 24 h, demonstrating a significant decrease in the CH mass transfer process when compared with free CH. The findings of this study outline the ability of gum arabic/maltodextrin matrices to entrap and preserve CH original properties after the spray-drying process and support the potential of the polymeric scaffold for protein delivery and tissue engineering.

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

    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...

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

    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

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

    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 ...

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

    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.

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

    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.

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

    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.

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

    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

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

    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

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

    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.

  17. A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics

    Teresa T. Bonello; Miro Janco; Jeff Hook; Alex Byun; Mark Appaduray; Irina Dedova; Sarah Hitchcock-DeGregori; Hardeman, Edna C.; Justine R. Stehn; Till Böcking; Gunning, Peter W.

    2016-01-01

    The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperativ...

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

    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.

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

    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.

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

    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

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

    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.

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

    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.

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

    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.

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

    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.

  5. A novel {sup 18}F-labeled two-helix scaffold protein for PET imaging of HER2-positive tumor

    Miao, Zheng; Ren, Gang; Jiang, Lei; Liu, Hongguang; Cheng, Zhen [Stanford University, Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Stanford Cancer Center, Bio-X Program, Stanford, CA (United States); Webster, Jack M.; Zhang, Rong; Syud, Faisal [General Electric Company, Global Research, Niskayuna, NY (United States); Namavari, Mohammad; Gambhir, Sanjiv S. [Stanford University, MIPS, Departments of Radiology and Bioengineering, Stanford Cancer Center, Bio-X Program, Stanford, CA (United States)

    2011-11-15

    Two-helix scaffold proteins ({proportional_to} 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 {sup 18}F-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 I{sub 2} oxidation to form a disulfide bridge. The cyclic peptide was then conjugated with a radiofluorination synthon, 4-{sup 18}F-fluorobenzyl aldehyde ({sup 18}F-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, {sup 18}F-FBO-MUT-DS, was performed at 37 C. {sup 18}F-FBO-MUT-DS with high specific activity (20-32 MBq/nmol, 88-140 {mu}Ci/{mu}g, 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 ({proportional_to}12% applied activity at 1 h) by incubation of {sup 18}F-FBO-MUT-DS with HER2 high-expressing SKOV3 ovarian cancer cells. The affinities (K{sub D}) 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 {sup 18}F-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 {mu}g of HER2 Affibody protein reduced the tumor uptake (6.9 vs 1.8%ID/g, p < 0.05). F-FBO-MUT-DS displays excellent HER2 targeting ability

  6. All Dact (Dapper/Frodo scaffold proteins dimerize and exhibit conserved interactions with Vangl, Dvl, and serine/threonine kinases

    Cheyette Benjamin NR

    2011-06-01

    Full Text Available Abstract Background The Dact family of scaffold proteins was discovered by virtue of binding to Dvl proteins central to Wnt and Planar Cell Polarity (PCP signaling. Subsequently Dact proteins have been linked to a growing list of potential partners implicated in β-catenin-dependent and β-catenin-independent forms of Wnt and other signaling. To clarify conserved and non-conserved roles for this protein family, we systematically compared molecular interactions of all three murine Dact paralogs by co-immunoprecipitation of proteins recombinantly expressed in cultured human embryonic kidney cells. Results Every Dact paralog readily formed complexes with the Vangl, Dvl, and CK1δ/ε proteins of species ranging from fruit flies to humans, as well as with PKA and PKC. Dact proteins also formed complexes with themselves and with each other; their conserved N-terminal leucine-zipper domains, which have no known binding partners, were necessary and sufficient for this interaction, suggesting that it reflects leucine-zipper-mediated homo- and hetero-dimerization. We also found weaker, though conserved, interactions of all three Dact paralogs with the catenin superfamily member p120ctn. Complex formation with other previously proposed partners including most other catenins, GSK3, LEF/TCF, HDAC1, and TGFβ receptors was paralog-specific, comparatively weak, and/or more sensitive to empirical conditions. Conclusions Combined with published functional evidence from targeted knock-out mice, these data support a conserved role for Dact proteins in kinase-regulated biochemistry involving Vangl and Dvl. This strongly suggests that a principal role for all Dact family members is in the PCP pathway or a molecularly related signaling cascade in vertebrates.

  7. [Cytoskeletal actin and its associated proteins. Some examples in Protista].

    Guillén, N; Carlier, M F; Brugerolle, G; Tardieux, I; Ausseil, J

    1998-06-01

    Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin

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

    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

  9. In vitro and in vivo evaluation of bone formation using solid freeform fabrication-based bone morphogenic protein-2 releasing PCL/PLGA scaffolds

    The aim of this study was to develop novel polycaprolactone/poly(lactic-co-glycolic acid) (PCL/PLGA) scaffolds with a heparin–dopamine (Hep–DOPA) conjugate for controlled release of bone morphogenic protein-2 (BMP-2) to enhance osteoblast activity in vitro and also bone formation in vivo. PCL/PLGA scaffolds were prepared by a solid freeform fabrication method. The PCL/PLGA scaffolds were functionalized with Hep–DOPA and then BMP-2 was sequentially coated onto the Hep–DOPA/PCL/PLGA scaffolds. The characterization and surface elemental composition of all scaffolds were evaluated by scanning electron microscope and x-ray photoelectron spectroscopy. The osteoblast activities on all scaffolds were assessed by cell proliferation, alkaline phosphatase (ALP) activity and calcium deposition in vitro. To demonstrate bone formation in vivo, plain radiograph, micro-computed tomography (micro-CT) evaluation and histological studies were performed after the implantation of all scaffolds on a rat femur defect. Hep–DOPA/PCL/PLGA had more controlled release of BMP-2, which was quantified by enzyme-linked immunosorbent assay, compared with Hep/PCL/PLGA. The in vitro results showed that osteoblast-like cells (MG-63 cells) grown on BMP-2/Hep–DOPA/PCL/PLGA had significantly enhanced ALP activity and calcium deposition compared with those on BMP-2/Hep/PCL/PLGA and PCL/PLGA. In addition, the plain radiograph, micro-CT evaluation and histological studies demonstrated that the implanted BMP-2/Hep–DOPA/PCL/PLGA on rat femur had more bone formation than BMP-2/Hep/PCL/PLGA and PCL/PLGA in vivo. (paper)

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

    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 ...

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

    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 ...

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

    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...

  13. Arabidopsis group Ie formins localize to specific cell membrane domains, interact with actin-binding proteins and cause defects in cell expansion upon aberrant expression

    Deeks, M.J.; Cvrčková, F.; Machesky, M. L.; Mikitova, V.; Ketelaar, T.; Žárský, Viktor; Davies, B.; Hussey, P.J.

    2005-01-01

    Roč. 168, č. 3 (2005), s. 529-540. ISSN 0028-646X R&D Projects: GA ČR GA204/02/1461; GA ČR GA204/05/0268 Institutional research plan: CEZ:AV0Z50380511 Keywords : actin * Arabidopsis * cytoskeleton Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.285, year: 2005

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

    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.

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

    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

  16. Scaffold protein enigma homolog 1 overcomes the repression of myogenesis activation by inhibitor of DNA binding 2.

    Nakatani, Miyuki; Ito, Jumpei; Koyama, Riko; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2016-05-27

    Enigma Homolog 1 (ENH1) is a scaffold protein for signaling proteins and transcription factors. Previously, we reported that ENH1 overexpression promotes the differentiation of C2C12 myoblasts. However, the molecular mechanism underlying the role of ENH1 in the C2C12 cells differentiation remains elusive. ENH1 was shown to inhibit the proliferation of neuroblastoma cells by sequestering Inhibitor of DNA binding protein 2 (Id2) in the cytosol. Id2 is a repressor of basic Helix-Loop-Helix transcription factors activity and prevents myogenesis. Here, we found that ENH1 overcome the Id2 repression of C2C12 cells myogenic differentiation and that ENH1 overexpression promotes mice satellite cells activation, the first step toward myogenic differentiation. In addition, we show that ENH1 interacted with Id2 in C2C12 cells and mice satellite cells. Collectively, our results suggest that ENH1 plays an important role in the activation of myogenesis through the repression of Id2 activity. PMID:27114303

  17. Receptor-Bound Targets of Selective Autophagy Use a Scaffold Protein to Activate the Atg1 Kinase.

    Kamber, Roarke A; Shoemaker, Christopher J; Denic, Vladimir

    2015-08-01

    Selective autophagy eliminates protein aggregates, damaged organelles, and other targets that otherwise accumulate and cause disease. Autophagy receptors mediate selectivity by connecting targets to the autophagosome membrane. It has remained unknown whether receptors perform additional functions. Here, we show that in yeast certain receptor-bound targets activate Atg1, the kinase that controls autophagosome formation. Specifically, we found that in nutrient-rich conditions, Atg1 is active only in a multisubunit complex comprising constitutive protein aggregates, their autophagy receptor, and a scaffold protein, Atg11. Development of a cell-free assay for Atg1-mediated phosphorylation enabled us to activate Atg1 with purified receptor-bound aggregates and Atg11. Another target, damaged peroxisomes, also activated Atg1 using Atg11 with a distinct receptor. Our work reveals that receptor-target complexes activate Atg1 to drive formation of selective autophagosomes. This regulatory logic is a key similarity between selective autophagy and bulk autophagy, which is initiated by a distinct Atg1 activation mechanism during starvation. PMID:26166702

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

    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

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

    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

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

    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.)

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

    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

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

    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

  3. Fibrin glue is a candidate scaffold for long-term therapeutic protein expression in spontaneously differentiated adipocytes in vitro

    Adipose tissue is expected to provide a source of cells for protein replacement therapies via auto-transplantation. However, the conditioning of the environment surrounding the transplanted adipocytes for their long-term survival and protein secretion properties has not been established. We have recently developed a preparation procedure for preadipocytes, ceiling culture-derived proliferative adipocytes (ccdPAs), as a therapeutic gene vehicle suitable for stable gene product secretion. We herein report the results of our evaluation of using fibrin glue as a scaffold for the transplanted ccdPAs for the expression of a transduced gene in a three-dimensional culture system. The ccdPAs secreted the functional protein translated from an exogenously transduced gene, as well as physiological adipocyte proteins, and the long viability of ccdPAs (up to 84 days) was dependent on the fibrinogen concentrations. The ccdPAs spontaneously accumulated lipid droplets, and their expression levels of the transduced exogenous gene with its product were maintained for at least 56 days. The fibrinogen concentration modified the adipogenic differentiation of ccdPAs and their exogenous gene expression levels, and the levels of exogenously transduced gene expression at the different fibrinogen concentrations were dependent on the extent of adipogenic differentiation in the gel. These results indicate that fibrin glue helps to maintain the high adipogenic potential of cultured adipocytes after passaging in a 3D culture system, and suggests that once they are successfully implanted at the transplantation site, the cells exhibit increased expression of the transduced gene with adipogenic differentiation.

  4. Recombinant human bone morphogenetic protein-2 released from polyurethane-based scaffolds promotes early osteogenic differentiation of human mesenchymal stem cells

    The purposes of this study were to determine the pharmacokinetics of recombinant human bone morphogenetic protein-2 (rhBMP-2) from a polyurethane (PUR)-based porous scaffold and to determine the biological responses of human mesenchymal stem cells (hMSCs) to the rhBMP-2 released from those scaffolds. The rhBMP-2 was incorporated into the PUR three-dimensional (3D) porous scaffolds and release profiles were determined using enzyme-linked immunosorbent assay. The bioactivity of the rhBMP-2 containing releasates was determined using hMSCs and compared with exogenous rhBMP-2. Release of rhBMP-2 from PUR-based systems was bi-phasic and characterized by an initial burst followed by a sustained release for up to 21 days. Expression of alkaline phosphatase activity by hMSCs treated with the rhBMP-2 releasates was significantly greater than the cells alone (control) throughout the time periods. Furthermore, after 14 days of culture, the hMSCs cultured with rhBMP-2 releasate had a greater amount of mineralization compared to exogenous rhBMP-2. Overall, the rhBMP-2 release from the PUR-based scaffolds was sustained for 21 days and the releasates appeared to be bioactive and promoted earlier osteogenic differentiation and mineralization of hMSCs than the exogenous rhBMP-2. (paper)

  5. 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

    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...

  6. Chemically-Conjugated Bone Morphogenetic Protein-2 on Three-Dimensional Polycaprolactone Scaffolds Stimulates Osteogenic Activity in Bone Marrow Stromal Cells

    Zhang, Huina; Migneco, Francesco; Lin, Chia-Ying; Hollister, Scott J.

    2010-01-01

    Poly(ε-caprolactone) (PCL) has received considerable attention in bone tissue engineering. However, the lack of osteoinductive ability of PCL limits its application. The aim of this study was to directly attach bone morphogenetic protein-2 (BMP-2) to PCL scaffolds by a crosslinking conjugation method and to investigate whether the bound BMP-2 maintained bioactivity in vitro. Immunofluorescent staining against BMP-2 and quantitative enzyme-linked immunosorbent assay measurements demonstrated t...

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

    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

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

    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.

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

    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

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

    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

  11. 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.

    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

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

    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

  13. Mitochondrial and Nucleolar Localization of Cysteine Desulfurase Nfs and the Scaffold Protein Isu in Trypanosoma brucei

    Kovářová, Julie; Horáková, Eva; Changmai, Piya; Vancová, Marie; Lukeš, Julius

    2014-01-01

    Roč. 13, č. 3 (2014), s. 353-362. ISSN 1535-9778 R&D Projects: GA ČR(CZ) GAP305/11/2179; GA MŠk LH12104; GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : transfer RNA * iron sulfur protein * blood stream forms Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.820, year: 2014

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

    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 ...

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

    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...

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

    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...

  17. 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

    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...

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

    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.

  19. Rad4 mainly functions in Chk1-mediated DNA damage checkpoint pathway as a scaffold protein in the fission yeast Schizosaccharomyces pombe.

    Ming Yue

    Full Text Available Rad4/Cut5 is a scaffold protein in the Chk1-mediated DNA damage checkpoint in S. pombe. However, whether it contains a robust ATR-activation domain (AAD required for checkpoint signaling like its orthologs TopBP1 in humans and Dpb11 in budding yeast has been incompletely clear. To identify the putative AAD in Rad4, we carried out an extensive genetic screen looking for novel mutants with an enhanced sensitivity to replication stress or DNA damage in which the function of the AAD can be eliminated by the mutations. Two new mutations near the N-terminus were identified that caused significantly higher sensitivities to DNA damage or chronic replication stress than all previously reported mutants, suggesting that most of the checkpoint function of the protein is eliminated. However, these mutations did not affect the activation of Rad3 (ATR in humans yet eliminated the scaffolding function of the protein required for the activation of Chk1. Several mutations were also identified in or near the recently reported AAD in the C-terminus of Rad4. However, all mutations in the C-terminus only slightly sensitized the cells to DNA damage. Interestingly, a mutant lacking the whole C-terminus was found resistant to DNA damage and replication stress almost like the wild type cells. Consistent with the resistance, all known Rad3 dependent phosphorylations of checkpoint proteins remained intact in the C-terminal deletion mutant, indicating that unlike that in Dpb11, the C-terminus of Rad4 does not contain a robust AAD. These results, together with those from the biochemical studies, show that Rad4 mainly functions as a scaffold protein in the Chk1, not the Cds1(CHK2 in humans, checkpoint pathway. It plays a minor role or is functionally redundant with an unknown factor in Rad3 activation.

  20. Discontinuous release of bone morphogenetic protein-2 loaded within interconnected pores of honeycomb-like polycaprolactone scaffold promotes bone healing in a large bone defect of rabbit ulna.

    Bae, Ji-Hoon; Song, Hae-Ryong; Kim, Hak-Jun; Lim, Hong-Chul; Park, Jung-Ho; Liu, Yuchun; Teoh, Swee-Hin

    2011-10-01

    The choice of an appropriate carrier and its microarchitectural design is integral in directing bone ingrowth into the defect site and determining its subsequent rate of bone formation and remodeling. We have selected a three-dimensional polycaprolactone (PCL) scaffold with an interconnected honeycomb-like porous structure to provide a conduit for vasculature ingrowth as well as an osteoconductive pathway to guide recruited cells responding to a unique triphasic release of osteoinductive bone morphogenetic proteins (BMP) from these PCL scaffolds. We hypothesize that the use of recombinant human bone morphogenetic protein 2 (rhBMP2)-PCL constructs promotes rapid union and bone regeneration of a large defect. Results of our pilot study on a unilateral 15 mm mid-diaphyseal segmental rabbit ulna defect demonstrated enhanced bone healing with greater amount of bone formation and bridging under plain radiography and microcomputed tomography imaging when compared with an empty PCL and untreated group after 8 weeks postimplantation. Quantitative measurements showed significantly higher bone volume fraction and trabecular thickness, with lower trabecular separation in the rhBMP2-treated groups. Histology evaluation also revealed greater mature bone formation spanning across the entire scaffold region compared with other groups, which showed no bone regeneration within the central defect zone. We highlight that it is the uniqueness of the scaffold having a highly porous network of channels that promoted vascular integration and allowed for cellular infiltration, leading to a discontinuous triphasic BMP2 release profile that mimicked the release profile during natural repair mechanisms in vivo. This study serves as preclinical evidence demonstrating the potential of combining osteoinductive rhBMP2 with our PCL constructs for the repair of large defects in a large animal model. PMID:21682591

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

    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.

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

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

    2012-01-01

    -containing cytoplasmic focal accumulations of the substituted protein to a degree that was correlated with the severity of the associated phenotypes. In contrast, study of mutations leading to substitutions in the FLNB rod domain that result in the same phenotypes as ABD mutations, demonstrated that with only one...

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

    Johansson, Henrik; Boesgaard, Michael Worch; Nørskov-Lauritsen, Lenea; Larsen, Inna; Kuhne, Sebastiaan; Gloriam, David E; Bräuner-Osborne, Hans; Sejer Pedersen, Daniel

    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......, and 34b as antagonists at the GPRC6A receptor in the low micromolar range and show that 7 and 34b display >9-fold selectivity for the GPRC6A receptor over related GPCRs, making 7 and 34b the most potent and selective antagonists for the GPRC6A receptor reported to date....

  4. 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

    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.

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

    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

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

    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

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

    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

  8. Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe–S cluster biogenesis regulation

    Ciesielski, Szymon J.; Schilke, Brenda; Marszalek, Jaroslaw; Craig, Elizabeth A.

    2016-01-01

    Iron–sulfur (Fe–S) clusters, essential protein cofactors, are assembled on the mitochondrial scaffold protein Isu and then transferred to recipient proteins via a multistep process in which Isu interacts sequentially with multiple protein factors. This pathway is in part regulated posttranslationally by modulation of the degradation of Isu, whose abundance increases >10-fold upon perturbation of the biogenesis process. We tested a model in which direct interaction with protein partners protects Isu from degradation by the mitochondrial Lon-type protease. Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe–S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu. Nfs1 and Jac1 variants known to be defective in interaction with Isu were also defective in protecting Isu from degradation. Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1. Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe–S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe–S cluster biogenesis does not meet cellular demands. PMID:26842892

  9. Semiotic scaffolding

    Hoffmeyer, Jesper

    2015-01-01

    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...

  10. Amyloid beta protein-induced zinc sequestration leads to synaptic loss via dysregulation of the ProSAP2/Shank3 scaffold

    Hof Patrick R

    2011-09-01

    Full Text Available Abstract Background Memory deficits in Alzheimer's disease (AD manifest together with the loss of synapses caused by the disruption of the postsynaptic density (PSD, a network of scaffold proteins located in dendritic spines. However, the underlying molecular mechanisms remain elusive. Since it was shown that ProSAP2/Shank3 scaffold assembly within the PSD is Zn2+-dependent and that the amyloid beta protein (Aβ is able to bind Zn2+, we hypothesize that sequestration of Zn2+ ions by Aβ contributes to ProSAP/Shank platform malformation. Results To test this hypothesis, we designed multiple in vitro and in vivo assays demonstrating ProSAP/Shank dysregulation in rat hippocampal cultures following Aβ oligomer accumulation. These changes were independent from alterations on ProSAP/Shank transcriptional level. However, application of soluble Aβ prevented association of Zn2+ ions with ProSAP2/Shank3 in a cell-based assay and decreased the concentration of Zn2+ clusters within dendrites. Zn2+ supplementation or saturation of Aβ with Zn2+ ions prior to cell treatment was able to counter the effects induced by Aβ on synapse density and ProSAP2/Shank3 levels at the PSD. Interestingly, intracellular Zn2+ levels in APP-PS1 mice and human AD hippocampus are reduced along with a reduction in synapse density and synaptic ProSAP2/Shank3 and Shank1 protein levels. Conclusions We conclude that sequestration of Zn2+ ions by Aβ significantly contributes to changes in ProSAP2/Shank3 platforms. These changes in turn lead to less consolidated (mature synapses reflected by a decrease in Shank1 protein levels at the PSD and decreased synapse density in hippocampal neurons.

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

    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.

  12. 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.

    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.

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

    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.

  14. Assembly of juxtaparanodes in myelinating DRG culture: Differential clustering of the Kv1/Caspr2 complex and scaffolding protein 4.1B.

    Hivert, Bruno; Pinatel, Delphine; Labasque, Marilyne; Tricaud, Nicolas; Goutebroze, Laurence; Faivre-Sarrailh, Catherine

    2016-05-01

    The precise distribution of ion channels at the nodes of Ranvier is essential for the efficient propagation of action potentials along myelinated axons. The voltage-gated potassium channels Kv1.1/1.2 are clustered at the juxtaparanodes in association with the cell adhesion molecules, Caspr2 and TAG-1 and the scaffolding protein 4.1B. In the present study, we set up myelinating cultures of DRG neurons and Schwann cells to look through the formation of juxtaparanodes in vitro. We showed that the Kv1.1/Kv1.2 channels were first enriched at paranodes before being restricted to distal paranodes and juxtaparanodes. In addition, the Kv1 channels displayed an asymmetric expression enriched at the distal juxtaparanodes. Caspr2 was strongly co-localized with Kv1.2 whereas the scaffolding protein 4.1B was preferentially recruited at paranodes while being present at juxtaparanodes too. Kv1.2/Caspr2 but not 4.1B, also transiently accumulated within the nodal region both in myelinated cultures and developing sciatic nerves. Studying cultures and sciatic nerves from 4.1B KO mice, we further showed that 4.1B is required for the proper targeting of Caspr2 early during myelination. Moreover, using adenoviral-mediated expression of Caspr-GFP and photobleaching experiments, we analyzed the stability of paranodal junctions and showed that the lateral stability of paranodal Caspr was not altered in 4.1B KO mice indicating that 4.1B is not required for the assembly and stability of the paranodal junctions. Thus, developing an adapted culture paradigm, we provide new insights into the dynamic and differential distribution of Kv1 channels and associated proteins during myelination. GLIA 2016;64:840-852. PMID:26840208

  15. Neuronal Networks on Nanocellulose Scaffolds.

    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

  16. Developmental Scaffolding

    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...

  17. Semiotic scaffolding

    Hoffmeyer, Jesper

    2014-01-01

    While organic life is the product of myriads of biochemical processes it usually escapes notice that the chemistry of life cannot be understood exclusively in terms of chemistry. What must be added is an understanding of the particular organized dynamics, which makes the integration of all these ...... brain and back again. One of the main structuring and enabling principles in the semiotic dynamics across levels has been called semiotic scaffolding a concept that relates to psychological catalyses in interesting ways to be furthe explored....

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

    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

  19. Mlp1 Acts as a Mitotic Scaffold to Spatially Regulate Spindle Assembly Checkpoint Proteins in Aspergillus nidulans

    De Souza, Colin P.; Hashmi, Shahr B.; Nayak, Tania; Oakley, Berl; Osmani, Stephen A.

    2009-01-01

    During open mitosis several nuclear pore complex (NPC) proteins have mitotic specific localizations and functions. We find that the Aspergillus nidulans Mlp1 NPC protein has previously unrealized mitotic roles involving spatial regulation of spindle assembly checkpoint (SAC) proteins. In interphase, An-Mlp1 tethers the An-Mad1 and An-Mad2 SAC proteins to NPCs. During a normal mitosis, An-Mlp1, An-Mad1, and An-Mad2 localize similarly on, and around, kinetochores until telophase when they trans...

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

    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.

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

    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.

  2. Engineered proteins with Pumilio/fem-3 mRNA binding factor scaffold to manipulate RNA metabolism.

    Wang, Yang; Wang, Zefeng; Tanaka Hall, Traci M

    2013-08-01

    Pumilio/fem-3 mRNA binding factor proteins are characterized by a sequence-specific RNA-binding domain. This unique single-stranded RNA recognition module, whose sequence specificity can be reprogrammed, has been fused with functional modules to engineer protein factors with various functions. We summarize the advances made with respect to developing RNA regulatory tools, as well as opportunities for the future. PMID:23731364

  3. Scaffolder - software for manual genome scaffolding

    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.

  4. Efficacy of whey protein gel networks as potential viability-enhancing scaffolds for cell immobilization of Lactobacillus rhamnosus GG.

    Doherty, S B; Gee, V L; Ross, R P; Stanton, C; Fitzgerald, G F; Brodkorb, A

    2010-03-01

    This study investigated cell immobilization of Lactobacillus rhamnosus GG in three separate protein products: native, denatured and hydrolysed whey protein isolate (WPI). Treatments were assessed for their ability to enhance probiotic survival during storage, heat stress and ex vivo gastric incubation. Spatial distribution of probiotic cells within immobilized treatments was evaluated by atomic force and confocal scanning laser microscopy, while cell viability was enumerated by plate count and flow cytometry (FACS). Microscopic analysis of denatured treatments revealed an oasis of immobilized cells, phase-separated from the surrounding protein matrix; an environmental characteristic analogous to hydrolysed networks. Cell immobilization in hydrolysed and denatured WPI enhanced survival by 6.1+/-0.1 and 5.8+/-0.1 log10 cycles, respectively, following 14 day storage at 37 degrees C and both treatments generated thermal protection at 57 degrees C (7.3+/-0.1 and 6.5+/-0.1 log(10) cfu/ml). Furthermore, denatured WPI enhanced probiotic protection (8.9+/-0.2 log(10) cfu/ml) following 3h gastric incubation at 37 degrees C. In conclusion, hydrolysed or denatured WPI were the most suitable matrices for cell immobilization, while native protein provided the weakest safeguard against thermal and acid stress, thus making it possible to envision whey protein gel networks as protective substrates for cell immobilization applications. PMID:20045713

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

    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.

  6. Enhanced healing of rat calvarial defects with sulfated chitosan-coated calcium-deficient hydroxyapatite/bone morphogenetic protein 2 scaffolds.

    Zhao, Jun; Shen, Gang; Liu, Changsheng; Wang, Shaoyi; Zhang, Wenjie; Zhang, Xiaochen; Zhang, Xiuli; Ye, Dongxia; Wei, Jie; Zhang, Zhiyuan; Jiang, Xinquan

    2012-01-01

    Calcium phosphate cements (CPCs), which are widely used in bone regeneration, possess good biocompatibility and osteoconductivity and have been demonstrated to be candidate carriers for bone growth factors. However, limited release of growth factors from CPCs and slow degradation of the materials are not desirable for certain clinical applications. Previous studies have shown that calcium-deficient hydroxyapatite (CDHA) from CPCs presents more rapid degradation rate than CPCs. In this study, a hybrid growth factor delivery system was prepared by using bone morphogenetic protein 2 (BMP-2) loaded CDHA porous scaffold with sulfated chitosan (SCS) coating for improved release profile. We tested the BMP-2 release characteristic of CDHA/BMP-2/SCS composite in vitro and its ability to repair rat calvarial bone defects. A higher percentage of BMP-2 was released when sulfated chitosan coating was present compared with CDHA/BMP-2 group. Eight weeks postoperation, the repaired crania were evaluated by microcomputed tomography, sequential fluorescent labeling, histological analysis, and immunohistochemistry. CDHA/BMP-2/SCS group promoted the most extensive new bone formation than CDHA/BMP-2 and CDHA groups. Our observations suggest that sulfated chitosan coating could enhance the release profile of CDHA/BMP-2 composite in vitro and promote new bone formation in vivo. The hybrid CDHA/BMP-2/SCS system is a promising growth factor delivery strategy for bone regeneration. PMID:21830854

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

    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...

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

    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.

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

    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...

  10. 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.

    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.

  11. Fused imidazoles as potential chemical scaffolds for inhibition of heat shock protein 70 and induction of apoptosis. Synthesis and biological evaluation of phenanthro[9,10-d]imidazoles and imidazo[4,5-f][1,10]phenanthrolines.

    Patel, Alpa; Sharp, Swee Y; Hall, Katelan; Lewis, William; Stevens, Malcolm F G; Workman, Paul; Moody, Christopher J

    2016-04-28

    The imidazole ring is widespread in biologically active compounds, and hence imidazole-containing scaffolds are useful starting points for drug discovery programmes. We report the synthesis of a series of novel imidazole-containing compounds fused with either phenanthrene or phenanthroline, which show enhanced growth inhibitory potency against human colon, breast and melanoma cancer cell lines, as well as evidence of inhibition of the molecular chaperone heat shock protein 70 (Hsp70) pathway in cells, as shown by depletion of downstream oncogenic client proteins of the Hsp90 chaperone pathway, and induction of apoptosis. PMID:27035072

  12. 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

    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

  13. 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

    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

  14. Postsynaptic scaffolds for nicotinic receptors on neurons

    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.

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

    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.

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

    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.

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

    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

  18. Mandibular defect reconstruction using three-dimensional polycaprolactone scaffold in combination with platelet-rich plasma and recombinant human bone morphogenetic protein-2: de novo synthesis of bone in a single case.

    Schuckert, Karl-Heinz; Jopp, Stefan; Teoh, Swee-Hin

    2009-03-01

    This publication describes the clinical case of a 71-year-old female patient. Using polycaprolactone (PCL) scaffold, platelet-rich plasma (PRP) and recombinant human bone morphogenetic protein-2 (rhBMP-2), a critical-sized defect in the anterior mandible was regenerated using de novo-grown bone. A bacterial infection had caused a periimplantitis in two dental implants leading to a large destruction in the anterior mandible. Both implants were removed under antibiotic prophylaxis. A PCL scaffold was prepared especially for this clinical case. In a second procedure with antibiotic prophylaxis, the bony defect was reopened. The PCL scaffold was fitted and charged with PRP and rhBMP-2 (1.2 mg). After complication-free wound healing, the radiological control demonstrated de novo-grown bone in the anterior mandible 6 months postoperatively. Dental implants were inserted in a third operation. A bone biopsy of the newly grown bone, as well as of the bordering local bone, was taken and histologically examined. The bone samples were identical and presented vital laminar bone. PMID:18767969

  19. The protein scaffold of the lipocalin odorant-binding protein is suitable for the design of new biosensors for the detection of explosive components

    The detection of hazard exposure is a current priority, including the detection of traces of explosive molecules in different environments like luggage storage rooms and public places, and is becoming a major requirement for homeland security. In the present study we carried out a preliminary investigation on the binding capacities of four forms of the lipocalin odorant-binding protein (OBP) for the detection of explosive components such as diphenylamine, dimethyl-phthalate, resorcinol and dinitrotoluene. The experimental results, showing that OBP binds these compounds with affinity constants ranging between 80 nM and 10.6 mM, indicate that this protein can be used as a probe for the realization of a biosensor to sense explosive compounds

  20. 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

    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.

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

    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

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

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

    2009-01-01

    find known active CDK2 scaffolds in a database. Additionally, SHOP was used for suggesting new inhibitors of p38 MAP kinase. Four p38 complexes were used to perform six scaffold searches. Several new scaffolds were suggested, and the resulting compounds were successfully docked into the query proteins....

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

    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.

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

    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

  5. Hierarchical Scaffolding With Bambus

    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...

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

    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

  7. Grafting of protein-protein binding sites

    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.

  8. Attribution of the discrepancy between ELISA and LC-MS/MS assay results of a PEGylated scaffold protein in post-dose monkey plasma samples due to the presence of anti-drug antibodies.

    Wang, Shujie J; Wu, Steven T; Gokemeijer, Jochem; Fura, Aberra; Krishna, Murli; Morin, Paul; Chen, Guodong; Price, Karen; Wang-Iverson, David; Olah, Timothy; Weiner, Russell; Tymiak, Adrienne; Jemal, Mohammed

    2012-01-01

    High-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) methods were developed for the quantification of a PEGylated scaffold protein drug in monkey plasma samples. The LC-MS/MS method was based on the extraction of the therapeutic protein with a water-miscible organic solvent and the subsequent trypsin digestion of the extract followed by the detection of a surrogate peptide. The assay was linear over a range of 10-3,000 ng/mL. The ELISA method utilized a therapeutic target-binding format in which the recombinant target antigen was used to capture the drug in the sample, followed by detection with an anti-PEG monoclonal antibody. The assay range was 30-2,000 ng/mL. A correlation study between the two methods was performed by measuring the drug concentrations in plasma samples from a single-dose pharmacokinetic (PK) study in cynomolgus monkeys following a 5-mg/kg subcutaneous administration (n = 4). In the early time points of the PK profile, the drug concentrations obtained by the LC-MS/MS method agreed very well with those obtained by the ELISA method. However, at later time points, the drug concentrations measured by the LC-MS/MS method were consistently higher than those measured by the ELISA method. The PK parameters calculated based on the concentration data showed that the two methods gave equivalent peak exposure (C(max)) at 24-48 h. However, the LC-MS/MS results exhibited about 1.53-fold higher total exposure (AUC(tot)) than the ELISA results. The discrepancy between the LC-MS/MS and ELISA results was investigated by conducting immunogenicity testing, anti-drug antibody (ADA) epitope mapping, and Western blot analysis of the drug concentrations coupled with Protein G separation. The results demonstrated the presence of ADA specific to the engineered antigen-binding region of the scaffold protein drug that interfered with the ability of the drug to bind to the target antigen used in the ELISA

  9. Silks as scaffolds for skin reconstruction.

    Reimers, Kerstin; Liebsch, Christina; Radtke, Christine; Kuhbier, Jörn W; Vogt, Peter M

    2015-11-01

    In this short review, we describe the use of high molecular weight proteins produced in the glands of several arthropods-commonly called silks-for the purpose to enhance human skin wound healing. To this end an extensive literature search has been performed, the publications have been categorized concerning silk preparation and application and summarized accordingly: Scaffolds to promote wound healing were prepared by processing the silks in different ways including solubilization of the protein fibers followed by casting or electrospinning. The silk scaffolds were additionally modified by coating or blending with the intention of further functionalization. In several approaches, the scaffolds were also vitalized with skin cells or stem cells. In vitro and in vivo models were implied to test for safety and efficiency. We conclude that silk scaffolds are characterized by an advantageous biocompatibility as well as an impressive versatility rendering them ideally suited for application in wounds. Nevertheless, further investigation is needed to exploit the full capacity of silk in different wound models and to achieve clinical transfer in time. PMID:25995140

  10. Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds

    Zalatan, Jesse G.; Lee, Michael E; Almeida, Ricardo; Gilbert, Luke A.; Whitehead, Evan H.; La Russa, Marie; Tsai, Jordan C; Weissman, Jonathan S.; Dueber, John E.; Qi, Lei S.; Lim, Wendell A.

    2014-01-01

    Eukaryotic cells execute complex transcriptional programs in which specific loci throughout the genome are regulated in distinct ways by targeted regulatory assemblies. We have applied this principle to generate synthetic CRISPR-based transcriptional programs in yeast and human cells. By extending guide RNAs to include effector protein recruitment sites, we construct modular scaffold RNAs that encode both target locus and regulatory action. Sets of scaffold RNAs can be used to generate synthe...

  11. 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

    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.

  12. Semiotic Scaffolding in Mathematics

    Johansen, Mikkel Willum; Misfeldt, Morten

    2015-01-01

    representational forms influenced the development of the theory of exponentiation. For the third case, we analyze the connection between the development of commutative diagrams and the development of both algebraic topology and category theory. Our main conclusions are that semiotic scaffolding indeed plays a role...

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

    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.

  14. Synergistic effect of surface modification and scaffold design of bioplotted 3-D poly-ε-caprolactone scaffolds in osteogenic tissue engineering.

    Declercq, Heidi A; Desmet, Tim; Berneel, Elke E M; Dubruel, Peter; Cornelissen, Maria J

    2013-08-01

    The hydrophobic nature and the regular scaffold architecture of bioplotted poly(ε-caprolactone) (PCL) scaffolds present some hurdles for homogeneous tissue formation and differentiation. The current hypothesis is that a synergistic effect of applied surface modification and scaffold design enhances colonization and osteogenic differentiation. First, PCL scaffolds with a 0/90° lay-down pattern (0/90) were plotted and subjected to an oxygen plasma (O2) or multistep surface modification, including post-argon 2-amino-ethylmethacrylate grafting (AEMA), followed by immobilization of gelatin type B (gelB) and physisorption of fibronectin (gelB Fn). Secondly, scaffolds of different designs were plotted (0/90° shift (0/90 S), 0/45° and 0/90° with narrow pores (0/90 NP)) and subjected to the double protein coating. Preosteoblasts were cultured on the scaffolds and the seeding efficiency, colonization and differentiation were studied. The data revealed that a biomimetic surface modification improved colonization (gelB Fn>gelB>AEMA>O2). Compact scaffold architectures (0/90 NP, 0/45, 0/90 S>0/90) positively influenced the seeding efficiency and differentiation. Interestingly, the applied surface modification had a greater impact on colonization than the scaffold design. In conclusion, the combination of a double protein coating with a compact design enhances tissue formation in the plotted PCL scaffolds. PMID:23669624

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

    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.

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

    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.

  17. Bioresorbable Scaffolds for Atheroregression: Understanding of Transient Scaffolding.

    Kharlamov, Alexander N

    2016-01-01

    This review focuses on the clinical and biological features of the bioresorbable scaffolds in interventional cardiology highlighting scientific achievements and challenges of the transient scaffolding with Absorb BVS. Special attention is granted to the vascular biology pathways which, involved in the resorption of scaffold, artery remodeling and mechanisms of Glagovian atheroregression setting the stage for subsequent clinical applications. Twenty five years ago Glagov described the phenomenon of limited external elastic membrane enlargement in response to an increase in plaque burden. We believe this threshold becomes the target for development of strategies that reverse atherosclerosis, and particularly transient scaffolding has a potential to be a tool to ultimately conquer atherosclerosis. PMID:26818488

  18. 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).

    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

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

    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\\

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

    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.

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

    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.

  2. 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

    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.

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

    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

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

    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.

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

    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

  6. Microstructure and properties of nano-fibrous PCL-b-PLLA scaffolds for cartilage tissue engineering

    L He

    2009-10-01

    Full Text Available Nano-fibrous scaffolds which could potentially mimic the architecture of extracellular matrix (ECM have been considered a good candidate matrix for cell delivery in tissue engineering applications. In the present study, a semicrystalline diblock copolymer, poly(e-caprolactone-block-poly(L-lactide (PCL-b-PLLA, was synthesized and utilized to fabricate nano-fibrous scaffolds via a thermally induced phase separation process. Uniform nano-fibrous networks were created by quenching a PCL-b-PLLA/THF homogenous solution to -20ºC or below, followed by further gelation for 2 hours due to the presence of PLLA and PCL microcrystals. However, knot-like structures as well as continuously smooth pellicles appeared among the nano-fibrous network with increasing gelation temperature. DSC analysis indicated that the crystallization of PCL segments was interrupted by rigid PLLA segments, resulting in an amorphous phase at high gelation temperatures. Combining TIPS (thermally induced phase separation with salt-leaching methods, nano-fibrous architecture and interconnected pore structures (144±36 mm in diameter with a high porosity were created for in vitro culture of chondrocytes. Specific surface area and protein adsorption on the surface of the nano-fibrous scaffold were three times higher than on the surface of the solid-walled scaffold. Chondrocytes cultured on the nano-fibrous scaffold exhibited a spherical condrocyte-like phenotype and secreted more cartilage-like extracellular matrix (ECM than those cultured on the solid-walled scaffold. Moreover, the protein and DNA contents of cells cultured on the nano-fibrous scaffold were 1.2-1.4 times higher than those on the solid-walled scaffold. Higher expression levels of collagen II and aggrecan mRNA were induced on the nano-fibrous scaffold compared to on the solid-walled scaffold. These findings demonstrated that scaffolds with a nano-fibrous architecture could serve as superior scaffolds for cartilage tissue

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

    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

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

    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.

  9. Semiotic scaffolding of multicellularity

    Hoffmeyer, Jesper

    2015-01-01

    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 of...... 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 and...

  10. The scaffolding protein IQGAP1 co-localizes with actin at the cytoplasmic face of the nuclear envelope: implications for cytoskeletal regulation

    Johnson, Michael A.; Henderson, Beric R.

    2012-01-01

    IQGAP1 is an important cytoskeletal regulator, known to act at the plasma membrane to bundle and cap actin filaments, and to tether the cortical actin meshwork to microtubules via plus-end binding proteins. Here we describe the novel subcellular localization of IQGAP1 at the cytoplasmic face of the nuclear envelope, where it co-located with F-actin. The IQGAP1 and F-actin staining overlapped that of microtubules at the nuclear envelope, revealing a pattern strikingly similar to that observed ...

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

    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

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

    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

  13. Collagen a natural scaffold for biology and engineering

    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...

  14. Manufacture of a weakly denatured collagen fiber scaffold with excellent biocompatibility and space maintenance ability

    Although collagen scaffolds have been used for regenerative medicine, they have insufficient mechanical strength. We made a weakly denatured collagen fiber scaffold from a collagen fiber suspension (physiological pH 7.4) through a process of freeze drying and denaturation with heat under low pressure (1 × 10−1 Pa). Heat treatment formed cross-links between the collagen fibers, providing the scaffold with sufficient mechanical strength to maintain the space for tissue regeneration in vivo. The scaffold was embedded under the back skin of a rat, and biocompatibility and space maintenance ability were examined after 2 weeks. These were evaluated by using the ratio of foreign body giant cells and thickness of the residual scaffold. A weakly denatured collagen fiber scaffold with moderate biocompatibility and space maintenance ability was made by freezing at −10 °C, followed by denaturation at 140 °C for 6 h. In addition, the direction of the collagen fibers in the scaffold was adjusted by cooling the suspension only from the bottom of the container. This process increased the ratio of cells that infiltrated into the scaffold. A weakly denatured collagen fiber scaffold thus made can be used for tissue regeneration or delivery of cells or proteins to a target site. (paper)

  15. Approaching rational epitope vaccine design for hepatitis C virus with meta-server and multivalent scaffolding

    He, Linling; Cheng, Yushao; Kong, Leopold; Azadnia, Parisa; Giang, Erick; Kim, Justin; Wood, Malcolm R.; Wilson, Ian A.; Law, Mansun; Zhu, Jiang

    2015-08-01

    Development of a prophylactic vaccine against hepatitis C virus (HCV) has been hampered by the extraordinary viral diversity and the poor host immune response. Scaffolding, by grafting an epitope onto a heterologous protein scaffold, offers a possible solution to epitope vaccine design. In this study, we designed and characterized epitope vaccine antigens for the antigenic sites of HCV envelope glycoproteins E1 (residues 314-324) and E2 (residues 412-423), for which neutralizing antibody-bound structures are available. We first combined six structural alignment algorithms in a “scaffolding meta-server” to search for diverse scaffolds that can structurally accommodate the HCV epitopes. For each antigenic site, ten scaffolds were selected for computational design, and the resulting epitope scaffolds were analyzed using structure-scoring functions and molecular dynamics simulation. We experimentally confirmed that three E1 and five E2 epitope scaffolds bound to their respective neutralizing antibodies, but with different kinetics. We then investigated a “multivalent scaffolding” approach by displaying 24 copies of an epitope scaffold on a self-assembling nanoparticle, which markedly increased the avidity of antibody binding. Our study thus demonstrates the utility of a multi-scale scaffolding strategy in epitope vaccine design and provides promising HCV immunogens for further assessment in vivo.

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

    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

  17. Self-assembly model, hepatocytes attachment and inflammatory response for silk fibroin/chitosan scaffolds

    Silk fibroin is an attractive natural fibrous protein for biomedical application due to its good biocompatibility and high tensile strength. Silk fibroin is apt to form a sheet-like structure during the freeze-drying process, which is not suitable for the scaffold of tissue engineering. In our former study, the adding of chitosan promoted the self-assembly of silk fibroin/chitosan (SFCS) into a three-dimensional (3D) homogeneous porous structure. In this study, a model of the self-assembly is proposed; furthermore, hepatocytes attachment and inflammatory response for the SFCS scaffold were examined. The rigid chain of chitosan may be used as a template for β-sheet formation of silk fibroin, and this may break the sheet structure of the silk fibroin scaffold and promote the formation of a 3D porous structure of the SFCS scaffold. Compared with the polylactic glycolic acid scaffold, the SFCS scaffold further facilitates the attachment of hepatocytes. To investigate the inflammatory response, SFCS scaffolds were implanted into the greater omentum of rats. From the results of implantation, we could demonstrate in vivo that the implantation of SFCS scaffolds resulted in only slight inflammation. Keeping the good histocompatibility and combining the advantages of both fibroin and chitosan, the SFCS scaffold could be a prominent candidate for soft tissue engineering, for example, in the liver.

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

    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

  19. Three-dimensional polycaprolactone scaffold via needleless electrospinning promotes cell proliferation and infiltration.

    Li, Dawei; Wu, Tong; He, Nanfei; Wang, Jing; Chen, Weiming; He, Liping; Huang, Chen; Ei-Hamshary, Hany A; Al-Deyab, Salem S; Ke, Qinfei; Mo, Xiumei

    2014-09-01

    Electrospinning has been widely used in fabrication of tissue engineering scaffolds. Currently, most of the electrospun nanofibers performed like a conventional two-dimensional (2D) membrane, which hindered their further applications. Moreover, the low production rate of the traditional needle-electrospinning (NE) also limited the commercialization. In this article, disc-electrospinning (DE) was utilized to fabricate a three-dimensional (3D) scaffold consisting of porous macro/nanoscale fibers. The morphology of the porous structure was investigated by scanning electron microscopy images, which showed irregular pores of nanoscale spreading on the surface of DE polycaprolactone (PCL) fibers. Protein adsorption assessment illustrated the porous structure could significantly enhance proteins pickup, which was 55% higher than that of solid fiber scaffolds. Fibroblasts were cultured on the scaffold. The results demonstrated that DE fiber scaffold could enhance initial cell attachment. In the 7 days of culture, fibroblasts grew faster on DE fiber scaffold in comparison with solid fiber, solvent cast (SC) film and TCP. Fibroblasts on DE fibers showed a stretched shape and integrated with the porous surface tightly. Cells were also found to migrate into the DE scaffold up to 800μm. Results supported the use of DE PCL fibers as a 3D tissue engineering scaffold in soft tissue regeneration. PMID:24996758

  20. Evolutionary and functional diversity of coronin proteins.

    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

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

    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. PMID:27018773

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

    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.

  3. Studies on the mitotic chromosome scaffold of Allium sativum

    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.

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

    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.

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

    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.

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

    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.

  7. Composite Scaffolds for Bone Tissue Engineering

    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.

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

    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

  9. Scaffolding in Assisted Instruction

    2007-01-01

    Full Text Available On-The-Job Training, developed as direct instruction, is one of the earliest forms of training. This method is still widely in use today because it requires only a person who knows how to do the task, and the tools the person uses to do the task. This paper is intended to be a study of the methods used in education in Knowledge Society, with more specific aspects in training the trainers; as a result of this approach, it promotes scaffolding in assisted instruction as a reflection of the digital age for the learning process. Training the trainers in old environment with default techniques and designing the learning process in assisted instruction, as an application of the Vygotskian concept of the zone of proximal development (ZPD to the area of computer literacy for the younger users, generate diversity in educational communities and requires standards for technology infrastructure, standards for the content, developed as a concepts map, and applications for personalized in-struction, based on ZPD theory.

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

    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

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

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

    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

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

    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

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

    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.

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

    Sweet, Lauren; Kang, Yunqing; 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

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

    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.

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

    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.

  18. Engineering microporosity in bacterial cellulose scaffolds.

    Bäckdahl, Henrik; Esguerra, Maricris; Delbro, Dick; Risberg, Bo; Gatenholm, Paul

    2008-08-01

    The scaffold is an essential component in tissue engineering. A novel method to prepare three-dimensional (3D) nanofibril network scaffolds with controlled microporosity has been developed. By placing paraffin wax and starch particles of various sizes in a growing culture of Acetobacter xylinum, bacterial cellulose scaffolds of different morphologies and interconnectivity were prepared. Paraffin particles were incorporated throughout the scaffold, while starch particles were found only in the outermost area of the resulting scaffold. The porogens were successfully removed after culture with bacteria and no residues were detected with electron spectroscopy for chemical analysis (ESCA) or Fourier transform infra-red spectroscopy (FT-IR). Resulting scaffolds were seeded with smooth muscle cells (SMCs) and investigated using histology and organ bath techniques. SMC were selected as the cell type since the main purpose of the resulting scaffolds is for tissue engineered blood vessels. SMCs attached to and proliferated on and partly into the scaffolds. PMID:18615821

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

    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

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

    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

  1. Composite Scaffolds for Bone Tissue Engineering

    Min Wang

    2006-01-01

    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 po...

  2. MCR synthesis of a tetracyclic tetrazole scaffold

    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

  3. Rethinking Scaffolding in the Information Age

    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…

  4. Biomaterials & scaffolds for tissue engineering

    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. Functional stability of endothelial cells on a novel hybrid scaffold for vascular tissue engineering

    Pankajakshan, Divya; Krishnan, Lissy K [Thrombosis Research Unit, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojapura, Trivandrum 695 012 (India); Krishnan V, Kalliyana, E-mail: lissykk@sctimst.ac.i [Division of Polymer Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojapura, Trivandrum 695 012 (India)

    2010-12-15

    Porous and pliable conduits made of biodegradable polymeric scaffolds offer great potential for the development of blood vessel substitutes but they generally lack signals for cell proliferation, survival and maintenance of a normal phenotype. In this study we have prepared and evaluated porous poly({epsilon}-caprolactone) (PCL) integrated with fibrin composite (FC) to get a biomimetic hybrid scaffold (FC PCL) with the biological properties of fibrin, fibronectin (FN), gelatin, growth factors and glycosaminoglycans. Reduced platelet adhesion on a human umbilical vein endothelial cell-seeded hybrid scaffold as compared to bare PCL or FC PCL was observed, which suggests the non-thrombogenic nature of the tissue-engineered scaffold. Analysis of real-time polymerase chain reaction (RT-PCR) after 5 days of endothelial cell (EC) culture on a hybrid scaffold indicated that the prothrombotic von Willebrand factor and plasminogen activator inhibitor (PAI) were quiescent and stable. Meanwhile, dynamic expressions of tissue plasminogen activator (tPA) and endothelial nitric oxide synthase indicated the desired cell phenotype on the scaffold. On the hybrid scaffold, shear stress could induce enhanced nitric oxide release, which implicates vaso-responsiveness of EC grown on the tissue-engineered construct. Significant upregulation of mRNA for extracellular matrix (ECM) proteins, collagen IV and elastin, in EC was detected by RT-PCR after growing them on the hybrid scaffold and FC-coated tissue culture polystyrene (FC TCPS) but not on FN-coated TCPS. The results indicate that the FC PCL hybrid scaffold can accomplish a remodeled ECM and non-thrombogenic EC phenotype, and can be further investigated as a scaffold for cardiovascular tissue engineering. (communication)

  6. Functional stability of endothelial cells on a novel hybrid scaffold for vascular tissue engineering

    Porous and pliable conduits made of biodegradable polymeric scaffolds offer great potential for the development of blood vessel substitutes but they generally lack signals for cell proliferation, survival and maintenance of a normal phenotype. In this study we have prepared and evaluated porous poly(ε-caprolactone) (PCL) integrated with fibrin composite (FC) to get a biomimetic hybrid scaffold (FC PCL) with the biological properties of fibrin, fibronectin (FN), gelatin, growth factors and glycosaminoglycans. Reduced platelet adhesion on a human umbilical vein endothelial cell-seeded hybrid scaffold as compared to bare PCL or FC PCL was observed, which suggests the non-thrombogenic nature of the tissue-engineered scaffold. Analysis of real-time polymerase chain reaction (RT-PCR) after 5 days of endothelial cell (EC) culture on a hybrid scaffold indicated that the prothrombotic von Willebrand factor and plasminogen activator inhibitor (PAI) were quiescent and stable. Meanwhile, dynamic expressions of tissue plasminogen activator (tPA) and endothelial nitric oxide synthase indicated the desired cell phenotype on the scaffold. On the hybrid scaffold, shear stress could induce enhanced nitric oxide release, which implicates vaso-responsiveness of EC grown on the tissue-engineered construct. Significant upregulation of mRNA for extracellular matrix (ECM) proteins, collagen IV and elastin, in EC was detected by RT-PCR after growing them on the hybrid scaffold and FC-coated tissue culture polystyrene (FC TCPS) but not on FN-coated TCPS. The results indicate that the FC PCL hybrid scaffold can accomplish a remodeled ECM and non-thrombogenic EC phenotype, and can be further investigated as a scaffold for cardiovascular tissue engineering. (communication)

  7. In Vitro and In Vivo Studies of BMP-2-Loaded PCL–Gelatin–BCP Electrospun Scaffolds

    Kim, Bo-Ram; Nguyen, Thuy Ba Linh; Min, Young-Ki; Lee, Byong-Taek

    2014-01-01

    To confirm the effect of recombinant human bone morphogenetic protein-2 (BMP-2) for bone regeneration, BMP-2-loaded polycaprolactone (PCL)–gelatin (Gel)–biphasic calcium phosphate (BCP) fibrous scaffolds were fabricated using the electrospinning method. The electrospinning process to incorporate BCP nanoparticles into the PCL–Gel scaffolds yielded an extracellular matrix-like microstructure that was a hybrid system composed of nano- and micro-sized fibers. BMP-2 was homogeneously loaded on th...

  8. Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection

    Tipper, Donald J.; Szomolanyi-Tsuda, Eva

    2016-01-01

    Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolde...

  9. 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.

    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

  10. Strategic Scaffolding for Scientific Inquiry

    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,…

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

    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.

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

    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

  13. Electrospun Scaffolds for Corneal Tissue Engineering: A Review

    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.

  14. Polyurethane-based scaffolds for myocardial tissue engineering.

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

    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

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

    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.

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

    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.

  18. Effect of sterilization on structural and material properties of 3-D silk fibroin scaffolds.

    Hofmann, Sandra; Stok, Kathryn S; Kohler, Thomas; Meinel, Anne J; Müller, Ralph

    2014-01-01

    The development of porous scaffolds for tissue engineering applications requires the careful choice of properties, as these influence cell adhesion, proliferation and differentiation. Sterilization of scaffolds is a prerequisite for in vitro culture as well as for subsequent in vivo implantation. The variety of methods used to provide sterility is as diverse as the possible effects they can have on the structural and material properties of the three-dimensional (3-D) porous structure, especially in polymeric or proteinous scaffold materials. Silk fibroin (SF) has previously been demonstrated to offer exceptional benefits over conventional synthetic and natural biomaterials in generating scaffolds for tissue replacements. This study sought to determine the effect of sterilization methods, such as autoclaving, heat-, ethylene oxide-, ethanol- or antibiotic-antimycotic treatment, on porous 3-D SF scaffolds. In terms of scaffold morphology, topography, crystallinity and short-term cell viability, the different sterilization methods showed only few effects. Nevertheless, mechanical properties were significantly decreased by a factor of two by all methods except for dry autoclaving, which seemed not to affect mechanical properties compared to the native control group. These data suggest that SF scaffolds are in general highly resistant to various sterilization treatments. Nevertheless, care should be taken if initial mechanical properties are of interest. PMID:24013025

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

    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

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

    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.

  1. Iota-carrageenan/chitosan/gelatin scaffold for the osteogenic differentiation of adipose-derived MSCs in vitro.

    Li, Junjie; Yang, Boguang; Qian, Yufeng; Wang, Qiyu; Han, Ruijin; Hao, Tong; Shu, Yao; Zhang, Yabin; Yao, Fanglian; Wang, Changyong

    2015-10-01

    In this study, we have developed ι-carrageenan/chitosan/gelatin (CCG) scaffold containing multiple functional groups (-NH2 , -OH, -COOH, and -SO3 H) to resemble the native extracellular matrix (ECM), using the ion-shielding technology and ultrasonic dispersion method. Fourier transform infrared spectroscopy (FTIR) of the CCG scaffolds suggests that the formation of CCG network involves electrostatic interactions between ι-carrageenan (ι-CA) and chitosan/gelatin, and the covalent cross-linking among amino groups of chitosan and/or gelatin. Scanning electron microscopic (SEM) observation reveals that the porous structure of scaffolds can be modulated by the ratio of ι-CA to chitosan/gelatin. The swelling ratio of the hydrogels increases as the ι-CA contents increase. Using differential scanning calorimetry, we found that the double helix structure of ι-CA is only stabilized at low contents of ι-CA in the CCG scaffolds (e.g., 5 wt %). The scaffolds containing 5% ι-CA showed the best protein adsorption capacity (4.46 ± 0.63 μg protein/mg scaffold) and elastic modulus (5.37 ± 1.03 MPa). In addition, the CCG scaffolds exhibit excellent support for adipose-derived mesenchymal stem cells (ADMSCs) attachment and proliferation, and they can improve the osteogenic differentiation and neovascularization capacities of ADMSCs. Overall, we conclude that the CCG may represent an ideal scaffold material for bone tissue engineering. PMID:25449538

  2. The CNK2 Scaffold Interacts with Vilse/ARHGAP39 and Modulates Rac Cycling During Morphogenesis of Dendritic Spines

    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...

  3. 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

    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

  4. Fabrication and evaluation of a sustained-release chitosan-based scaffold embedded with PLGA microspheres

    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

  5. Fabrication and evaluation of a sustained-release chitosan-based scaffold embedded with PLGA microspheres

    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

  6. Construction of a 3D rGO-collagen hybrid scaffold for enhancement of the neural differentiation of mesenchymal stem cells

    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

  7. Ceramic Identity Contributes to Mechanical Properties and Osteoblast Behavior on Macroporous Composite Scaffolds

    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.

  8. Tropomyosin is localized in the nuclear matrix and chromosome scaffold of physarum polycephalum

    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.

  9. Biologically improved nanofibrous scaffolds for cardiac tissue engineering

    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

  10. Biologically improved nanofibrous scaffolds for cardiac tissue engineering

    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

  11. Molecular Recognition within Synaptic Scaffolds

    Erlendsson, Simon

    function. At the molecular level PICK1 contains both a BAR and a PDZ domain making it quite unique. Especially the specificity and promiscuity of the PICK1 PDZ domain seems to be more complicated than normally seen for PDZ domains. Also, the ability of PICK1 to form dimeric structures via its central BAR...... by the spatial architecture of the synapse itself. In this thesis, the molecular scaffolding mechanisms of PICK1 have been investigated in both isolated and near native conditions. Our findings have significantly benefitted the general understanding of how PICK1 and PDZ domain scaffolding works. In the first...... later in evolution to accommodate increasingly diverse PDZ domain ligands. Our findings provide basis for development of new and more specific peptide inhibitors. In the second study, we utilized SAXS, NMR spectroscopy, MD simulations and various other biochemical methods, to construct a full...

  12. Construction of a 3D rGO-collagen hybrid scaffold for enhancement of the neural differentiation of mesenchymal stem cells.

    Guo, Weibo; Wang, Shu; Yu, Xin; Qiu, Jichuan; Li, Jianhua; Tang, Wei; Li, Zhou; Mou, Xiaoning; Liu, Hong; Wang, Zhonglin

    2016-01-21

    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. PMID:26750302

  13. Effect of low-temperature ethylene oxide and electron beam sterilization on the in vitro and in vivo function of reconstituted extracellular matrix-derived scaffolds.

    Proffen, Benedikt L; Perrone, Gabriel S; Fleming, Braden C; Sieker, Jakob T; Kramer, Joshua; Hawes, Michael L; Murray, Martha M

    2015-10-01

    Reconstituted extracellular matrix (ECM)-derived scaffolds are commonly utilized in preclinical tissue engineering studies as delivery vehicles for cells and growth factors. Translation into clinical use requires identifying a sterilization method that effectively removes bacteria but does not harm scaffold function. To determine effectiveness of sterilization and impact on ECM scaffold integrity and function, low-temperature ethylene oxide and 15 kGy electron beam irradiation techniques were evaluated. Scaffold sterility was assessed in accordance to United States Pharmacopeia Chapter 71. Scaffold matrix degradation was determined in vitro using enzymatic resistance tests and gel electrophoresis. Scaffold mechanics including elastic modulus, yield stress and collapse modulus were tested. Lastly, 14 Yorkshire pigs underwent ACL transection and bio-enhanced ACL repair using sterilized scaffolds. Histologic response of ligament, synovium, and lymph nodes was compared at 4, 6, and 8 weeks. Ethylene oxide as well as electron beam irradiation yielded sterile scaffolds. Scaffold resistance to enzymatic digestion and protein integrity slightly decreased after electron beam irradiation while ethylene oxide altered scaffold matrix. Scaffold elastic modulus and yield stress were increased after electron beam treatment, while collapse modulus was increased after ethylene oxide treatment. No significant changes in ACL dimensions, in vivo scaffold resorption rate, or histologic response of synovium, ligament, and lymph nodes with either terminal sterilization technique were detectable. In conclusion, this study identifies two methods to terminally sterilize an ECM scaffold. In vitro scaffold properties were slightly changed without significantly influencing the biologic responses of the surrounding tissues in vivo. This is a critical step toward translating new tissue engineering strategies to clinical trials. PMID:26088294

  14. Scaffolding With and Through Videos

    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 nee...... 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....

  15. Fabrication of multi-biofunctional gelatin-based electrospun fibrous scaffolds for enhancement of osteogenesis of mesenchymal stem cells.

    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

  16. Carbohydrate mimetics and scaffolds: sweet spots in medicinal chemistry.

    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

  17. Preparation of poly(ethylene glycol/polylactide hybrid fibrous scaffolds for bone tissue engineering

    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

  18. Mandibular Jaw Bone Regeneration Using Human Dental Cell-Seeded Tyrosine-Derived Polycarbonate Scaffolds.

    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

  19. Cell–scaffold interaction within engineered tissue

    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.

  20. In vitro performance of 13-93 bioactive glass fiber and trabecular scaffolds with MLO-A5 osteogenic cells.

    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

  1. RhBMP-2 microspheres-loaded chitosan/collagen scaffold enhanced osseointegration: an experiment in dog.

    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

  2. The effect of the fibre orientation of electrospun scaffolds on the matrix production of rabbit annulus fibrosus-derived stem cells

    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.

  3. Application of Wnt Pathway Inhibitor Delivering Scaffold for Inhibiting Fibrosis in Urethra Strictures: In Vitro and in Vivo Study

    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

  4. Cell alignment induced by anisotropic electrospun fibrous scaffolds alone has limited effect on cardiomyocyte maturation.

    Han, Jingjia; Wu, Qingling; Xia, Younan; Wagner, Mary B; Xu, Chunhui

    2016-05-01

    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. PMID:27131761

  5. Cell alignment induced by anisotropic electrospun fibrous scaffolds alone has limited effect on cardiomyocyte maturation

    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.

  6. Cell alignment induced by anisotropic electrospun fibrous scaffolds alone has limited effect on cardiomyocyte maturation

    Han, Jingjia; Wu, Qingling; Xia, Younan; Wagner, Mary B; Xu, Chunhui

    2016-01-01

    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. PMID:27131761

  7. SHOP: scaffold hopping by GRID-based similarity searches

    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...... scaffolds were in the 31 top-ranked scaffolds. SHOP also identified new scaffolds with substantially different chemotypes from the queries. Docking analysis indicated that the new scaffolds would have similar binding modes to those of the respective query scaffolds observed in X-ray structures. The...

  8. Application of Collagen Scaffold in Tissue Engineering: Recent Advances and New Perspectives

    Chanjuan Dong

    2016-02-01

    Full Text Available Collagen is the main structural protein of most hard and soft tissues in animals and the human body, which plays an important role in maintaining the biological and structural integrity of the extracellular matrix (ECM and provides physical support to tissues. Collagen can be extracted and purified from a variety of sources and offers low immunogenicity, a porous structure, good permeability, biocompatibility and biodegradability. Collagen scaffolds have been widely used in tissue engineering due to these excellent properties. However, the poor mechanical property of collagen scaffolds limits their applications to some extent. To overcome this shortcoming, collagen scaffolds can be cross-linked by chemical or physical methods or modified with natural/synthetic polymers or inorganic materials. Biochemical factors can also be introduced to the scaffold to further improve its biological activity. This review will summarize the structure and biological characteristics of collagen and introduce the preparation methods and modification strategies of collagen scaffolds. The typical application of a collagen scaffold in tissue engineering (including nerve, bone, cartilage, tendon, ligament, blood vessel and skin will be further provided. The prospects and challenges about their future research and application will also be pointed out.

  9. Mapping of the interaction domains of the Crimean-Congo hemorrhagic fever virus nucleocapsid protein.

    Levingston Macleod, Jesica M; Marmor, Hannah; García-Sastre, Adolfo; Frias-Staheli, Natalia

    2015-03-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the genus Nairovirus of the family Bunyaviridae, that can cause severe haemorrhagic fever in humans, with mortality rates above 30  %. CCHFV is the most widespread of the tick-borne human viruses and it is endemic in areas of central Asia, the Middle East, Africa and southern Europe. Its viral genome consists of three negative-sense RNA segments. The large segment (L) encodes a viral RNA-dependent RNA polymerase (L protein), the small segment (S) encodes the nucleocapsid protein (N protein) and the medium segment (M) encodes the envelope proteins. The N protein of bunyaviruses binds genomic RNA, forming the viral ribonucleoprotein (RNP) complex. The L protein interacts with these RNP structures, allowing the initiation of viral replication. The N protein also interacts with actin, although the regions and specific residues involved in these interactions have not yet been described. Here, by means of immunoprecipitation and immunofluorescence assays, we identified the regions within the CCHFV N protein implicated in homo-oligomerization and actin binding. We describe the interaction of the N protein with the CCHFV L protein, and identify the N- and C-terminal regions within the L protein that might be necessary for the formation of these N-L protein complexes. These results may guide the development of potent inhibitors of these complexes that could potentially block CCHFV replication. PMID:25389186

  10. Mapping of the interaction domains of the Crimean–Congo hemorrhagic fever virus nucleocapsid protein

    Macleod, Jesica M. Levingston; Marmor, Hannah; Frias-Staheli, Natalia

    2015-01-01

    Crimean–Congo hemorrhagic fever virus (CCHFV) is a member of the genus Nairovirus of the family Bunyaviridae, that can cause severe haemorrhagic fever in humans, with mortality rates above 30 %. CCHFV is the most widespread of the tick-borne human viruses and it is endemic in areas of central Asia, the Middle East, Africa and southern Europe. Its viral genome consists of three negative-sense RNA segments. The large segment (L) encodes a viral RNA-dependent RNA polymerase (L protein), the small segment (S) encodes the nucleocapsid protein (N protein) and the medium segment (M) encodes the envelope proteins. The N protein of bunyaviruses binds genomic RNA, forming the viral ribonucleoprotein (RNP) complex. The L protein interacts with these RNP structures, allowing the initiation of viral replication. The N protein also interacts with actin, although the regions and specific residues involved in these interactions have not yet been described. Here, by means of immunoprecipitation and immunofluorescence assays, we identified the regions within the CCHFV N protein implicated in homo-oligomerization and actin binding. We describe the interaction of the N protein with the CCHFV L protein, and identify the N- and C-terminal regions within the L protein that might be necessary for the formation of these N–L protein complexes. These results may guide the development of potent inhibitors of these complexes that could potentially block CCHFV replication. PMID:25389186

  11. Cell penetration to nanofibrous scaffolds

    Rampichová, Michala; Buzgo, Matej; Chvojka, J.; Prosecká, Eva; Kofroňová, Olga; Amler, Evžen

    2014-01-01

    Roč. 8, č. 1 (2014), s. 36-41. ISSN 1933-6918 Grant ostatní: GA UK(CZ) 384311; GA UK(CZ) 626012; GA UK(CZ) 270513; GA UK(CZ) 330611; GA UK(CZ) 648112; GA MZd(CZ) NT12156; GA MŠk(CZ) project IPv6 Institutional support: RVO:68378041 ; RVO:61388971 Keywords : fibrous scaffold * mesenchymal stem cell s * Forcespinning (R) Subject RIV: FP - Other Medical Disciplines Impact factor: 4.505, year: 2014

  12. A practice scaffolding interactive platform

    Bundsgaard, Jeppe

    2009-01-01

    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......, 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...... foundations, and gives an example of a PracSIP....

  13. Metacognitive scaffolding in an innovative learning arrangement

    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

  14. Lithographically defined 3-dimensional graphene scaffolds

    Burckel, D. Bruce; Xiao, Xiaoyin; Polsky, Ronen

    2015-09-01

    Interferometrically defined 3D photoresist scaffolds are formed through a series of three successive two-beam interference exposures, a post exposure bake and development. Heating the resist scaffold in a reducing atmosphere to > 1000 °C, results in the conversion of the resist structure into a carbon scaffold through pyrolysis, resulting in a 3D sp3- bonded glassy carbon scaffold which maintains the same in-plane morphology as the resist despite significant shrinkage. The carbon scaffolds are readily modified using a variety of deposition methods such as electrochemical, sputtering and CVD/ALD. Remarkably, sputtering metal into scaffolds with ~ 5 unit cells tall results in conformal coating of the scaffold with the metal. When the metal is a transition metal such as nickel, the scaffold can be re-annealed, during which time the carbon diffuses through the nickel, emerging on the exterior of the nickel as sp2-bonded carbon, termed 3D graphene. This paper details the fabrication, characterization and some potential applications for these structures.

  15. The Effect of Sterilization Methods on the Physical Properties of Silk Sericin Scaffolds

    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)...

  16. Fabrication and evaluation of a sustained-release chitosan-based scaffold embedded with PLGA microspheres.

    Song, Kedong; Liu, Yingchao; Macedo, Hugo M; Jiang, Lili; Li, Chao; Mei, Guanyu; Liu, Tianqing

    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(-2) mg/mL. This novel chitosan-based scaffold embedded with PLGA microspheres has proven to be a promising technique

  17. Proton transfer in organic scaffolds

    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.

  18. Dual stimuli responsive poly(N-isopropylacrylamide) coated chitosan scaffolds for controlled release prepared from a non residue technology

    Temtem, M.; Barroso, T.; Casimiro, T.; Mano, J. F.; Aguiar Ricardo, Ana

    2012-01-01

    biomaterials for delivery of bioactive substances that can be controlled by external stimuli. Herein we report the production of smart partially biodegradable scaffolds that exhibit pH- and temperatureresponsive behavior and their effects on the release of a model protein and a drug of low molecular weight. Chitosan (CHT) scaffolds (pH sensitive) were coated/impregnated with a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm), by in situ synthesis of PNIPAAm withi...

  19. Cdk2 Silencing via a DNA/PCL Electrospun Scaffold Suppresses Proliferation and Increases Death of Breast Cancer Cells

    Achille, Clément; Sundaresh, Sowmya; Chu, Benjamin; Hadjiargyrou, Michael

    2012-01-01

    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 polycaprolac...

  20. Potential of a PLA-PEO-PLA-based scaffold for skin tissue engineering: in vitro evaluation.

    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

  1. The Genetic Engineering of Motor Proteins

    Hartz, Rachael M.

    Molecular motors are a remarkable feature within living organisms that are responsible for directional mechanical motion, which is driven by adenosine triphosphate (ATP) hydrolysis. Actin-binding molecular motors are of specific interest in the field of nanotechnology as filamentous actin is capable of carrying cargo, such as quantum dots, while it is translocated along a motor coated surface. The binding regions of motor proteins, which are known to interact with actin, such as Myosin, have been thoroughly examined and identified. Rapid genetic engineering of the ATP-hydrolyzing enzyme, adenosine kinase, to incorporate these binding regions is possible through the use of site- directed mutagenesis. The sequences, which were mutated into the ADK wt gene, were incorporated in an unstructured loop region. During the phosphate transfer, the mutants switch between open and closed conformational states. The binding affinity of the sequences to the actin is altered during this conformational switch, thus causing the motor to move along actin filament. The ADK mutants and their interaction with filamentous actin was monitored by an in vitro motility assay. Two different mutants of ADK were found to have retained enzymatic functionality after the mutagenesis as well as function as actin-based motor proteins.

  2. SYNTHETIC HYDROGELS AS SCAFFOLDS FOR MANIPULATING ENDOTHELIUM CELL BEHAVIORS

    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

  3. Scaffolding Strategies in Electronic Performance Support Systems: Types and Challenges

    Cagiltay, Kursat

    2006-01-01

    In the study described in this paper, the major components of an electronic performance support system are described and the use of scaffolding techniques within such electronic environments is explored. Four different types of scaffolding are discussed: "conceptual" (supportive) scaffolding, "metacognitive" (reflective) scaffolding, "procedural"…

  4. Titanate nanotube coatings on biodegradable photopolymer scaffolds

    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

  5. Titanate nanotube coatings on biodegradable photopolymer scaffolds

    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.

  6. Tolerogenic responses of CD206+, CD83+, FOXP3+, and CTLA-4 to sericin/polyvinyl alcohol/glycerin scaffolds relevant to IL-33 and HSP60 activity.

    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

  7. Computational Exploration of Molecular Scaffolds in Medicinal Chemistry.

    Hu, Ye; Stumpfe, Dagmar; Bajorath, Jürgen

    2016-05-12

    The scaffold concept is widely applied in medicinal chemistry. Scaffolds are mostly used to represent core structures of bioactive compounds. Although the scaffold concept has limitations and is often viewed differently from a chemical and computational perspective, it has provided a basis for systematic investigations of molecular cores and building blocks, going far beyond the consideration of individual compound series. Over the past 2 decades, alternative scaffold definitions and organization schemes have been introduced and scaffolds have been studied in a variety of ways and increasingly on a large scale. Major applications of the scaffold concept include the generation of molecular hierarchies, structural classification, association of scaffolds with biological activities, and activity prediction. This contribution discusses computational approaches for scaffold generation and analysis, with emphasis on recent developments impacting medicinal chemistry. A variety of scaffold-based studies are discussed, and a perspective on scaffold methods is provided. PMID:26840095

  8. Electrospinning Auricular Shaped Scaffolds for Tissue Engineering

    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...

  9. Ionic solutes impact collagen scaffold bioactivity.

    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

  10. Repair of osteochondral defects with adipose stem cells and a dual growth factor-releasing scaffold in rabbits.

    Im, Gun-Il; Lee, Jin Ho

    2010-02-01

    The purpose of this work was to evaluate the in vivo effectiveness of a TGF-beta(2) and bone morphogenetic protein (BMP)-7-immobilized porous polycaprolactone (PCL)/F127 scaffold to enhance the healing of cartilage defect. An osteochondral defect was created on the patellar groove of the right distal femur of 12 rabbits and managed by one of the following methods: filling it with the scaffold only (Group I); the scaffold seeded with adipose stem cells (ASCs) (Group II); a TGF-beta(2) and BMP-7-immobilized scaffold (Group III); and a TGF-beta(2) and BMP-7-immobilized scaffold seeded with ASCs (Group IV). Each group had three rabbits. Nine weeks after the implantation, the implanted scaffolds were filled with yellowish, dense tissue, and had distinct margins with adjacent normal cartilage. The histological findings showed infiltration of foreign-body giant cells and blood vessel, more prominently in Groups III and IV. The presence of growth factor significantly increased the ICRS Macroscopic Score (p = 0.045) while the presence of ASC did not. The ICRS Visual Histological Score was not significantly affected by the presence of either growth factors or ASCs, showing similar values in all groups. In conclusion, the use of TGF-beta(2) and BMP-7-immobilized PCL/F127 scaffolds improved gross appearances of the osteochondral defects while not actually leading to better histological results and induced a greater degree of foreign body reaction. PMID:19957354

  11. Dermal fibroblast infiltration of poly(ε-caprolactone) scaffolds fabricated by melt electrospinning in a direct writing mode

    Melt electrospinning in a direct writing mode is a recent additive manufacturing approach to fabricate porous scaffolds for tissue engineering applications. In this study, we describe porous and cell-invasive poly (ε-caprolactone) scaffolds fabricated by combining melt electrospinning and a programmable x–y stage. Fibers were 7.5 ± 1.6 µm in diameter and separated by interfiber distances ranging from 8 to 133 µm, with an average of 46 ± 22 µm. Micro-computed tomography revealed that the resulting scaffolds had a highly porous (87%), three-dimensional structure. Due to the high porosity and interconnectivity of the scaffolds, a top-seeding method was adequate to achieve fibroblast penetration, with cells present throughout and underneath the scaffold. This was confirmed histologically, whereby a 3D fibroblast-scaffold construct with full cellular penetration was produced after 14 days in vitro. Immunohistochemistry was used to confirm the presence and even distribution of the key dermal extracellular matrix proteins, collagen type I and fibronectin. These results show that melt electrospinning in a direct writing mode can produce cell invasive scaffolds, using simple top-seeding approaches. (paper)

  12. A Simple Approach for an Eggshell-Based 3D-Printed Osteoinductive Multiphasic Calcium Phosphate Scaffold.

    Dadhich, Prabhash; Das, Bodhisatwa; Pal, Pallabi; Srivas, Pavan K; Dutta, Joy; Ray, Sabyasachi; Dhara, Santanu

    2016-05-18

    Natural origin bioceramics are widely used for bone grafts. In the present study, an eggshell-derived bioceramic scaffold is fabricated by 3D printing as a potential bone-graft analogue. The eggshell, a biological waste material, was mixed with a specific ratio of phosphoric acid and chitosan to form a precursor toward the fabrication of an osteoinductive multiphasic calcium phosphate scaffold via a coagulation-assisted extrusion and sintering for a multiscalar hierarchical porous structure with improved mechanical properties. Physicochemical characterization of the formed scaffolds was carried out for phase analysis, surface morphology, and mechanical properties. A similar scaffold was prepared using a chemically synthesized calcium phosphate powder that was compared with the natural origin one. The higher surface area associated with the interconnected porosity along with multiple phases of the natural origin scaffold facilitated higher cell adhesion and proliferation compared to the chemically synthesized one. Further, the natural origin scaffold displayed relatively higher cell differentiation activity, as is evident by protein and gene expression studies. On subcutaneous implantation for 30 days, promising vascular tissue in-growth was observed, circumventing a major foreign body response. Collagen-rich vascular extracellular matrix deposition and osteocalcin secretion indicated bonelike tissue formation. Finally, the eggshell-derived multiphasic calcium phosphate scaffold displayed improvement in the mechanical properties with higher porosity and osteoinductivity compared to the chemically derived apatite and unveiled a new paradigm for utilization of biological wastes in bone-graft application. PMID:26853051

  13. S/MARt DB: a database on scaffold/matrix attached regions

    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...

  14. Biological functionality of extracellular matrix-ornamented three-dimensional printed hydroxyapatite scaffolds.

    Kumar, A; Nune, K C; Misra, R D K

    2016-06-01

    Three-dimensional (3D) printing is considered an ideally suitable method to fabricate patient specific implantable devices. The approach enabled to produce a porous scaffold with tailored physical, mechanical, and biological properties because of the flexibility to tune the scaffold architecture. The objective of the study described was to elucidate the determining role of cell-laid extracellular matrix (ECM) in impacting biological response. In this regard, to mimic the natural ECM environment or the attributes of the native tissue, a natural ECM analogue surface was produced on the 3D printed and sintered hydroxyapatite (HA) scaffold surface by the mineralized ECM of the osteoblast. This involved the growth of osteoblast on 3D printed scaffolds, followed by differentiation to deposit the mineralized ECM on the biomaterial surface. The cells were removed from the mineralized matrix using freeze-thaw cycles to obtain a decellularized extracellular matrix (dECM) on the biomaterial surface. Subsequently, seeding of osteoblast on dECM-ornamented HA scaffolds led to 3D growth with enhanced expression of prominent proteins, actin and vinculin. Based on preliminary observations of present study, it was underscored that HA scaffolds-ornamented with dECM provided an optimized microenvironment conducive to the growth of 3D structural tissue and favorably promoted biological functionality because of the availability of an environment that promoted cell-cell and cell-scaffold interaction. The primary advantage of dECM is that it enabled constructive remodeling and promoted the formation of tissue in lieu of less functional tissue. The study opens-up a new path for printing of 3D structures suitable to treat segmental bone defects. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1343-1351, 2016. PMID:26799466

  15. In vivo xenogeneic scaffold fate is determined by residual antigenicity and extracellular matrix preservation.

    Wong, Maelene L; Wong, Janelle L; Vapniarsky, Natalia; Griffiths, Leigh G

    2016-06-01

    The immunological potential of animal-derived tissues and organs is the critical hurdle to increasing their clinical implementation. Glutaraldehyde-fixation cross-links proteins in xenogeneic tissues (e.g., bovine pericardium) to delay immune rejection, but also compromises the regenerative potential of the resultant biomaterial. Unfixed xenogeneic biomaterials in which xenoantigenicity has been ameliorated and native extracellular matrix (ECM) architecture has been maintained have the potential to overcome limitations of current clinically utilized glutaraldehyde-fixed biomaterials. The objective of this work was to determine how residual antigenicity and ECM architecture preservation modulate recipient immune and regenerative responses towards unfixed bovine pericardium (BP) ECM scaffolds. Disruption of ECM architecture during scaffold generation, with either SDS-decellularization or glutaraldehyde-fixation, stimulated recipient foreign body response and resultant fibrotic encapsulation following leporine subpannicular implantation. Conversely, BP scaffolds subjected to stepwise removal of hydrophilic and lipophilic antigens using amidosulfobetaine-14 (ASB-14) maintained native ECM architecture and thereby avoided fibrotic encapsulation. Removal of hydrophilic and lipophilic antigens significantly decreased local and systemic graft-specific, adaptive immune responses and subsequent calcification of BP scaffolds compared to scaffolds undergoing hydrophile removal only. Critically, removal of antigenic components and preservation of ECM architecture with ASB-14 promoted full-thickness recipient non-immune cellular repopulation of the BP scaffold. Further, unlike clinically utilized fixed BP, ASB-14-treated scaffolds fostered rapid intimal and medial vessel wall regeneration in a porcine carotid patch angioplasty model. This work highlights the importance of residual antigenicity and ECM architecture preservation in modulating recipient immune and regenerative

  16. Sequence and comparative genomic analysis of actin-related proteins.

    Muller, Jean; Oma, Yukako; Vallar, Laurent; Friederich, Evelyne; Poch, Olivier; Winsor, Barbara

    2005-12-01

    Actin-related proteins (ARPs) are key players in cytoskeleton activities and nuclear functions. Two complexes, ARP2/3 and ARP1/11, also known as dynactin, are implicated in actin dynamics and in microtubule-based trafficking, respectively. ARP4 to ARP9 are components of many chromatin-modulating complexes. Conventional actins and ARPs codefine a large family of homologous proteins, the actin superfamily, with a tertiary structure known as the actin fold. Because ARPs and actin share high sequence conservation, clear family definition requires distinct features to easily and systematically identify each subfamily. In this study we performed an in depth sequence and comparative genomic analysis of ARP subfamilies. A high-quality multiple alignment of approximately 700 complete protein sequences homologous to actin, including 148 ARP sequences, allowed us to extend the ARP classification to new organisms. Sequence alignments revealed conserved residues, motifs, and inserted sequence signatures to define each ARP subfamily. These discriminative characteristics allowed us to develop ARPAnno (http://bips.u-strasbg.fr/ARPAnno), a new web server dedicated to the annotation of ARP sequences. Analyses of sequence conservation among actins and ARPs highlight part of the actin fold and suggest interactions between ARPs and actin-binding proteins. Finally, analysis of ARP distribution across eukaryotic phyla emphasizes the central importance of nuclear ARPs, particularly the multifunctional ARP4. PMID:16195354

  17. Biomimetic nanoclay scaffolds for bone tissue engineering

    Ambre, Avinash Harishchandra

    Tissue engineering offers a significant potential alternative to conventional methods for rectifying tissue defects by evoking natural regeneration process via interactions between cells and 3D porous scaffolds. Imparting adequate mechanical properties to biodegradable scaffolds for bone tissue engineering is an important challenge and extends from molecular to macroscale. This work focuses on the use of sodium montmorillonite (Na-MMT) to design polymer composite scaffolds having enhanced mechanical properties along with multiple interdependent properties. Materials design beginning at the molecular level was used in which Na-MMT clay was modified with three different unnatural amino acids and further characterized using Fourier Transform Infrared (FTIR) spectroscopy, X-ray diffraction (XRD). Based on improved bicompatibility with human osteoblasts (bone cells) and intermediate increase in d-spacing of MMT clay (shown by XRD), 5-aminovaleric acid modified clay was further used to prepare biopolymer (chitosan-polygalacturonic acid complex) scaffolds. Osteoblast proliferation in biopolymer scaffolds containing 5-aminovaleric acid modified clay was similar to biopolymer scaffolds containing hydroxyapatite (HAP). A novel process based on biomineralization in bone was designed to prepare 5-aminovaleric acid modified clay capable of imparting multiple properties to the scaffolds. Bone-like apatite was mineralized in modified clay and a novel nanoclay-HAP hybrid (in situ HAPclay) was obtained. FTIR spectroscopy indicated a molecular level organic-inorganic association between the intercalated 5-aminovaleric acid and mineralized HAP. Osteoblasts formed clusters on biopolymer composite films prepared with different weight percent compositions of in situ HAPclay. Human MSCs formed mineralized nodules on composite films and mineralized extracellular matrix (ECM) in composite scaffolds without the use of osteogenic supplements. Polycaprolactone (PCL), a synthetic polymer, was

  18. Segmental bone regeneration using rhBMP-2-loaded collagen/chitosan microspheres composite scaffold in a rabbit model

    The reconstruction of segmental bone defects remains an urgent problem in the orthopaedic field, and bone morphogenetic protein-2 (BMP-2) is known for its potent osteoinductive properties in bone regeneration. In this study, chitosan microspheres (CMs) were prepared and combined with absorbable collagen sponge to maintain controlled-release recombinant human bone morphogenetic protein-2 (rhBMP-2). The rhBMP-2-loaded composite scaffolds were implanted into 15 mm radius defects of rabbits and the bone-repair ability was evaluated systematically. CMs were spherical in shape and had a polyporous surface, according to SEM images. The complex scaffold exhibited an ideal releasing profile in vitro. The micro-computed tomographic analysis revealed that the rhBMP-2-loaded composite scaffold not only bridged the defects as early as 4 weeks, but also healed the defects and presented recanalization of the bone-marrow cavity at 12 weeks. These results were confirmed by x-ray. When compared with other control groups, the composite scaffold group remarkably enhanced new bone formation and mechanical properties, as evidenced by bone mineral content evaluation, histological observations and biomechanical testing. Moreover, the biocompatibility and appropriate degradation of the composite scaffold could be obtained. All of these results clearly demonstrated that the composite scaffold is a promising carrier of BMP-2 for the treatment of segmental bone defects. (paper)

  19. Magnetic nanocomposite scaffolds combined with static magnetic field in the stimulation of osteoblastic differentiation and bone formation.

    Yun, Hyung-Mun; Ahn, Su-Jin; Park, Kyung-Ran; Kim, Mi-Joo; Kim, Jung-Ju; Jin, Guang-Zhen; Kim, Hae-Won; Kim, Eun-Cheol

    2016-04-01

    Magnetism has recently been implicated to play significant roles in the regulation of cell responses. Allowing cells to experience a magnetic field applied externally or scaffolding them in a material with intrinsic magnetic properties has been a possible way of utilizing magnetism. Here we aim to investigate the combined effects of the external static magnetic field (SMF) with magnetic nanocomposite scaffold made of polycaprolactone/magnetic nanoparticles on the osteoblastic functions and bone formation. The SMF synergized with the magnetic scaffolds in the osteoblastic differentiation of primary mouse calvarium osteoblasts, including the expression of bone-associated genes (Runx2 and Osterix) and alkaline phosphatase activity. The synergism was demonstrated in the activation of integrin signaling pathways, such as focal adhesion kinase, paxillin, RhoA, mitogen-activated protein kinase, and nuclear factor-kappaB, as well as in the up-regulation of bone morphogenetic protein-2 and phosphorylation of Smad1/5/8. Furthermore, the SMF/magnetic scaffold-stimulated osteoblasts promoted the angiogenic responses of endothelial cells, including the expression of vascular endothelial growth factor and angiogenin-1 genes and the formation of capillary tubes. When the magnetic scaffolds were implanted in mouse calvarium defects, the application of SMF significantly enhanced the new bone formation at 6 weeks, as revealed by the histological and micro-computed tomographic analyses. Current findings suggest that the combinatory application of external (SMF) and internal (scaffold) magnetism can be a promising tool to regenerative engineering of bone. PMID:26854394

  20. Composite Scaffolds for Cartilage Tissue Engineering

    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 function 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 cruc...

  1. Macroporous nanowire nanoelectronic scaffolds for synthetic tissues

    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...

  2. Effects of Therapeutic Radiation on Polymeric Scaffolds

    Cooke, Shelley Lynn

    2014-01-01

    High levels of ionizing radiation are known to cause degradation and/or cross-linking in polymers. Lower levels of ionizing radiation, such as x-rays, are commonly used in the treatment of cancers. Material characterization has not been fully explored for polymeric materials exposed to therapeutic radiation levels. This study investigated the effects of therapeutic radiation on three porous scaffolds: polycaprolactone (PCL), polyurethane (PU) and gelatin. Porous scaffolds were fabricate...

  3. The influence of biological motifs and dynamic mechanical stimulation in hydrogel scaffold systems on the phenotype of chondrocytes.

    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

  4. Chitosan-collagen porous scaffold and bone marrow mesenchymal stem cell transplantation for ischemic stroke

    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.

  5. Nuclear scaffold attachment sites within ENCODE regions associate with actively transcribed genes.

    Mignon A Keaton

    Full Text Available The human genome must be packaged and organized in a functional manner for the regulation of DNA replication and transcription. The nuclear scaffold/matrix, consisting of structural and functional nuclear proteins, remains after extraction of nuclei and anchors loops of DNA. In the search for cis-elements functioning as chromatin domain boundaries, we identified 453 nuclear scaffold attachment sites purified by lithium-3,5-iodosalicylate extraction of HeLa nuclei across 30 Mb of the human genome studied by the ENCODE pilot project. The scaffold attachment sites mapped predominately near expressed genes and localized near transcription start sites and the ends of genes but not to boundary elements. In addition, these regions were enriched for RNA polymerase II and transcription factor binding sites and were located in early replicating regions of the genome. We believe these sites correspond to genome-interactions mediated by transcription factors and transcriptional machinery immobilized on a nuclear substructure.

  6. Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

    Pullikuth, Ashok K.; Catling, Andrew D.

    2007-01-01

    Cell migration is critical for many physiological processes and is often misregulated in developmental disorders and pathological conditions including cancer and neurodegeneration. MAPK signaling and the Rho family of proteins are known regulators of cell migration that exert their influence on cellular cytoskeleton during cell adhesion and migration. Here we review data supporting the view that localized ERK signaling mediated through recently identified scaffold proteins may regulate cell m...

  7. Neocellularization and neovascularization of nanosized bioactive glass-coated decellularized trabecular bone scaffolds

    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.

  8. Osteogenic effect of controlled released rhBMP-2 in 3D printed porous hydroxyapatite scaffold.

    Wang, Hai; Wu, Gui; Zhang, Jing; Zhou, Kui; Yin, Bo; Su, Xinlin; Qiu, Guixing; Yang, Guang; Zhang, Xianglin; Zhou, Gang; Wu, Zhihong

    2016-05-01

    Recently, 3D printing as effective technology has been highlighted in the biomedical field. Previously, a porous hydroxyapatite (HA) scaffold with the biocompatibility and osteoconductivity has been developed by this method. However, its osteoinductivity is limited. The main purpose of this study was to improve it by the introduction of recombinant human bone morphogenetic protein-2 (rhBMP-2). This scaffold was developed by coating rhBMP-2-delivery microspheres with collagen. These synthesized scaffolds were characterized by Scanning Electron Microscopy (SEM), a delivery test in vitro, cell culture, and the experiments in vivo by a Micro-computed tomography (μCT) scan and histological evaluation of VanGieson staining. SEM results indicated the surface of scaffolds were more fit for the adhesion of hMSCs to coat collagen/rhBMP-2 microspheres. Biphasic release of rhBMP-2 could continue for more than 21 days, and keep its osteoinductivity to induce osteogenic differentiation of hMSCs in vitro. In addition, the experiments in vivo showed that the scaffold had a good bone regeneration capacity. These findings demonstrate that the HA/Collagen/Chitosan Microspheres system can simultaneously achieve localized long-term controlled release of rhBMP-2 and bone regeneration, which provides a promising route for improving the treatment of bone defects. PMID:26896655

  9. Nanocomposite scaffold fabrication by incorporating gold nanoparticles into biodegradable polymer matrix: Synthesis, characterization, and photothermal effect.

    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

  10. The differential effect of scaffold composition and architecture on chondrocyte response to mechanical stimulation.

    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

  11. Biomimetic Concealing of PLGA Microspheres in a 3D Scaffold to Prevent Macrophage Uptake.

    Minardi, Silvia; Corradetti, Bruna; Taraballi, Francesca; Sandri, Monica; Martinez, Jonathan O; Powell, Sebastian T; Tampieri, Anna; Weiner, Bradley K; Tasciotti, Ennio

    2016-03-01

    Scaffolds functionalized with delivery systems for the release of growth factors is a robust strategy to enhance tissue regeneration. However, after implantation, macrophages infiltrate the scaffold, eventually initiating the degradation and clearance of the delivery systems. Herein, it is hypothesized that fully embedding the poly(d,l-lactide-co-glycolide acid) microspheres (MS) in a highly structured collagen-based scaffold (concealing) can prevent their detection, preserving the integrity of the payload. Confocal laser microscopy reveals that non-embedded MS are easily internalized; when concealed, J774 and bone marrow-derived macrophages (BMDM) cannot detect them. This is further demonstrated by flow cytometry, as a tenfold decrease is found in the number of MS engulfed by the cells, suggesting that collagen can cloak the MS. This correlates with the amount of nitric oxide and tumor necrosis factor-α produced by J774 and BMDM in response to the concealed MS, comparable to that found for non-functionalized collagen scaffolds. Finally, the release kinetics of a reporter protein is preserved in the presence of macrophages, only when MS are concealed. The data provide detailed strategies for fabricating three dimensional (3D) biomimetic scaffolds able to conceal delivery systems and preserve the therapeutic molecules for release. PMID:26797709

  12. Identification of the secreted watery saliva proteins of the rice brown planthopper, Nilaparvata lugens (Stål) by transcriptome and Shotgun LC-MS/MS approach.

    Liu, Xiaoqing; Zhou, Hanyu; Zhao, Jing; Hua, Hongxia; He, Yueping

    2016-06-01

    The rice brown planthopper, Nilaparvata lugens (Stål), a major rice insect pest in Asia, is a vascular bundle-feeder that ejects gelling and watery saliva during the feeding process. Although major proteins in the salivary glands of N. lugens have been identified using 2D PAGE, very little is known about the secreted saliva of this insect. In this study, we identified the major proteins in the secreted watery saliva of N. lugens, via collecting from a sucrose diet that adult planthoppers had fed upon through a membrane of stretched parafilm, and using shotgun LC-MS/MS analysis with reference to transcriptome database of salivary glands of N. lugens. A total of 107 proteins were identified in the watery saliva of N. lugens, over 80% of which showed significant similarity to known proteins. When annotated by the Blast2GO suite, 29 proteins had catalytic activity and 24 proteins were binding proteins. The saliva enzymes included oxidoreductases, hydrolases, phosphatases, peptidases (proteases), kinases, transferases, and lyases. Binding proteins in N. lugens watery saliva included ATP-binding, lipophorin, calcium-binding, actin-binding and DNA-, RNA-, and chromatin-binding proteins. Other non-enzymatic proteins, such as ubiquitins, heat shock proteins, ribosomal proteins, and immunoglobulin proteins were also found in N. lugens watery saliva. This is the first study to identify, characterize and list the proteins in watery saliva of N. lugens, which might be involved in planthopper-rice interactions. PMID:27080912

  13. Bioimprinted Polymer Scaffolds for Selective Recognition of RGD Peptides

    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.

  14. Antioxidant effects of betulin on porcine chondrocyte behavior in gelatin/C6S/C4S/HA modified tricopolymer scaffold

    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.

  15. DNA-scaffolded nanoparticle structures

    Hoegberg, Bjoern; Olin, Haakan [Department of Engineering Physics and Mathematics, Mid Sweden University, SE-851 70 Sundsvall, Sweden (Sweden)

    2007-03-15

    DNA self-assembly is a powerful route to the production of very small, complex structures. When used in combination with nanoparticles it is likely to become a key technology in the production of nanoelectronics in the future. Previously, demonstrated nanoparticle assemblies have mainly been periodic and highly symmetric arrays, unsuited as building blocks for any complex circuits. With the invention of DNA-scaffolded origami reported earlier this year (Rothemund P W K 2006 Nature 440 (7082) 297-302), a new route to complex nanostructures using DNA has been opened. Here, we give a short review of the field and present the current status of our experiments were DNA origami is used in conjunction with nanoparticles. Gold nanoparticles are functionalized with thiolated single stranded DNA. Strands that are complementary to the gold particle strands can be positioned on the self-assembled DNA-structure in arbitrary patterns. This property should allow an accurate positioning of the particles by letting them hybridize on the lattice. We report on our recent experiments on this system and discuss open problems and future applications.

  16. Biodegradable Polymer-Based Scaffolds for Bone Tissue Engineering

    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.

  17. Elastomeric PGS scaffolds in arterial tissue engineering.

    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

  18. Incorporation of Fucoidan in β-Tricalcium phosphate-Chitosan scaffold prompts the differentiation of human bone marrow stromal cells into osteogenic lineage

    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

  19. The effect of 3D nanofibrous scaffolds on the chondrogenesis of induced pluripotent stem cells and their application in restoration of cartilage defects.

    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.

  20. Simvastatin Release from Poly(lactide-co-glycolide Membrane Scaffolds

    Julian B. Chaudhuri

    2010-12-01

    Full Text Available Statins, a group of potent inhibitors of 3-hydroxy-3-methylglutaryl Coenzyme A reductase in cholesterol biosynthesis pathway, have been widely used as a cholesterol lowering drug. The plieotrophic effect of statins on bone metabolism in long-term usage has been begun to be studied during recent years and several in vitro and in vivo studies have demonstrated the ability of statins to promote expression of bone morphogenetic protein-2 (BMP-2, inhibition of osteoclast differentiation and reduction of osteoporotic fractures risk. The high liver specificity and low oral bioavailability of statins, leading to poor peripheral distribution, are the main obstacles to benefit anabolic effects of hydrophobic statins on bone formation. Therefore, developing new administration roots for direct delivery to achieve optimum concentration in the bone microenvironment is of interest. Here we present and compare two approaches of combining statins with bone tissue engineering scaffolds. Simvastatin was combined with a poly(lactide-co-glycolide (PLGA membrane scaffold for diffusion-controlled release by dissolving simvastatin (dis-sim in the membrane casting dope, and for degradation-controlled release by covalently bonding saponifiedsimvastatin (sap-sim to the PLGA in the spinning dope. Rheological and concentration-dependent membrane morphology changes were observed with saponifiedsimvastatin, suggesting ester bond cleavage and covalent bonding of the statin to the PLGA, but not with dissolved simvastatin. Dissolved simvastatin membranes showed a logarithmic decay release profile while the saponifiedsimvastatin membranes showed constant release. It can be concluded that the covalent bonding of simvastatinto PLGA scaffolds is showing potential for use as a controlled releasescaffold for bone tissue engineering.

  1. TGF-β1-Enhanced TCP-Coated Sensate Scaffolds Can Detect Bone Bonding

    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...

  2. Sustained Release of Cx43 Antisense Oligodeoxynucleotides from Coated Collagen Scaffolds Promotes Wound Healing.

    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

  3. In vitro osteoclastogenesis on textile chitosan scaffold

    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.

  4. Heterogeneity of Scaffold Biomaterials in Tissue Engineering

    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.

  5. Fabrication and characterization of bioactive β-Ca{sub 2}SiO{sub 4}/PHBV composite scaffolds

    Wang, Nana [College of Materials Science and Engineering, Hunan University, Changsha 410082 (China); Zhou, Zheng, E-mail: zhouzheng@hnu.edu.cn [College of Biology, Hunan University, Changsha 410082 (China); Xia, Leilei; Dai, Yao [College of Materials Science and Engineering, Hunan University, Changsha 410082 (China); Liu, Hairong, E-mail: liuhairong@hnu.edu.cn [College of Materials Science and Engineering, Hunan University, Changsha 410082 (China)

    2013-05-01

    A key challenge in tissue engineering is the construction of a scaffold with adequate properties which would mimic extracellular matrix (ECM) to induce the cells' efficient adhesion, proliferation and proper differentiation. Novel β-Ca{sub 2}SiO{sub 4}/PHBV composite scaffolds were fabricated by integrating β-Ca{sub 2}SiO{sub 4} nanoparticles with PHBV backbone via a modified solvent casting-particulates leaching method, which generates interconnected porous structure and the high porosity, about 87%, of these scaffolds. Compared with PHBV scaffolds, β-Ca{sub 2}SiO{sub 4}/PHBV composite scaffolds facilitate the adhesion of human osteoblast-like MG-63 cells due to their increased hydrophilicity. The β-Ca{sub 2}SiO{sub 4}/PHBV composite scaffolds containing 2.5 or 5% β-Ca{sub 2}SiO{sub 4} nanoparticles significantly enhance the proliferation of MG-63 cells by stimulating the transcription of the transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7) genes. These scaffolds also induce early differentiation via promoting the transcription of alkaline phosphatase (ALP). The results suggest the potential application of β-Ca{sub 2}SiO{sub 4}/PHBV composites in bone tissue engineering. - Graphical abstract: The β-Ca{sub 2}SiO{sub 4}/PHBV composite scaffolds with multiple bioactivity. Panel a shows the ESEM micrographs of the synthesized β-Ca{sub 2}SiO{sub 4} nanoparticles; panels b and c are ESEM micrographs of MG-63 cell adhesion on β-Ca{sub 2}SiO{sub 4}/PHBV scaffolds for 4 h: (b) pure PHBV scaffold; (c) composite scaffold with 2.5 wt.% β-Ca{sub 2}SiO{sub 4} nanoparticles; panel d presents the influence of β-Ca{sub 2}SiO{sub 4}/PHBV scaffolds to the proliferation of MG-63 cells; panel e shows the β-Ca{sub 2}SiO{sub 4}/PHBV composite scaffolds that influence the transcription of genes listed. Highlights: ► β-Ca{sub 2}SiO{sub 4}/PHBV composite scaffold was fabricated by integrating β-Ca{sub 2}SiO{sub 4} nanoparticles. ►

  6. Three-Dimensional Polydopamine Functionalized Coiled Microfibrous Scaffolds Enhance Human Mesenchymal Stem Cells Colonization and Mild Myofibroblastic Differentiation.

    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

  7. Mechanical and in vitro performance of 13-93 bioactive glass scaffolds prepared by a polymer foam replication technique.

    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

  8. Scaffold-mediated BMP-2 minicircle DNA delivery accelerated bone repair in a mouse critical-size calvarial defect model.

    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. Constitution and in vivo test of micro-porous tubular scaffold for esophageal tissue engineering.

    Hou, Lei; Jin, Jiachang; Lv, Jingjing; Chen, Ling; Zhu, Yabin; Liu, Xingyu

    2015-11-01

    Current clinical techniques in treating long-gap esophageal defects often lead to complications and high morbidity. Aiming at long-gap synthetic esophageal substitute, we had synthesized a biodegradable copolymer, poly(L-lactide-co-caprolactone) (PLLC), with low glass transition temperature. In this work, we developed a tubular PLLC porous scaffold using a self-designed tubular mold and thermal induced phase separation (TIPS) method. In order to enhance the interaction between tissue and scaffold, fibrin, a natural fibrous protein derived from blood fibrinogen, was coated on the scaffold circumferential surface. The fibrin density was measured to be 1.23 ± 0.04 mg/cm(2). Primary epithelial cell culture demonstrated the improved in vitro biocompatibility. In animal study with partial scaffold implantation, in situ mucosa regeneration was observed along the degradation of the scaffold. These indicate that fibrin incorporated PLLC scaffold can greatly improve epithelial regeneration in esophagus repair, therefore serve as a good candidate for long-term evaluation of post-implantation at excision site. PMID:26208515

  10. Effects of scaffold architecture on cranial bone healing.

    Berner, A; Woodruff, M A; Lam, C X F; Arafat, M T; Saifzadeh, S; Steck, R; Ren, J; Nerlich, M; Ekaputra, A K; Gibson, I; Hutmacher, D W

    2014-04-01

    In the present study, polycaprolactone-tricalcium phosphate (PCL/TCP) scaffolds with two different fibre laydown patterns, which were coated with hydroxyapatite and gelatine, were used as an approach for optimizing bone regeneration in a critical-sized calvarial defect. After 12 weeks, bone regeneration was quantified using microcomputed tomography (micro-CT) analysis, biomechanical testing, and histological evaluation. Notably, the experimental groups with coated scaffolds showed lower bone formation and lower biomechanical properties within the defect compared to the uncoated scaffolds. Surprisingly, the different laydown pattern of the fibres resulted in different bone formation and biomechanical properties: the 0°/60°/120° scaffolds revealed lower bone formation and biomechanical properties compared to the 0°/90° scaffolds in all the experimental groups. Therefore, future bone regeneration strategies utilizing scaffolds should consider scaffold architecture as an important factor during the scaffold optimization stages in order to move closer to a clinical application. PMID:24183512

  11. Peptide functionalized polyhydroxyalkanoate nanofibrous scaffolds enhance Schwann cells activity

    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

  12. Effect of different calcium phosphate scaffold ratios on odontogenic differentiation of human dental pulp cells

    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.

  13. Nanocomposite scaffold fabrication by incorporating gold nanoparticles into biodegradable polymer matrix: Synthesis, characterization, and photothermal effect

    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.

  14. Microsphere-based scaffolds encapsulating tricalcium phosphate and hydroxyapatite for bone regeneration.

    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

  15. Effect of different calcium phosphate scaffold ratios on odontogenic differentiation of human dental pulp cells

    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

  16. Nanocomposite scaffold fabrication by incorporating gold nanoparticles into biodegradable polymer matrix: Synthesis, characterization, and photothermal effect

    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. A novel technique for the production of electrospun scaffolds with tailored three-dimensional micro-patterns employing additive manufacturing

    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)

  18. An in vivo study on the effect of scaffold geometry and growth factor release on the healing of bone defects.

    Yilgor, P; Yilmaz, G; Onal, M B; Solmaz, I; Gundogdu, S; Keskil, S; Sousa, R A; Reis, R L; Hasirci, N; Hasirci, V

    2013-09-01

    The hypothesis of this study was that the extent of bone regeneration could be enhanced by using scaffolds with appropriate geometry, and that such an effect could be further increased by mimicking the natural timing of appearance of bone morphogenetic proteins BMP-2 and BMP-7 after fracture. Bioplotted poly(ε-caprolactone) (PCL) disks with four different fibre organizations were used to study the effect of 3D scaffold architecture on the healing of bone defects in a rat pelvis model. Moreover, one PCL construct was further modified by introducing a nanoparticulate sequential BMP-2/BMP-7 delivery system into this scaffold. Scaffolds and functionalized construct along with free nanocapsules were implanted using a rat iliac crest defect model. Six weeks post-implantation, the defects were evaluated by CT scan and histology. Analysis revealed that the basic architecture, having the highest pore volume for tissue ingrowth, presented the highest bone formation as determined by the bone mineral density (BMD) within the defect (144.2 ± 7.1); about four-fold higher than that of the empty defect (34.9 ± 10.7). It also showed the highest histological analysis scores with a high amount of bone formation within the defect, within the scaffold pores and along the outer surfaces of the scaffold. The basic scaffold carrying the BMP-2/BMP-7 delivery system showed significantly higher bone formation than the growth factor-free basic scaffold at 6 weeks (BMD 206.8 ± 15.7). Histological analysis also revealed new bone formation in close to or in direct contact with the construct interface. This study indicates the importance of open and interconnecting pore geometry on the better healing of bone defects, and that this effect could be further increased by supplying growth factors, as is the case in nature. PMID:22396311

  19. Scaffolds for central nervous system tissue engineering

    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.

  20. Scaffolding for Three-Dimensional Embryonic Vasculogenesis

    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.

  1. Nano/macro porous bioactive glass scaffold

    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

  2. The fundamental of tissue engineering: new scaffolds

    The ability to regenerate new bone for skeletal use is a major clinical need. In this study, two novel porous calcium phosphate materials pure HA and biphasic HA/β-Tricalcium phosphate (HA/β-TCP) were evaluated as potential scaffolds for cell-seeded bone substitutes using human osteoblast-like cells (HOS) and primary human mesenchymal stem cells (hMSCs). A high rate of proliferation was observed on both scaffolds. A greater increase in alkaline phosphatase (ALP - an indicator of osteoblast differentiation) was observed on HA//β-TCP compared to HA. This observation indicates that HA/TCP may play a role in inducing osteoblastic differentiation. Although further evaluation is required both materials show potential as innovative synthetic substitutes for tissue engineered scaffolds. (Author)

  3. Knowledge scaffolding visualizations: A guiding framework

    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.

  4. Hemocompatible surface of electrospun nanofibrous scaffolds by ATRP modification

    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

  5. Fabrication and characterization of multiscale electrospun scaffolds for cartilage regeneration

    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 ...

  6. Hemocompatible surface of electrospun nanofibrous scaffolds by ATRP modification

    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

  7. Effect of Polycaprolactone Scaffold Permeability on Bone Regeneration In Vivo

    Mitsak, Anna G.; Kemppainen, Jessica M.; Harris, Matthew T.; Hollister, Scott J

    2011-01-01

    Successful bone tissue engineering depends on the scaffold's ability to allow nutrient diffusion to and waste removal from the regeneration site, as well as provide an appropriate mechanical environment. Since bone is highly vascularized, scaffolds that provide greater mass transport may support increased bone regeneration. Permeability encompasses the salient features of three-dimensional porous scaffold architecture effects on scaffold mass transport. We hypothesized that higher permeabilit...

  8. SCAFFOLDING IN CONNECTIVIST MOBILE LEARNING ENVIRONMENT

    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.

  9. Bio-functionalized PCL nanofibrous scaffolds for nerve tissue engineering

    Surface properties of scaffolds such as hydrophilicity and the presence of functional groups on the surface of scaffolds play a key role in cell adhesion, proliferation and migration. Different modification methods for hydrophilicity improvement and introduction of functional groups on the surface of scaffolds have been carried out on synthetic biodegradable polymers, for tissue engineering applications. In this study, alkaline hydrolysis of poly (ε-caprolactone) (PCL) nanofibrous scaffolds was carried out for different time periods (1 h, 4 h and 12 h) to increase the hydrophilicity of the scaffolds. The formation of reactive groups resulting from alkaline hydrolysis provides opportunities for further surface functionalization of PCL nanofibrous scaffolds. Matrigel was attached covalently on the surface of an optimized 4 h hydrolyzed PCL nanofibrous scaffolds and additionally the fabrication of blended PCL/matrigel nanofibrous scaffolds was carried out. Chemical and mechanical characterization of nanofibrous scaffolds were evaluated using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, contact angle, scanning electron microscopy (SEM) and tensile measurement. In vitro cell adhesion and proliferation study was carried out after seeding nerve precursor cells (NPCs) on different scaffolds. Results of cell proliferation assay and SEM studies showed that the covalently functionalized PCL/matrigel nanofibrous scaffolds promote the proliferation and neurite outgrowth of NPCs compared to PCL and hydrolyzed PCL nanofibrous scaffolds, providing suitable substrates for nerve tissue engineering.

  10. Patterns of Scaffolding in Computer-Mediated Collaborative Inquiry

    Lakkala, Minna; Muukkonen, Hanni; Hakkarainen, Kai

    2005-01-01

    There is wide agreement on the importance of scaffolding for student learning. Yet, models of individual and face-to-face scaffolding are not necessarily applicable to educational settings in which a group of learners is pursuing a process of inquiry mediated by technology. The scaffolding needed for such a process may be examined from three…

  11. Scaffolds of polycaprolactone with hydroxyapatite fibers

    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)

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

    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

  13. Role of chondroitin sulphate tethered silk scaffold in cartilaginous disc tissue regeneration.

    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

  14. S-SCAM/MAGI-2 is an essential synaptic scaffolding molecule for the GluA2-containing maintenance pool of AMPA receptors

    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...

  15. Translocation of an 89-kDa periplasmic protein is associated with Holospora infection

    The symbiotic bacterium Holospora obtusa infects the macronucleus of the ciliate Paramecium caudatum. After ingestion by its host, an infectious form of Holospora with an electron-translucent tip passes through the host digestive vacuole and penetrates the macronuclear envelope with this tip. To investigate the underlying molecular mechanism of this process, we raised a monoclonal antibody against the tip-specific 89-kDa protein, sequenced this partially, and identified the corresponding complete gene. The deduced protein sequence carries two actin-binding motifs. Indirect immunofluorescence microscopy shows that during escape from the host digestive vacuole, the 89-kDa proteins translocates from the inside to the outside of the tip. When the bacterium invades the macronucleus, the 89-kDa protein is left behind at the entry point of the nuclear envelope. Transmission electron microscopy shows the formation of fine fibrous structures that co-localize with the antibody-labeled regions of the bacterium. Our findings suggest that the 89-kDa protein plays a role in Holospora's escape from the host digestive vacuole, the migration through the host cytoplasm, and the invasion into the macronucleus

  16. Stem Cells Grown in Osteogenic Medium on PLGA, PLGA/HA, and Titanium Scaffolds for Surgical Applications

    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.

  17. Fluorescent composite scaffolds made of nanodiamonds/polycaprolactone

    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.

  18. Comparison of TALEN scaffolds in Xenopus tropicalis

    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.

  19. Composite tissue engineering on polycaprolactone nanofiber scaffolds.

    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

  20. Acellular organ scaffolds for tumor tissue engineering

    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.

  1. Scaffolding in Connectivist Mobile Learning Environment

    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…

  2. Scaffolding Practices that Enhance Mathematics Learning

    Anghileri, Julia

    2006-01-01

    It is over 25 years since Wood, Bruner and Ross (1976, "Journal of Child Psychology and Psychiatry," 17, 89-100) introduced the idea of "scaffolding" to represent the way children's learning can be supported. Despite problems, this metaphor has enduring attraction in the way it emphasises the intent to support a sound foundation with increasing…

  3. ACTIBIND, an actin-binding fungal T2-RNase with antiangiogenic and anticarcinogenic characteristics.

    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...

  4. Development of a scaffold displaying exoloops of RXFP1.

    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

  5. In vivo characterization of the scaffold activity of flotillin on the membrane kinase KinC of Bacillus subtilis

    Schneider, Johannes; Mielich-Süss, Benjamin; Böhme, Richard; Lopez, Daniel

    2015-01-01

    Summary Scaffold proteins are ubiquitous chaperones that bind to proteins and facilitate the physical interaction of the components of signal transduction pathways or multi-enzymatic complexes. In this study, we used a biochemical approach to dissect the molecular mechanism of a membrane-associated scaffold protein, FloT, a flotillin-homolog protein that is localized in Functional Membrane Microdomains of the bacterium Bacillus subtilis. This study provides unambiguous evidence that FloT physically binds to and interacts with the membrane-bound sensor kinase KinC. This sensor kinase activates biofilm formation in B. subtilis in response to the presence of the self-produced signal surfactin. Furthermore, we have characterized the mechanism by which the interaction of FloT with KinC benefits the activity of KinC. Two separate and synergistic effects constitute this mechanism: first, the scaffold activity of FloT promotes more efficient self-interaction of KinC and facilitates dimerization into its active form. Second, the selective binding of FloT to KinC prevents the occurrence of unspecific aggregation between KinC and other proteins that may generate dead-end intermediates that could titrate the activity of KinC. Flotillin proteins appear to play an important role in prokaryotes in promoting effective binding of signaling proteins with their correct protein partners. PMID:26297017

  6. Functionalization of a Collagen-Hydroxyapatite Scaffold with Osteostatin to Facilitate Enhanced Bone Regeneration.

    Quinlan, Elaine; Thompson, Emmet M; Matsiko, Amos; O'Brien, Fergal J; López-Noriega, Adolfo

    2015-12-01

    Defects within bones caused by trauma and other pathological complications may often require the use of a range of therapeutics to facilitate tissue regeneration. A number of approaches have been widely utilized for the delivery of such therapeutics via physical encapsulation or chemical immobilization suggesting significant promise in the healing of bone defects. The study focuses on the chemical immobilization of osteostatin, a pentapeptide of the parathyroid hormone (PTHrP107-111), within a collagen-hydroxyapatite scaffold. The chemical attachment method via crosslinking supports as little as 4% release of the peptide from the scaffolds after 21 d whereas non-crosslinking leads to 100% of the peptide being released by as early as 4 d. In vitro characterization demonstrates that this cross-linking method of immobilization supports a pro-osteogenic effect on osteoblasts. Most importantly, when implanted in a critical-sized calvarial defect within a rat, these scaffolds promote significantly greater new bone volume and area compared to nonfunctionalized scaffolds (**p < 0.01) and an empty defect control (***p < 0.001). Collectively, this study suggests that such an approach of chemical immobilization offers greater spatiotemporal control over growth factors and can significantly modulate tissue regeneration. Such a system may be adopted for a range of different proteins and thus offers the potential for the treatment of various complex pathologies that require localized mediation of drug delivery. PMID:26414944

  7. Highly porous and interconnected starch-based scaffolds: Production, characterization and surface modification

    A convenient and straightforward process for preparation of highly porous and interconnected fiber mesh scaffolds with 50 wt.% content of starch is described. The proposed methodology avoids some of the previous encountered problems associated with the processing of starch-based materials such as thermal degradation, starch entrapment in the material bulk and inability to control/minimise the thickness of the fibers obtained by melt spinning, or low porosity and lack of interconnectivity for the scaffolds obtained by extrusion or injection moulding with blowing agent. Topographical characterisation of the obtained fibers revealed rough surface commonly related with increased cell attachment and growth. The in vitro tests with osteoblast cell line confirmed this trend and we observed higher cell number with increasing of the culture time. These results were also associated with protein adsorption from a complex solution where predominant adsorption of vitronectin over fibronectin was detected. Finally, a model modification by plasma was also carried out in order to confirm the versatility of these scaffolds by the possibility to further upgrade them via surface functionalisation. The in vitro tests confirmed that osteoblast-like cells proliferate faster on the modified scaffolds, which allows shortening the time needed for culturing prior to implantation.

  8. Design and testing of biological scaffolds for delivering reparative cells to target sites in the lung.

    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

  9. Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function.

    Lerit, Dorothy A; Jordan, Holly A; Poulton, John S; Fagerstrom, Carey J; Galletta, Brian J; Peifer, Mark; Rusan, Nasser M

    2015-07-01

    Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes. A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle. Here, we investigate the mechanisms that spatially restrict and temporally coordinate centrosome scaffold formation. Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome. We identify an unprecedented role for Pericentrin-like protein (PLP), which localizes to the tips of extended Cnn flares, to maintain robust interphase centrosome activity and promote the formation of interphase MT asters required for normal nuclear spacing, centrosome segregation, and compartmentalization of the syncytial embryo. Our data reveal that Cnn and PLP directly interact at two defined sites to coordinate the cell cycle-dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability. PMID:26150390

  10. Astrocytes Grown in Alvetex(®) Three Dimensional Scaffolds Retain a Non-reactive Phenotype.

    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

  11. Biomechanical properties of Achilles tendon repair augmented with a bioadhesive-coated scaffold

    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...

  12. Nature's lessons in design: nanomachines to scaffold, remodel and shape membrane compartments.

    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...

  13. Biohybrid Fibro-Porous Vascular Scaffolds: Effect of Crosslinking on Properties

    Thomas, Vinoy; Nozik, Danna; Patel, Harsh; Singh, Raj K.; Vohra, Yogesh K.

    2015-01-01

    Tubular grafts were fabricated from blends of polycaprolactone (PCL) and poly(glycolide -co-caprolactone) (PGC) polymers and coated with an extracellular matrix containing collagens, laminin, and proteoglycans, but not growth factors (HuBiogel™). Multifunctional scaffolds from polymer blends and membrane proteins provide the necessary biomechanics and biological functions for tissue regeneration. Two crosslinking agents, a natural crosslinker namely genipin (Gp) and a carbodiimide reagent nam...

  14. Alginate hydrogel enriched with enamel matrix derivative to target osteogenic cell differentiation in TiO2 scaffolds

    Pullisaar, Helen; Verket, Anders; Szoke, Krisztina; Tiainen, Hanna; Haugen, Håvard J; Brinchmann, Jan E; Reseland, Janne E; Østrup, Esben

    2015-01-01

    various donors on titanium dioxide (TiO2) scaffolds coated with an alginate hydrogel enriched with enamel matrix derivative. Cells were harvested for quantitative reverse transcription polymerase chain reaction on days 14 and 21, and medium was collected on days 2, 14, and 21 for protein analyses. Neither...

  15. The host response to allogeneic and xenogeneic biological scaffold materials.

    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. Preparation of bioactive porous HA/PCL composite scaffolds

    Zhao, J.; Guo, L. Y.; Yang, X. B.; Weng, J.

    2008-12-01

    Porous hydroxyapatite (HA) bioceramic scaffold has been widely attracted the attention to act as a three-dimensional (3D) template for cell adhesion, proliferation, differentiation and thus promoting bone and cartilage regeneration because of its osteoinduction. However, the porous bioceramic scaffold is fragile so that it is not suitable to be applied in clinic for bone repair or replacement. Therefore, it is significant to improve the mechanical property of porous HA bioceramics while the interconnected structure is maintained for tissue ingrowth in vivo. In the present research, a porous composite scaffold composed of HA scaffold and polycaprolactone (PCL) lining was fabricated by the method of polymer impregnating to produce HA scaffold coated with PCL lining. Subsequently, the composite scaffolds were deposited with biomimetic coating for improving the bioactivity. The HA/PCL composite scaffolds with improved mechanical property and bioactivity is expected to be a promising bone substitute in tissue engineering applications.

  17. Preparation of bioactive porous HA/PCL composite scaffolds

    Zhao, J.; Guo, L.Y.; Yang, X.B. [Key Laboratory of Advanced Technologies of Materials (Ministry of Education), School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Weng, J. [Key Laboratory of Advanced Technologies of Materials (Ministry of Education), School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)], E-mail: jweng@swjtu.cn

    2008-12-30

    Porous hydroxyapatite (HA) bioceramic scaffold has been widely attracted the attention to act as a three-dimensional (3D) template for cell adhesion, proliferation, differentiation and thus promoting bone and cartilage regeneration because of its osteoinduction. However, the porous bioceramic scaffold is fragile so that it is not suitable to be applied in clinic for bone repair or replacement. Therefore, it is significant to improve the mechanical property of porous HA bioceramics while the interconnected structure is maintained for tissue ingrowth in vivo. In the present research, a porous composite scaffold composed of HA scaffold and polycaprolactone (PCL) lining was fabricated by the method of polymer impregnating to produce HA scaffold coated with PCL lining. Subsequently, the composite scaffolds were deposited with biomimetic coating for improving the bioactivity. The HA/PCL composite scaffolds with improved mechanical property and bioactivity is expected to be a promising bone substitute in tissue engineering applications.

  18. Preparation of bioactive porous HA/PCL composite scaffolds

    Porous hydroxyapatite (HA) bioceramic scaffold has been widely attracted the attention to act as a three-dimensional (3D) template for cell adhesion, proliferation, differentiation and thus promoting bone and cartilage regeneration because of its osteoinduction. However, the porous bioceramic scaffold is fragile so that it is not suitable to be applied in clinic for bone repair or replacement. Therefore, it is significant to improve the mechanical property of porous HA bioceramics while the interconnected structure is maintained for tissue ingrowth in vivo. In the present research, a porous composite scaffold composed of HA scaffold and polycaprolactone (PCL) lining was fabricated by the method of polymer impregnating to produce HA scaffold coated with PCL lining. Subsequently, the composite scaffolds were deposited with biomimetic coating for improving the bioactivity. The HA/PCL composite scaffolds with improved mechanical property and bioactivity is expected to be a promising bone substitute in tissue engineering applications

  19. Polyelectrolyte-complex nanostructured fibrous scaffolds for tissue engineering

    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 μm are observed in the scaffolds. The thickness of the fibers was found to be in the range of 1-2 μm. 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.

  20. Synergistic Effect of Carbon Nanotubes and Graphene on Diopside Scaffolds

    Liu, Tingting; Wu, Ping; Gao, Chengde; Feng, Pei; Xiao, Tao; Deng, Youwen; Shuai, Cijun; Peng, Shuping

    2016-01-01

    A synergetic effect between carbon nanotubes (CNTs) and graphene on diopside (Di) scaffolds was demonstrated. 3D network architecture in the matrix was formed through the 1D CNTs inlaid among the 2D graphene platelets (GNPs). The mechanical properties of the CNTs/GNPs/Di scaffolds were significantly improved compared with the CNTs/Di scaffolds and GNPs/Di scaffolds. In addition, the scaffolds exhibited excellent apatite-forming ability, a modest degradation rate, and stable mechanical properties in simulated body fluid (SBF). Moreover, cell culturing tests indicated that the scaffolds supported the cells attachment and proliferation. Taken together, the CNTs/GNPs/Di scaffolds offered great potential for bone tissue engineering.

  1. Poly(lactic-co-glycolic Acid/Nanohydroxyapatite Scaffold Containing Chitosan Microspheres with Adrenomedullin Delivery for Modulation Activity of Osteoblasts and Vascular Endothelial Cells

    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.

  2. Identification of the nuclear matrix and chromosome scaffold in dinoflagellate Crypthecodinium cohnii

    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. Effect of hydroxyapatite-containing microspheres embedded into three-dimensional magnesium phosphate scaffolds on the controlled release of lysozyme and in vitro biodegradation

    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

  4. Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection.

    Tipper, Donald J; Szomolanyi-Tsuda, Eva

    2016-01-01

    Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%. PMID:27213160

  5. Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection

    Donald J. Tipper

    2016-01-01

    Full Text Available Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs. YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP or U65-Apolipoprotein A1 (ApoA1 subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%.

  6. Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection

    Tipper, Donald J.; Szomolanyi-Tsuda, Eva

    2016-01-01

    Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%.

  7. The Unique-5 and -6 Motifs of ZO-1 Regulate Tight Junction Strand Localization and Scaffolding Properties

    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. Biomimetic component coating on 3D scaffolds using high bioactivity of mesoporous bioactive ceramics

    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

  9. Polycaprolactone nanofiber interspersed collagen type-I scaffold for bone regeneration: a unique injectable osteogenic scaffold

    There is an increasing demand for an injectable cell coupled three-dimensional (3D) scaffold to be used as bone fracture augmentation material. To address this demand, a novel injectable osteogenic scaffold called PN-COL was developed using cells, a natural polymer (collagen type-I), and a synthetic polymer (polycaprolactone (PCL)). The injectable nanofibrous PN-COL is created by interspersing PCL nanofibers within pre-osteoblast cell embedded collagen type-I. This simple yet novel and powerful approach provides a great benefit as an injectable bone scaffold over other non-living bone fracture stabilization polymers, such as polymethylmethacrylate and calcium content resin-based materials. The advantages of injectability and the biomimicry of collagen was coupled with the structural support of PCL nanofibers, to create cell encapsulated injectable 3D bone scaffolds with intricate porous internal architecture and high osteoconductivity. The effects of PCL nanofiber inclusion within the cell encapsulated collagen matrix has been evaluated for scaffold size retention and osteocompatibility, as well as for MC3T3-E1 cells osteogenic activity. The structural analysis of novel bioactive material proved that the material is chemically stable enough in an aqueous solution for an extended period of time without using crosslinking reagents, but it is also viscous enough to be injected through a syringe needle. Data from long-term in vitro proliferation and differentiation data suggests that novel PN-COL scaffolds promote the osteoblast proliferation, phenotype expression, and formation of mineralized matrix. This study demonstrates for the first time the feasibility of creating a structurally competent, injectable, cell embedded bone tissue scaffold. Furthermore, the results demonstrate the advantages of mimicking the hierarchical architecture of native bone with nano- and micro-size formation through introducing PCL nanofibers within macron-size collagen fibers and in

  10. Polycaprolactone nanofiber interspersed collagen type-I scaffold for bone regeneration: a unique injectable osteogenic scaffold.

    Baylan, Nuray; Bhat, Samerna; Ditto, Maggie; Lawrence, Joseph G; Lecka-Czernik, Beata; Yildirim-Ayan, Eda

    2013-08-01

    There is an increasing demand for an injectable cell coupled three-dimensional (3D) scaffold to be used as bone fracture augmentation material. To address this demand, a novel injectable osteogenic scaffold called PN-COL was developed using cells, a natural polymer (collagen type-I), and a synthetic polymer (polycaprolactone (PCL)). The injectable nanofibrous PN-COL is created by interspersing PCL nanofibers within pre-osteoblast cell embedded collagen type-I. This simple yet novel and powerful approach provides a great benefit as an injectable bone scaffold over other non-living bone fracture stabilization polymers, such as polymethylmethacrylate and calcium content resin-based materials. The advantages of injectability and the biomimicry of collagen was coupled with the structural support of PCL nanofibers, to create cell encapsulated injectable 3D bone scaffolds with intricate porous internal architecture and high osteoconductivity. The effects of PCL nanofiber inclusion within the cell encapsulated collagen matrix has been evaluated for scaffold size retention and osteocompatibility, as well as for MC3T3-E1 cells osteogenic activity. The structural analysis of novel bioactive material proved that the material is chemically stable enough in an aqueous solution for an extended period of time without using crosslinking reagents, but it is also viscous enough to be injected through a syringe needle. Data from long-term in vitro proliferation and differentiation data suggests that novel PN-COL scaffolds promote the osteoblast proliferation, phenotype expression, and formation of mineralized matrix. This study demonstrates for the first time the feasibility of creating a structurally competent, injectable, cell embedded bone tissue scaffold. Furthermore, the results demonstrate the advantages of mimicking the hierarchical architecture of native bone with nano- and micro-size formation through introducing PCL nanofibers within macron-size collagen fibers and in

  11. Cdk2 silencing via a DNA/PCL electrospun scaffold suppresses proliferation and increases death of breast cancer cells.

    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.

  12. Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

    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.

  13. Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

    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-ε-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.

  14. Fabrication and characterization of multiscale electrospun scaffolds for cartilage regeneration

    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)

  15. Modeling of porous scaffold deformation induced by medium perfusion.

    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

  16. Functionalisation of PLLA nanofiber scaffolds using a possible cooperative effect between collagen type I and BMP-2: impact on colonization and bone formation in vivo.

    Schofer, Markus D; Tünnermann, Lisa; Kaiser, Hendric; Roessler, Philip P; Theisen, Christina; Heverhagen, Johannes T; Hering, Jacqueline; Voelker, Maximilian; Agarwal, Seema; Efe, Turgay; Fuchs-Winkelmann, Susanne; Paletta, Jürgen R J

    2012-09-01

    The reconstruction of large bone defects after injury or tumor resection often requires the use of bone substitution. Artificial scaffolds based on synthetic biomaterials can overcome disadvantages of autologous bone grafts, like limited availability and donor side morbidity. Among them, scaffolds based on nanofibers offer great advantages. They mimic the extracellular matrix, can be used as a carrier for growth factors and allow the differentiation of human mesenchymal stem cells. Differentiation is triggered by a series of signaling processes, including integrin and bone morphogenetic protein (BMP), which act in a cooperative manner. The aim of this study was to analyze whether these processes can be remodeled in artificial poly-(l)-lactide acid (PLLA) based nanofiber scaffolds in vivo. Electrospun matrices composed of PLLA-collagen type I or BMP-2 incorporated PLLA-collagen type I were implanted in calvarial critical size defects in rats. Cranial CT-scans were taken 4, 8 and 12 weeks after implantation. Specimens obtained after euthanasia were processed for histology and immunostainings on osteocalcin, BMP-2 and Smad5. After implantation the scaffolds were inhomogeneously colonized and cells were only present in wrinkle- or channel-like structures. Ossification was detected only in focal areas of the scaffold. This was independent of whether BMP-2 was incorporated in the scaffold. However, cells that migrated into the scaffold showed an increased ratio of osteocalcin and Smad5 positive cells compared to empty defects. Furthermore, in case of BMP-2 incorporated PLLA-collagen type I scaffolds, 4 weeks after implantation approximately 40 % of the cells stained positive for BMP-2 indicating an autocrine process of the ingrown cells. These findings indicate that a cooperative effect between BMP-2 and collagen type I can be transferred to PLLA nanofibers and furthermore, that this effect is active in vivo. However, this had no effect on bone formation. The reason for

  17. Ectopic osteogenic tissue formation by MC3T3-E1 cell-laden chitosan/hydroxyapatite composite scaffold.

    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

  18. Combination therapy with BMP-2 and BMSCs enhances bone healing efficacy of PCL scaffold fabricated using the 3D plotting system in a large segmental defect model.

    Kang, Sun-Woong; Bae, Ji-Hoon; Park, Su-A; Kim, Wan-Doo; Park, Mi-Su; Ko, You-Jin; Jang, Hyon-Seok; Park, Jung-Ho

    2012-07-01

    The three-dimensional (3D) plotting system is a rapidly-developing scaffold fabrication method for bone tissue engineering. It yields a highly porous and inter-connective structure without the use of cytotoxic solvents. However, the therapeutic effects of a scaffold fabricated using the 3D plotting system in a large segmental defect model have not yet been demonstrated. We have tested two hypotheses: whether the bone healing efficacy of scaffold fabricated using the 3D plotting system would be enhanced by bone marrow-derived mesenchymal stem cell (BMSC) transplantation; and whether the combination of bone morphogenetic protein-2 (BMP-2) administration and BMSC transplantation onto the scaffold would act synergistically to enhance bone regeneration in a large segmental defect model. The use of the combined therapy did increase bone regeneration further as compared to that with monotherapy in large segmental bone defects. PMID:22447098

  19. 29 CFR (non - mandatory) Appendix A to Subpart L of Part 1926-Scaffold Specifications

    2010-07-01

    ...) Needle beam scaffolds. (v) Multi-level suspension scaffolds. (w) Mobile scaffolds. (x) Repair bracket... in the American Softwood Lumber Standard of the U.S. Department of Commerce. (i) Allowable spans... are being given for these scaffolds. (w) Mobile Scaffolds. Stability test as described in the ANSI...

  20. Evidence for regulated interleukin-4 expression in chondrocyte-scaffolds under in vitro inflammatory conditions.

    Muhammad Farooq Rai

    Full Text Available OBJECTIVE: To elucidate the anti-inflammatory and anabolic effects of regulated expression of IL-4 in chondrocyte-scaffolds under in vitro inflammatory conditions. METHODS: Mature articular chondrocytes from dogs (n = 3 were conditioned through transient transfection using pcDNA3.1.cIL-4 (constitutive or pCOX-2.cIL-4 (cytokine-responsive plasmids. Conditioned cells were seeded in alginate microspheres and rat-tail collagen type I matrix (CaReS® to generate two types of tissue-engineered 3-dimensional scaffolds. Inflammatory arthritis was simulated in the packed chondrocytes through exogenous addition of recombinant canine (rc IL-1β (100 ng/ml plus rcTNFα (50 ng/ml in culture media for 96 hours. Harvested cells and culture media were analyzed by various assays to monitor the anti-inflammatory and regenerative (anabolic properties of cIL-4. RESULTS: cIL-4 was expressed from COX-2 promoter exclusively on the addition of rcIL-1β and rcTNFα while its expression from CMV promoter was constitutive. The expressed cIL-4 downregulated the mRNA expression of IL-1β, TNFα, IL-6, iNOS and COX-2 in the cells and inhibited the production of NO and PGE(2 in culture media. At the same time, it up-regulated the expression of IGF-1, IL-1ra, COL2a1 and aggrecan in conditioned chondrocytes in both scaffolds along with a diminished release of total collagen and sGAG into the culture media. An increased amount of cIL-4 protein was detected both in chondrocyte cell lysate and in concentrated culture media. Neutralizing anti-cIL-4 antibody assay confirmed that the anti-inflammatory and regenerative effects seen are exclusively driven by cIL-4. There was a restricted expression of IL-4 under COX-2 promoter possibly due to negative feedback loop while it was over-expressed under CMV promoter (undesirable. Furthermore, the anti-inflammatory /anabolic outcomes from both scaffolds were reproducible and the therapeutic effects of cIL-4 were both scaffold- and

  1. Improved Human Bone Marrow Mesenchymal Stem Cell Osteogenesis in 3D Bioprinted Tissue Scaffolds with Low Intensity Pulsed Ultrasound Stimulation.

    Zhou, Xuan; Castro, Nathan J; Zhu, Wei; Cui, Haitao; Aliabouzar, Mitra; Sarkar, Kausik; Zhang, Lijie Grace

    2016-01-01

    3D printing and ultrasound techniques are showing great promise in the evolution of human musculoskeletal tissue repair and regeneration medicine. The uniqueness of the present study was to combine low intensity pulsed ultrasound (LIPUS) and advanced 3D printing techniques to synergistically improve growth and osteogenic differentiation of human mesenchymal stem cells (MSC). Specifically, polyethylene glycol diacrylate bioinks containing cell adhesive Arginine-Glycine-Aspartic acid-Serene (RGDS) peptide and/or nanocrystalline hydroxyapatite (nHA) were used to fabricate 3D scaffolds with different geometric patterns via novel table-top stereolithography 3D printer. The resultant scaffolds provide a highly porous and interconnected 3D environment to support cell proliferation. Scaffolds with small square pores were determined to be the optimal geometric pattern for MSC attachment and growth. The optimal LIPUS working parameters were determined to be 1.5 MHz, 20% duty cycle with 150 mW/cm(2) intensity. Results demonstrated that RGDS peptide and nHA containing 3D printed scaffolds under LIPUS treatment can greatly promote MSC proliferation, alkaline phosphatase activity, calcium deposition and total protein content. These results illustrate the effectiveness of the combination of LIPUS and biomimetic 3D printing scaffolds as a valuable combinatorial tool for improved MSC function, thus make them promising for future clinical and various regenerative medicine application. PMID:27597635

  2. Human unrestricted somatic stem cells loaded in nanofibrous PCL scaffold and their healing effect on skin defects.

    Bahrami, Hoda; Keshel, Saeed Heidari; Chari, Aliakbar Jafari; Biazar, Esmaeil

    2016-09-01

    Unrestricted somatic stem cells (USSCs) loaded in nanofibrous polycaprolactone (PCL) scaffolds can be used for skin regeneration when grafted onto full-thickness skin defects of rats. Nanofibrous PCL scaffolds were designed by the electrospinning method and crosslinked with laminin protein. Afterwards, the scaffolds were evaluated by scanning electron microscopy, and physical and mechanical assays. In this study, nanofibrous PCL scaffolds loaded with USSCs were grafted onto the skin defects. The wounds were subsequently investigated 21 days after grafting. Results of mechanical and physical analyses showed good resilience and compliance to movement as a skin graft. In animal models; study samples exhibited the most pronounced effect on wound closure, with statistically significant improvement in wound healing being seen at 21 days post-operatively. Histological examinations of healed wounds from all samples showed a thin epidermis plus recovered skin appendages in the dermal layer for samples with cell. Thus, the graft of nanofibrous PCL scaffolds loaded with USSC showed better results during the healing process of skin defects in rat models. PMID:26140614

  3. Gelatin Tight-Coated Poly(lactide-co-glycolide Scaffold Incorporating rhBMP-2 for Bone Tissue Engineering

    Juan Wang

    2015-03-01

    Full Text Available Surface coating is the simplest surface modification. However, bioactive molecules can not spread well on the commonly used polylactone-type skeletons; thus, the surface coatings of biomolecules are typically unstable due to the weak interaction between the polymer and the bioactive molecules. In this study, a special type of poly(lactide-co-glycolide (PLGA-based scaffold with a loosened skeleton was fabricated by phase separation, which allowed gelatin molecules to more readily diffuse throughout the structure. In this application, gelatin modified both the internal substrate and external surface. After cross-linking with glutaraldehyde, the surface layer gelatin was tightly bound to the diffused gelatin, thereby preventing the surface layer gelatin coating from falling off within 14 days. After gelatin modification, PLGA scaffold demonstrated enhanced hydrophilicity and improved mechanical properties (i.e., increased compression strength and elastic modulus in dry and wet states. Furthermore, a sustained release profile of recombinant human bone morphogenetic protein-2 (rhBMP-2 was achieved in the coated scaffold. The coated scaffold also supported the in vitro attachment, proliferation, and osteogenesis of rabbit bone mesenchymal stem cells (BMSCs, indicating the bioactivity of rhBMP-2. These results collectively demonstrate that the cross-linked-gelatin-coated porous PLGA scaffold incorporating bioactive molecules is a promising candidate for bone tissue regeneration.

  4. Bioprinted Scaffolds for Cartilage Tissue Engineering.

    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

  5. In vitro cell-biological performance and structural characterization of selective laser sintered and plasma surface functionalized polycaprolactone scaffolds for bone regeneration

    Van Bael, Simon, E-mail: simon.vanbael@mech.kuleuven.be [Department of Mechanical Engineering, Division of Production Engineering, Machine Design and Automation, Katholieke Universiteit Leuven, Celestijnenlaan 300b, 3001 Leuven (Belgium); Department of Mechanical Engineering, Division of Biomechanics and Engineering Design, Katholieke Universiteit Leuven, Celestijnenlaan 300c, bus 2419, 3001 Heverlee (Belgium); Prometheus, Division of Skeletal Tissue Engineering, Katholieke Universiteit Leuven, O and N 1, Herestraat 49, bus 813, 3000 Leuven (Belgium); Desmet, Tim [Polymer Chemistry and Biomaterials Research Group, Ghent University, Krijgslaan 281 S4 Bis, Ghent, 9000 (Belgium); Research Unit Plasma Technology (RUPT), Department of Applied Physics, Faculty of Engineering, Ghent University, Jozef Plateaustraat 22, 9000 Ghent (Belgium); Chai, Yoke Chin [Prometheus, Division of Skeletal Tissue Engineering, Katholieke Universiteit Leuven, O and N 1, Herestraat 49, bus 813, 3000 Leuven (Belgium); Pyka, Gregory [Prometheus, Division of Skeletal Tissue Engineering, Katholieke Universiteit Leuven, O and N 1, Herestraat 49, bus 813, 3000 Leuven (Belgium); Department of Metallurgy and Materials Engineering, Katholieke Universiteit Leuven, Kasteelpark Arenberg 44, bus 2450, 3001 Leuven (Belgium); Dubruel, Peter [Polymer Chemistry and Biomaterials Research Group, Ghent University, Krijgslaan 281 S4 Bis, Ghent, 9000 (Belgium); Research Unit Plasma Technology (RUPT), Department of Applied Physics, Faculty of Engineering, Ghent University, Jozef Plateaustraat 22, 9000 Ghent (Belgium); Kruth, Jean-Pierre [Department of Mechanical Engineering, Division of Production Engineering, Machine Design and Automation, Katholieke Universiteit Leuven, Celestijnenlaan 300b, 3001 Leuven (Belgium); Schrooten, Jan [Prometheus, Division of Skeletal Tissue Engineering, Katholieke Universiteit Leuven, O and N 1, Herestraat 49, bus 813, 3000 Leuven (Belgium)

    2013-08-01

    In the present study a structural characterization and in vitro cell-biological evaluation was performed on polycaprolactone (PCL) scaffolds that were produced by the additive manufacturing technique selective laser sintering (SLS), followed by a plasma-based surface modification technique, either non-thermal oxygen plasma or double protein coating, to functionalize the PCL scaffold surfaces. In the first part of this study pore morphology by means of 2D optical microscopy, surface chemistry by means of hydrophilicity measurement and X-ray photoelectron spectroscopy, strut surface roughness by means of 3D micro-computed tomography (CT) imaging and scaffold mechanical properties by means of compression testing were evaluated before and after the surface modifications. The results showed that both surface modifications increased the PCL scaffold hydrophilicity without altering the morphological and mechanical properties. In the second part of this study the in vitro cell proliferation and differentiation of human osteoprogenitor cells, over 14 days of culture in osteogenic and growth medium were investigated. The O{sub 2} plasma modification gave rise to a significant lower in vitro cell proliferation compared to the untreated and double protein coated scaffolds. Furthermore the double protein coating increased in vitro cell metabolic activity and cell differentiation compared to the untreated and O{sub 2} plasma PCL scaffolds when OM was used. - Highlights: • Polycaprolactone scaffolds are produced with selective laser sintering. • 2 types of plasma based surface functionalization were applied. • Plasma had no significant effect on strut roughness and pore morphology. • Plasma improved surface hydrophilicity. • In vitro cell differentiation increased with plasma protein coated functionalization.

  6. In vitro cell-biological performance and structural characterization of selective laser sintered and plasma surface functionalized polycaprolactone scaffolds for bone regeneration

    In the present study a structural characterization and in vitro cell-biological evaluation was performed on polycaprolactone (PCL) scaffolds that were produced by the additive manufacturing technique selective laser sintering (SLS), followed by a plasma-based surface modification technique, either non-thermal oxygen plasma or double protein coating, to functionalize the PCL scaffold surfaces. In the first part of this study pore morphology by means of 2D optical microscopy, surface chemistry by means of hydrophilicity measurement and X-ray photoelectron spectroscopy, strut surface roughness by means of 3D micro-computed tomography (CT) imaging and scaffold mechanical properties by means of compression testing were evaluated before and after the surface modifications. The results showed that both surface modifications increased the PCL scaffold hydrophilicity without altering the morphological and mechanical properties. In the second part of this study the in vitro cell proliferation and differentiation of human osteoprogenitor cells, over 14 days of culture in osteogenic and growth medium were investigated. The O2 plasma modification gave rise to a significant lower in vitro cell proliferation compared to the untreated and double protein coated scaffolds. Furthermore the double protein coating increased in vitro cell metabolic activity and cell differentiation compared to the untreated and O2 plasma PCL scaffolds when OM was used. - Highlights: • Polycaprolactone scaffolds are produced with selective laser sintering. • 2 types of plasma based surface functionalization were applied. • Plasma had no significant effect on strut roughness and pore morphology. • Plasma improved surface hydrophilicity. • In vitro cell differentiation increased with plasma protein coated functionalization

  7. Scaffolding In Connectivist Mobile Learning Environment

    Ozlem OZAN

    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: 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 desc...

  8. Nanofiber Scaffold Gradients for Interfacial Tissue Engineering

    Ramalingam, Murugan; Young, Marian F.; Thomas, Vinoy; Sun, Limin; Chow, Laurence C.; Tison, Christopher K.; Chatterjee, Kaushik; Miles, William C.; Simon, Carl G.

    2012-01-01

    We have designed a 2-spinnerette device that can directly electrospin nanofiber scaffolds containing a gradient in composition that can be used to engineer interfacial tissues such as ligament and tendon. Two types of nanofibers are simultaneously electrospun in an overlapping pattern to create a nonwoven mat of nanofibers containing a composition gradient. The approach is an advance over previous methods due to its versatility - gradients can be formed from any materials that can be electros...

  9. Metagenomic scaffolds enable combinatorial lignin transformation

    Strachan, Cameron R.; Singh, Rahul; VanInsberghe, David; Ievdokymenko, Kateryna; Budwill, Karen; Mohn, William W.; Eltis, Lindsay D.; Steven J Hallam

    2014-01-01

    Plant biomass conversion into biofuels and chemicals can reduce human reliance on petroleum and promote sustainable biorefining processes. The structural polymer lignin can comprise up to 40% of plant biomass, but resists decomposition into valuable monoaromatic compounds. In this study, we devised a previously unidentified biosensor responsive to lignin transformation products. We used this biosensor in a functional screen to recover metagenomic scaffolds sourced from coal bed bacterial comm...

  10. Injectable Hydrogel Scaffold from Decellularized Human Lipoaspirate

    Young, D. Adam; Ibrahim, Dina O.; Hu, Diane; Christman, Karen L.

    2010-01-01

    Soft tissue fillers are rapidly gaining popularity for aesthetic improvements or repair of adipose tissue deficits. Several injectable biopolymers have been investigated for this purpose but often face rapid resorption or limited adipogenesis, and do not mimic the native adipose extracellular matrix (ECM). We have generated an injectable adipose matrix scaffold by efficiently removing both the cellular and lipid contents of human lipoaspirate. The decellularized material retained a complex co...

  11. Mobile Learning: location, collaboration and scaffolding inquiry

    Scanlon, Eileen

    2014-01-01

    Critiques of mobile learning pedagogy are concerned with whether such approaches are technology led. This chapter discusses how the particular features of mobile learning can be harnessed to provide new learning opportunities in relation to collaboration, inquiry and location-based learning. Technology supported inquiry learning is a situation rich with possibilities for collaboration. In particular, mobile learning offers new possibilities for scaffolding collaboration together with its othe...

  12. Membrane supported scaffold : architectures for tissue engineering

    Bettahalli, Narasimha Murthy Srivatsa

    2011-01-01

    Tissue engineering aims at restoring or regenerating a damaged tissue. Often the tissue recreation occurs by combining cells, derived from a patient biopsy, onto a 3D porous matrix, functioning as a scaffold. One of the current limitations of tissue engineering is the inability to provide sufficient nutrient and oxygen supply in developing 3D in-vitro culture. In human body the vasculature is embedded into almost every tissues and organs. They transport blood, and thus nutrients and waste pro...

  13. Design of graded biomimetic osteochondral composite scaffolds

    Tampieri, Anna; Sandri, Monica; Landi, Elena; Pressato, Daniele; Francioli, Silvia; Quarto, Rodolfo; Martin, Ivan

    2008-01-01

    With the ultimate goal to generate suitable materials for the repair of osteochondral defects, in this work we aimed at developing composite osteochondral scaffolds organized in different integrated layers, with features which are biomimetic for articular cartilage and subchondral bone and can differentially support formation of such tissues. A biologically inspired mineralization process was first developed to nucleate Mg-doped hydroxyapatite crystals on type I collagen fibers during their s...

  14. Work Related Musculoskeletal Disorders in Scaffolders

    Elders, Leo

    2003-01-01

    textabstractIn many occupational populations, musculoskeletal disorders constitute an important source of morbidity, sickness absence, and disability and attribute to a substantial social and economic burden for society. This is certainly applicable to scaffolders, the study population in this thesis. Although many research efforts have been undertaken, relationships between various work-related risk factors and different stages of musculoskeletal disorders still remain unclear. A theoretical...

  15. Comparison of scaffold-enhanced albumin and n-butyl-cyanoacrylate adhesives for joining of tissue in a porcine model

    McNally-Heintzelman, Karen M.; Riley, Jill N.; Heintzelman, Douglas L.

    2003-06-01

    An ex vivo study was conducted to compare the tensile strength of tissue samples repaired using three different techniques: (i) application of a scaffold-enhanced light-activated albumin protein solder, (ii) application of a scaffold-enhanced n-butyl-cyanoacrylate adhesive, and (iii) repair via conventional suture technique. Biodegradable polymer scaffolds of controlled porosity were fabricated with poly(L-lactic-co-glycolic acid) and salt particles using a solvent-casting and particulate-leaching technique. Group I porous 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. Group II scaffolds were doped with n-butyl-cyanoacrylate, and required no light-activation. No stay sutures were required for Group I or II experiments. Group III repairs were performed using a single 4-0 suture. Thirteen organs were tested ranging from skin to liver to the small intestine, as well as the coronary, pulmonary, carotid, femoral and splenic arteries. Acute breaking strengths were measured and the data were analyzed by Student"s T-test. Using the protein solder of Group I, repairs formed on the ureter were most successful followed by small intestine, sciatic nerve, spleen, atrium, kidney, muscle, skin and ventricle. The strongest vascular repairs were achieved in the carotid artery and femoral artery. Overall, the tensile strength of Group III repairs performed via suture techniques were equivalent in magnitude to that of Group I repairs, however, a larger variance was observed in the suture repair group. Group II repairs utilizing the cyanoacrylate-doped scaffold all performed extremely well. Bonds formed using the Group II adhesive were approximately 30% stronger than Group I and III organ repairs and approximately 20% stronger than Group I and III vascular repairs. Application of the polymer scaffold assists in tissue alignment and reduces

  16. Protein-Protein Interactions at the Adrenergic Receptors

    Cotecchia, Susanna; Stanasila, Laura; Diviani, Dario

    2012-01-01

    The adrenergic receptors are among the best characterized G protein-coupled receptors (GPCRs) and knowledge on this receptor family has provided several important paradigms about GPCR function and regulation. One of the most recent paradigms initially supported by studies on adrenergic receptors is that both βarrestins and G protein-coupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effect...

  17. Affective Scaffolds, Expressive Arts, and Cognition

    Maiese, Michelle

    2016-01-01

    Some theorists have argued that elements of the surrounding world play a crucial role in sustaining and amplifying both cognition and emotion. Such insights raise an interesting question about the relationship between cognitive and affective scaffolding: in addition to enabling the realization of specific affective states, can an affective niche also enable the realization of certain cognitive capacities? In order to gain a better understanding of this relationship between affective niches and cognition, I will examine the use of expressive arts in the context of psychotherapy and peacebuilding. In these settings, environmental resources and interpersonal scaffolds not only evoke emotion and encourage the adoption of particular bodily affective styles, but also support the development of capacities for self-awareness and interpersonal understanding. These affective scaffolds play a crucial role in therapy and peacebuilding, in fact, insofar as they facilitate the development of self-knowledge, enhance capacities associated with social cognition, and build positive rapport and trust among participants. I will argue that this is because affectivity is linked to the way that subjects frame and attend to their surroundings. Insofar as the regulation and modification of emotion goes hand in hand with opening up new interpretive frames and establishing new habits of mind, the creation of an affective niche can contribute significantly to various modes of cognition. PMID:27014164

  18. Cardiac tissue engineering in magnetically actuated scaffolds

    Cardiac tissue engineering offers new possibilities for the functional and structural restoration of damaged or lost heart tissue by applying cardiac patches created in vitro. Engineering such functional cardiac patches is a complex mission, involving material design on the nano- and microscale as well as the application of biological cues and stimulation patterns to promote cell survival and organization into a functional cardiac tissue. Herein, we present a novel strategy for creating a functional cardiac patch by combining the use of a macroporous alginate scaffold impregnated with magnetically responsive nanoparticles (MNPs) and the application of external magnetic stimulation. Neonatal rat cardiac cells seeded within the magnetically responsive scaffolds and stimulated by an alternating magnetic field of 5 Hz developed into matured myocardial tissue characterized by anisotropically organized striated cardiac fibers, which preserved its features for longer times than non-stimulated constructs. A greater activation of AKT phosphorylation in cardiac cell constructs after applying a short-term (20 min) external magnetic field indicated the efficacy of magnetic stimulation to actuate at a distance and provided a possible mechanism for its action. Our results point to a synergistic effect of magnetic field stimulation together with nanoparticulate features of the scaffold surface as providing the regenerating environment for cardiac cells driving their organization into functionally mature tissue. (paper)

  19. Multilayer micromolding of degradable polymer tissue engineering scaffolds

    Gallego, Daniel; Ferrell, Nicholas; Sun, Yang [Biomedical Engineering Department, Ohio State University, 1080 Carmack Road, 270 Bevis Hall, Columbus (OH), 43210 (United States); Hansford, Derek J. [Biomedical Engineering Department, Ohio State University, 1080 Carmack Road, 270 Bevis Hall, Columbus (OH), 43210 (United States)], E-mail: hansford.4@osu.edu

    2008-04-01

    Precise surface geometrical morphologies have been shown to improve cellular proliferation, adhesion, and functionality. It has been found that cells respond strongly to feature dimensions a fraction of their size. In this paper, soft lithography techniques were applied to microfabricate polydimethylsiloxane molds with precisely controlled micro-scale patterns. Three-dimensional polycaprolactone (PCL) scaffolds were fabricated using a multilayer micromolding (MMM) method. Proper heating and stamping parameters were developed for micromolding PCL. This process allowed control of the size, shape, and spacing of support structures within the scaffold. The micromolding of multiple layers with independent features allowed for alignment between layers. The high porosity, abundant interconnections, and sharp features were inherent advantages of the scaffolds. Human osteosarcoma cells were seeded in the 3-D scaffolds for cell growth testing. Fluorescent microscopy and scanning electron micrographs showed that cells responded well to the 3-D scaffolds and the scaffolds regulated cell morphology and adhesion.

  20. PREPARATION OF BIOACTIVE NANOSTRUCTURE SCAFFOLD WITH IMPROVED COMPRESSIVE STRENGTH

    R. EMADI

    2011-03-01

    Full Text Available Highly porous scaffolds with open structure are today the best candidates for bone substitution to ensure bone oxygenation and angiogenesis. In this study, we developed a new route to enhance the compressive strength of porous hydroxyapatite scaffold made of natural bone. Briefly, the spongy bone of an adult bovine was extracted, annealed, and coated by a nanostructure bioactive glass layer to be subsequently sintered at different temperatures. The apatite formation ability on the surfaces of the coated scaffolds was investigated by standard procedures. Our results showed that the scaffold and coating microstructure consisted of the grains smaller than 100 nm. These nanostructures improved the compressive strength and bioactivity of highly porous scaffold. The results showed that with increasing the sintering temperature, the compressive strength of scaffolds increased while their in vitro bioactivity decreased.

  1. Further Development of Scaffolds for Regeneration of Nerves

    Sakamoto, Jeffrey; Tuszynski, Mark

    2009-01-01

    Progress has been made in continuing research on scaffolds for the guided growth of nerves to replace damaged ones. The scaffolds contain pores that are approximately cylindrical and parallel, with nearly uniform widths ranging from tens to hundreds of microns. At the earlier stage of development, experimental scaffolds had been made from agarose hydrogel. Such a scaffold was made in a multistep process in which poly(methyl methacrylate) [PMMA] fibers were used as templates for the pores. The process included placement of a bundle of the PMMA fibers in a tube, filling the interstices in the tube with a hot agarose solution, cooling to turn the solution into a gel, and then immersion in acetone to dissolve the PMMA fibers. The scaffolds were typically limited to about 25 pores per scaffold, square cross sections of no more than about 1.5 by 1.5 mm, and lengths of no more than about 2 mm.

  2. Multilayer micromolding of degradable polymer tissue engineering scaffolds

    Precise surface geometrical morphologies have been shown to improve cellular proliferation, adhesion, and functionality. It has been found that cells respond strongly to feature dimensions a fraction of their size. In this paper, soft lithography techniques were applied to microfabricate polydimethylsiloxane molds with precisely controlled micro-scale patterns. Three-dimensional polycaprolactone (PCL) scaffolds were fabricated using a multilayer micromolding (MMM) method. Proper heating and stamping parameters were developed for micromolding PCL. This process allowed control of the size, shape, and spacing of support structures within the scaffold. The micromolding of multiple layers with independent features allowed for alignment between layers. The high porosity, abundant interconnections, and sharp features were inherent advantages of the scaffolds. Human osteosarcoma cells were seeded in the 3-D scaffolds for cell growth testing. Fluorescent microscopy and scanning electron micrographs showed that cells responded well to the 3-D scaffolds and the scaffolds regulated cell morphology and adhesion

  3. Functionalized Ormosil Scaffolds Processed by Direct Laser Polymerization for Application in Tissue Engineering

    Matei, A.; Schou, Jørgen; Canulescu, Stela;

    The N,N’-(methacryloyloxyethyl triehtoxy silyl propyl carbamoyl-oxyhexyl)-urea hybrid methacrylate for applications in tissue engineering was synthesized and afterwards polymerized by direct laser polymerization using femtosecond laser pulses with the aim of using it for further applications...... in tissue engineering. The as-obtained scaffolds were modified either by low pressure argon plasma treatment or by using two different proteins (lysozyme, fibrinogen). For improved adhesion, the proteins were deposited by matrix assisted pulsed laser evaporation. The functionalized structures were tested...

  4. Functionalized ormosil scaffolds processed by direct laser polymerization for application in tissue engineering

    Matei, A., E-mail: andreeapurice@nipne.ro [Petru Poni Institute of Macromolecular Chemistry, Grigore Ghica Voda 41A, 6600 Iasi (Romania); National Institute for Lasers, Plasma and Radiation Physics, Atomistilor 409, 77125 Bucharest-Magurele (Romania); Schou, J.; Canulescu, S. [DTU Fotonik, Risø Campus, Technical University of Denmark, DK-4000 Roskilde (Denmark); Zamfirescu, M.; Albu, C.; Mitu, B. [National Institute for Lasers, Plasma and Radiation Physics, Atomistilor 409, 77125 Bucharest-Magurele (Romania); Buruiana, E.C.; Buruiana, T. [Petru Poni Institute of Macromolecular Chemistry, Grigore Ghica Voda 41A, 6600 Iasi (Romania); Mustaciosu, C.; Petcu, I. [Department of Environmental and Life Physics, National Institute for Physics and Nuclear Engineering ‘Horia Hulubei’, Bucharest (Romania); Dinescu, M. [National Institute for Lasers, Plasma and Radiation Physics, Atomistilor 409, 77125 Bucharest-Magurele (Romania)

    2013-08-01

    Synthesized N,N′-(methacryloyloxyethyl triehtoxy silyl propyl carbamoyl-oxyhexyl)-urea hybrid methacrylate was polymerized by direct laser polymerization using femtosecond laser pulses with the aim of using it for subsequent applications in tissue engineering. The as-obtained scaffolds were modified either by low pressure argon plasma treatment or by covering the structures with two different proteins (lysozyme, fibrinogen). For improved adhesion, the proteins were deposited by matrix assisted pulsed laser evaporation technique. The functionalized structures were tested in mouse fibroblasts culture and the cells morphology, proliferation, and attachment were analyzed.

  5. The Effect of Sterilization Methods on the Structural and Chemical Properties of Fibrin Microthread Scaffolds.

    Grasman, Jonathan M; O'Brien, Megan P; Ackerman, Kevin; Gagnon, Keith A; Wong, Gregory M; Pins, George D

    2016-06-01

    A challenge for the design of scaffolds in tissue engineering is to determine a terminal sterilization method that will retain the structural and biochemical properties of the materials. Since commonly used heat and ionizing energy-based sterilization methods have been shown to alter the material properties of protein-based scaffolds, the effects of ethanol and ethylene oxide (EtO) sterilization on the cellular compatibility and the structural, chemical, and mechanical properties of uncrosslinked, UV crosslinked, or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) crosslinked fibrin microthreads in neutral (EDCn) or acidic (EDCa) buffers are evaluated. EtO sterilization significantly reduces the tensile strength of uncrosslinked microthreads. Surface chemistry analyses show that EtO sterilization induces alkylation of EDCa microthreads leading to a significant reduction in myoblast attachment. The material properties of EDCn microthreads do not appear to be affected by the sterilization method. These results significantly enhance the understanding of how sterilization or crosslinking techniques affect the material properties of protein scaffolds. PMID:26847494

  6. Silver Nano Scaffold Formation by Flowers of Hibiscus Rosa Sinensis

    S.Shabana

    2013-06-01

    Full Text Available Hibiscusrosa sinensis grows for their showy flowers and are used as landscape shrubs. Hibiscusrosa sinensis also has too many medicinal properties. Flowers are rich in polyphenols, flavonoids and anthocyanins. Inourstudy the flower extracts of Hibiscus rosa sinensis has been used for the synthesis of silver nano scaffold formation and its reduction by the flowers. The synthesis of silver nanoparticles occurred under the exposure of the flower extract to 1mM (AgNO3, Silver nitrate aqueous solution. During this process the complete reduction of nanoparticles was observed nearly 48hours of reaction at 30°C under vigorous shaking conditions. The colour change is noted in the reaction mixture and observed during the process of incubation period, it may be due to the formation of the silver nanoparticles which are able to produce the colour in the reaction mixture may be due to their specific properties of surface Plasmon resonance. The colour change was made confirmed by visualization and the characterization by FT-IR, UV-Visible Spectroscopy. Thus we conclude that the reduction process and capping may be having occurred due to the presence of many flavonoids, terpenoids, anthocyanins, some aminoacids and proteins. In conclusion, Hibiscus rosa sinensis flower extract appears capable of forming silver nanoparticles. It can be concluded that, Hibiscus rosa sinensis flower extract was able to form the silver nano scaffold and may be probably due to its antioxidants potential, some flavonoids, terpenes etc. This study is a preliminary effort and requires further investigation at different levels.

  7. PDZ-scaffolding proteins : mediators of the olfactosome

    Baumgart, Sabrina

    2012-01-01

    Die olfaktorische Signaltransduktionskaskade, welche eine enorme Diversität an olfaktorischen Rezeptoren aufweist, ist ein hoch komplexes Proteinnetzwerk, welches es Lebewesen ermöglicht tausende verschiedene Duftstoffe wahrzunehmen und zu unterscheiden. Obwohl die meisten Kernkomponenten dieses Signalweges bereits bekannt sind, ist bis heute unklar, wie diese Signalproteine in räumliche Nähe zueinander gebracht werden. Die vorliegende Arbeit hatte die Aufklärung dieser Problemati...

  8. Preparation and characterization of gelatin scaffold containing microorganism fermented cellulose

    Lim, Youn Mook; Gwon, Hui Jeong; Park, Jong Seok; Nho, Young Chang; Lee, Byeong Heon [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of); Kim, Mi Yeong; Lee, Jong Dae; Song, Sung Gi [Quegenbiotech, Co., Incheon (Korea, Republic of)

    2010-12-15

    Cellulose, chitin, chitosan and hyaluronic acid are well known as polysaccharides. These polysaccharides have many effects on cell growth and differentiation. Cell activation increases with increasing the polysaccharides concentration. In this study, gelatin scaffold containing microorganism fermented cellulose, citrus gel were prepared by using irradiation technique. Physical properties of the scaffolds were investigated as a function of the concentrations of gelatin and citrus gel and the cell attachment, cell morphology and inflammation of the scaffolds also were characterized for regeneration of skin tissue.

  9. Chitosan Scaffolds Containing Hyaluronic Acid for Cartilage Tissue Engineering

    Correia, Clara R.; Moreira Teixeira, Liliana S.; Moroni, Lorenzo; Reis, Rui L.; Blitterswijk, van, C.A.; Karperien, Marcel; Mano, João F.

    2011-01-01

    Scaffolds derived from natural polysaccharides are very promising in tissue engineering applications and regenerative medicine, as they resemble glycosaminoglycans in the extracellular matrix (ECM). In this study, we have prepared freeze-dried composite scaffolds of chitosan (CHT) and hyaluronic acid (HA) in different weight ratios containing either no HA (control) or 1%, 5%, or 10% of HA. We hypothesized that HA could enhance structural and biological properties of CHT scaffolds. To test thi...

  10. Accelerating Vascularization in Polycaprolactone Scaffolds by Endothelial Progenitor Cells

    Singh, Shivani; Wu, Benjamin M.; Dunn, James C.Y.

    2011-01-01

    Vascularization is a major challenge in tissue engineering. The purpose of this study is to expedite the formation of blood vessels in porous polycaprolactone (PCL) scaffolds by the delivery of endothelial progenitor cells (EPCs). To establish a pro-angiogenic and pro-vasculogenic microenvironment, we employed EPCs seeded in PCL scaffold with surface-immobilized heparin and vascular endothelial growth factor (VEGF). EPCs seeded on scaffolds with VEGF exhibited phosphorylation of the receptor....

  11. Partially Nanofibrous Architecture of 3D Tissue Engineering Scaffolds

    Wei, Guobao; Ma, Peter X.

    2009-01-01

    An ideal tissue-engineering scaffold should provide suitable pores and appropriate pore surface to induce desired cellular activities and to guide 3D tissue regeneration. In the present work, we have developed macroporous polymer scaffolds with varying pore wall architectures from smooth (solid), microporous, partially nanofibrous, to entirely nanofibrous ones. All scaffolds are designed to have well-controlled interconnected macropores, resulting from leaching sugar sphere template. We exami...

  12. Magnetic scaffolds enriched with bioactive nanoparticles for tissue engineering.

    Skaat, Hadas; Ziv-Polat, Ofra; Shahar, Abraham; Last, David; Mardor, Yael; Margel, Shlomo

    2012-03-01

    Novel magnetic fibrin hydrogel scaffolds for cell implantation and tissue engineering are reported. The magnetic scaffolds are produced by the interaction between thrombin-conjugated maghemite nanoparticles of narrow size distribution and fibrinogen. These scaffolds, enriched with growth factor conjugated fluorescent maghemite nanoparticles, provide a supporting 3D environment for massive proliferation of various cell types, and can be successfully visualized by MRI. PMID:23184719

  13. Synthesis and characterization of gelatin based polyester urethane scaffold

    S Sarkar; A Chourasia; S Maji; S Sadhukhan; S Kumar; B Adhikari

    2006-10-01

    For tissue engineering purpose two gelatin based polyester urethane scaffolds of different compositions were prepared from lactic acid, polyethylene glycol 400 (PEG 400) and characterized by FTIR, XRD for their mechanical and morphological properties using SEM and optical microscopic analyses. Degradation and swelling studies of gelatin based polyester urethane scaffolds in phosphate buffer saline (PBS) were performed. Human keratinocyte cells were cultured within these scaffolds, which showed good cell adherence and proliferation.

  14. Teaching physics novices at university: A case for stronger scaffolding

    Christine Lindstrøm

    2011-01-01

    In 2006 a new type of tutorial, called Map Meeting, was successfully trialled with novice first year physics students at the University of Sydney, Australia. Subsequently, in first semester 2007 a large-scale experiment was carried out with 262 students who were allocated either to the strongly scaffolding Map Meetings or to the less scaffolding Workshop Tutorials, which have been run at the University of Sydney since 1995. In this paper we describe what makes Map Meetings more scaffolding th...

  15. Morphological and Cell Growth Assessment in Near Dense Hydroxyapatite Scaffold

    Florencia Edith Wiria; Bee Yen Tay; Elaheh Ghassemieh

    2013-01-01

    This paper reports the preliminary results on the morphology of low porosity hydroxyapatite scaffold and its compatibility as a substrate for osteoblast cells. Although having low porosity, the hydroxyapatite scaffold was found to be capable of sustaining cell growth and thus assisting bone ingrowth. Due to the low porosity nature, the scaffold provides higher strength and therefore more suitable for applications with load-bearing requirements such as spinal spacer. The hydroxyapatite scaffol...

  16. Collagen-Hyaluronic Acid Scaffolds for Adipose Tissue Engineering

    Davidenko, Natalia; Campbell, J J; Thian, E. S.; C. J. Watson; Cameron, Ruth Elizabeth

    2010-01-01

    Three-dimensional (3-D) in vitro models of the mammary gland require a scaffold matrix that supports the development of adipose stroma within a robust freely permeable matrix. 3-D porous collagen-hyaluronic acid (HA: 7.5% and 15%) scaffolds were produced by controlled freeze-drying technique and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. All scaffolds displayed uniform, interconnected pore structure (total porosity approximately 85%). Physical and chemical...

  17. Cryopreservation of Cell/Scaffold Tissue-Engineered Constructs

    Costa, Pedro F.; Dias, Ana F.; Reis, Rui L.; Gomes, Manuela E.

    2012-01-01

    The aim of this work was to study the effect of cryopreservation over the functionality of tissue-engineered constructs, analyzing the survival and viability of cells seeded, cultured, and cryopreserved onto 3D scaffolds. Further, it also evaluated the effect of cryopreservation over the properties of the scaffold material itself since these are critical for the engineering of most tissues and in particular, tissues such as bone. For this purpose, porous scaffolds, namely fiber meshes based o...

  18. Chitosan-collagen porous scaffold and bone marrow mesenchymal stem cell transplantation for ischemic stroke

    Feng Yan; Wei Yue; Yue-lin Zhang; Guo-chao Mao; Ke Gao; Zhen-xing Zuo; Ya-jing Zhang; Hui Lu

    2015-01-01

    In this study, we successfully constructed a composite of bone marrow mesenchymal stem cells and a chitosan-collagen scaffoldin 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 hi-tosan-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 ifbrillary acidic protein and a low level of expression of neuron-spe-ciifc enolase were visible in BrdU-labeled bone marrow mesenchymal stem cells. These ifndings suggest that transplantation of a composite of bone marrow mesenchymal stem cells and a chi-tosan-collagen scaffold has a neuroprotective effect following ischemic stroke.

  19. The key role of the scaffold on the efficiency of dendrimer nanodrugs.

    Caminade, Anne-Marie; Fruchon, Séverine; Turrin, Cédric-Olivier; Poupot, Mary; Ouali, Armelle; Maraval, Alexandrine; Garzoni, Matteo; Maly, Marek; Furer, Victor; Kovalenko, Valeri; Majoral, Jean-Pierre; Pavan, Giovanni M; Poupot, Rémy

    2015-01-01

    Dendrimers are well-defined macromolecules whose highly branched structure is reminiscent of many natural structures, such as trees, dendritic cells, neurons or the networks of kidneys and lungs. Nature has privileged such branched structures for increasing the efficiency of exchanges with the external medium; thus, the whole structure is of pivotal importance for these natural networks. On the contrary, it is generally believed that the properties of dendrimers are essentially related to their terminal groups, and that the internal structure plays the minor role of an 'innocent' scaffold. Here we show that such an assertion is misleading, using convergent information from biological data (human monocytes activation) and all-atom molecular dynamics simulations on seven families of dendrimers (13 compounds) that we have synthesized, possessing identical terminal groups, but different internal structures. This work demonstrates that the scaffold of nanodrugs strongly influences their properties, somewhat reminiscent of the backbone of proteins. PMID:26169490

  20. Heterocyclic Scaffolds in the Design of Peptidomimetic Integrin Ligands: Synthetic Strategies, Structural Aspects, and Biological Activity.

    De Marco, Rossella; Mazzotti, Giacomo; Greco, Arianna; Gentilucci, Luca

    2016-01-01

    The integrin receptors represent valuable targets for therapeutic interventions; being overexpressed in many pathological states, their inhibition can be effective to treat a number of severe diseases. Since integrin functions are mediated by interactions with ECM protein ligands, the inhibition can be achieved by interfering with such interactions using small mimetics of the integrin-ligand recognition motifs (e.g. RGD, LDV, etc.). In this review, we focus on the antagonists with peptideheterocycle hybrid structures. The introduction of well-designed scaffolds has met considerable success in the rational design of highly stable, bioavailable, and conformationally defined antagonists. Two main approaches are discussed herein. The first approach is the use of scaffolds external to the main recognition motifs, aimed at improving conformational definition. In the second approach, heterocyclic cores are introduced within the recognition motifs, giving access to libraries of 3D diverse candidate antagonists. PMID:26265351

  1. Yeast Interacting Proteins Database: YLR423C, YLR423C [Yeast Interacting Proteins Database

    Full Text Available YLR423C ATG17 Scaffold protein responsible for phagophore assembly site organization ; regulatory ... d protein responsible for phagophore assembly site organization ; regulatory subunit of an autophagy-specific compl ... d protein responsible for phagophore assembly site organization ; regulatory subunit of an autophagy-specific compl ...

  2. Novel biodegradable porous scaffold applied to skin regeneration.

    Hui-Min Wang

    Full Text Available Skin wound healing is an important lifesaving issue for massive lesions. A novel porous scaffold with collagen, hyaluronic acid and gelatin was developed for skin wound repair. The swelling ratio of this developed scaffold was assayed by water absorption capacity and showed a value of over 20 g water/g dried scaffold. The scaffold was then degraded in time- and dose-dependent manners by three enzymes: lysozyme, hyaluronidase and collagenase I. The average pore diameter of the scaffold was 132.5±8.4 µm measured from SEM images. With human skin cells growing for 7 days, the SEM images showed surface fractures on the scaffold due to enzymatic digestion, indicating the biodegradable properties of this scaffold. To simulate skin distribution, the human epidermal keratinocytes, melanocytes and dermal fibroblasts were seeded on the porous scaffold and the cross-section immunofluorescent staining demonstrated normal human skin layer distributions. The collagen amount was also quantified after skin cells seeding and presented an amount 50% higher than those seeded on culture wells. The in vivo histological results showed that the scaffold ameliorated wound healing, including decreasing neutrophil infiltrates and thickening newly generated skin compared to the group without treatments.

  3. Fabrication of polylactide nanocomposite scaffolds for bone tissue engineering applications

    Mkhabela, Vuyiswa J.; Ray, Suprakas Sinha [Department of Applied Chemistry, University of Johannesburg, Doornfontein 2028 (South Africa); DST/CSIR National Centre for Nanostructured Materials, Council for Scientific and Industrial Research, Pretoria 0001 (South Africa)

    2015-05-22

    Highly porous three-dimensional polylactide (PLA) scaffolds were obtained from PLA incorporated with different amounts of chitosan-modified montmorillonite (CS-MMT), through solvent casting and particulate leaching method. The processed scaffolds were tested in vitro for their possible application in bone tissue engineering. Scaffolds were characterized by Focused Ion Beam Scanning Electron Microscopy (FIB SEM), Fourier Transform Infra-Red (FTIR), and X-Ray Diffraction (XRD) to study their structure and intermolecular interactions. Bioresorbability tests in simulated body fluid (pH 7.4) were conducted to assess the response of the scaffolds in a simulated physiological condition. The FIB SEM images of the scaffolds showed a porous architecture with gradual change in morphology with increasing CS-MMT concentration. FTIR analysis revealed the presence of both PLA and CS-MMT particles on the surface of the scaffolds. XRD showed that the crystalline unit cell type was the same for all the scaffolds, and crystallinity decreased with an increase in CS-MMT concentration. The scaffolds were found to be bioresorbable, with rapid bioresorbability on the scaffolds with a high CS-MMT concentration.

  4. Investigation of fabrication and environmental effects on bioceramic bone scaffolds

    Vivanco Morales, Juan Francisco

    2011-12-01

    Bioactive ceramic materials like tricalcium phosphates (TCP) have been emerging as viable material alternatives to the current therapies of bone scaffolding to target fracture healing and osteoporosis. Once scaffolds are implanted at the defect site they should provide mechanical and biological functions, ultimately serving to facilitate with surrounding native tissue. Optimal osteogenic signal expression and subsequent differentiation of cells seeded on the scaffold in both in vivo and in vitro conditions is known to be influenced by scaffold properties and biomechanical environmental conditions. Thus, the objective of this research was to investigate the effect of fabrication and environmental variables on the properties of bioceramic scaffolds for bone tissue engineering applications. Specifically, the effect of sintering temperature in the range of 950°C -1150°C of a cost-effective on a large scale manufacturing process, on the physical and mechanical properties of bioceramic bone scaffolds, was investigated. In addition, the effect of a controlled environment was investigated by implementing a bioreactor and bone loading system to study the response of ex vivo trabecular bone to compressive load while perfused with culture medium. Collectively, this thesis demonstrates that: (1) the sintering temperature to fabricate bioceramic scaffolds can be tuned to structural properties, and (2) the use of a controlled mechanical and biochemical environment can enhance bone tissue development. These findings support the development of clinically successful bioceramic scaffolds that may stimulate bone regeneration and scaffold integration while providing structural integrity.

  5. Low elastic modulus titanium–nickel scaffolds for bone implants

    Li, Jing; Yang, Hailin; Wang, Huifeng; Ruan, Jianming, E-mail: jianming@csu.edu.cn

    2014-01-01

    The superelastic nature of repeating the human bones is crucial to the ideal artificial biomedical implants to ensure smooth load transfer and foster the ingrowth of new bone tissues. Three dimensional interconnected porous TiNi scaffolds, which have the tailorable porous structures with micro-hole, were fabricated by slurry immersing with polymer sponge and sintering method. The crystallinity and phase composition of scaffolds were studied by X-ray diffraction. The pore morphology, size and distribution in the scaffolds were characterized by scanning electron microscopy. The porosity ranged from 65 to 72%, pore size was 250–500 μm. Compressive strength and elastic modulus of the scaffolds were ∼ 73 MPa and ∼ 3GPa respectively. The above pore structural and mechanical properties are similar to those of cancellous bone. In the initial cell culture test, osteoblasts adhered well to the scaffold surface during a short time, and then grew smoothly into the interconnected pore channels. These results indicate that the porous TiNi scaffolds fabricated by this method could be bone substitute materials. - Highlights: • A novel approach for the fabrication of porous TiNi scaffolds • Macroporous structures are replicated from the polymer sponge template. • The pore characteristics and mechanical properties of TiNi scaffolds agree well with the requirement of trabecular bone. • Cytocompatibility of TiNi scaffolds is assessed, and it closely associated with pore property.

  6. Fabrication of polylactide nanocomposite scaffolds for bone tissue engineering applications

    Mkhabela, Vuyiswa J.; Ray, Suprakas Sinha

    2015-05-01

    Highly porous three-dimensional polylactide (PLA) scaffolds were obtained from PLA incorporated with different amounts of chitosan-modified montmorillonite (CS-MMT), through solvent casting and particulate leaching method. The processed scaffolds were tested in vitro for their possible application in bone tissue engineering. Scaffolds were characterized by Focused Ion Beam Scanning Electron Microscopy (FIB SEM), Fourier Transform Infra-Red (FTIR), and X-Ray Diffraction (XRD) to study their structure and intermolecular interactions. Bioresorbability tests in simulated body fluid (pH 7.4) were conducted to assess the response of the scaffolds in a simulated physiological condition. The FIB SEM images of the scaffolds showed a porous architecture with gradual change in morphology with increasing CS-MMT concentration. FTIR analysis revealed the presence of both PLA and CS-MMT particles on the surface of the scaffolds. XRD showed that the crystalline unit cell type was the same for all the scaffolds, and crystallinity decreased with an increase in CS-MMT concentration. The scaffolds were found to be bioresorbable, with rapid bioresorbability on the scaffolds with a high CS-MMT concentration.

  7. A post-synaptic scaffold at the origin of the animal kingdom.

    Onur Sakarya

    Full Text Available BACKGROUND: The evolution of complex sub-cellular structures such as the synapse requires the assembly of multiple proteins, each conferring added functionality to the integrated structure. Tracking the early evolution of synapses has not been possible without genomic information from the earliest branching animals. As the closest extant relatives to the Eumetazoa, Porifera (sponges represent a pivotal group for understanding the evolution of nervous systems, because sponges lack neurons with clearly recognizable synapses, in contrast to eumetazoan animals. METHODOLOGY/PRINCIPAL FINDINGS: We show that the genome of the demosponge Amphimedon queenslandica possesses a nearly complete set of post-synaptic protein homologs whose conserved interaction motifs suggest assembly into a complex structure. In the critical synaptic scaffold gene, dlg, residues that make hydrogen bonds and van der Waals interactions with the PDZ ligand are 100% conserved between sponge and human, as is the motif organization of the scaffolds. Expression in Amphimedon of multiple post-synaptic gene homologs in larval flask cells further supports the existence of an assembled structure. Among the few post-synaptic genes absent from Amphimedon, but present in Eumetazoa, are receptor genes including the entire ionotropic glutamate receptor family. CONCLUSIONS/SIGNIFICANCE: Highly conserved protein interaction motifs and co-expression in sponges of multiple proteins whose homologs interact in eumetazoan synapses indicate that a complex protein scaffold was present at the origin of animals, perhaps predating nervous systems. A relatively small number of crucial innovations to this pre-existing structure may represent the founding changes that led to a post-synaptic element.

  8. Degradation and osteogenic potential of a novel poly(lactic acid/nano-sized β-tricalcium phosphate scaffold

    Cao L

    2012-11-01

    Full Text Available Lu Cao,1,2 Ping-Guo Duan,1,2 Hui-Ren Wang,1,2 Xi-Lei Li,1,2 Feng-Lai Yuan,3 Zhong-Yong Fan,4 Su-Ming Li,5 Jian Dong1,21Department of Orthopedic Surgery, Zhongshan Hospital, Fudan University, Shanghai, China; 2State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai, China; 3Affiliated Third Hospital of Nantong University, Wuxi, Jiangsu, China; 4Department of Materials Science, Fudan University, Shanghai, China; 5Max Mousseron Institute on Biomolecules, Montpellier I University, Montpellier, FranceAbstract: The purpose of this study was to investigate the influence of nano-sized β-tricalcium phosphate (β-TCP on the biological performance of poly (lactic acid (PLA composite scaffolds by using in vitro degradation and an in vivo model of heterotopic bone formation. Nano-sized βTCP (nβ-TCP was prepared with a wet grinding method from micro-sized β-TCP (mβ-TCP, and composite scaffolds containing 0, 10, 30, or 50 wt% nβ-TCP or 30 wt% mβ -TCP were generated using a freeze-drying method. Degradation was assessed by monitoring changes in microstructure, pH, weight, and compressive strength over a 26-week period of hydrolysis. Composite scaffolds were processed into blocks, and implanted into muscular pockets of rabbits after loading with recombinant human bone morphogenetic protein-2 (rhBMP-2. New bone formation was evaluated based on histological and immunohistochemical analysis 2, 4, and 8 weeks after implantation. The in vitro results indicated that the buffering effect of nβ-TCP was stronger than mβ-TCP, which was positively correlated with the content of nβ-TCP. The in vivo findings demonstrated that nβ-TCP enhanced the osteoconductivity of the scaffolds. Although composite scaffolds containing 30% nβ-TCP exhibited similar osteoconductivity to 50% nβ-TCP, they had better mechanical properties than the 50% nβ-TCP scaffolds. This study supports the potential application of a composite scaffold containing 30

  9. Protein turnover during in vitro tissue engineering.

    Li, Qiyao; Chang, Zhen; Oliveira, Gisele; Xiong, Maiyer; Smith, Lloyd M; Frey, Brian L; Welham, Nathan V

    2016-03-01

    Repopulating acellular biological scaffolds with phenotypically appropriate cells is a promising approach for regenerating functional tissues and organs. Under this tissue engineering paradigm, reseeded cells are expected to remodel the scaffold by active protein synthesis and degradation; however, the rate and extent of this remodeling remain largely unknown. Here, we present a technique to measure dynamic proteome changes during in vitro remodeling of decellularized tissue by reseeded cells, using vocal fold mucosa as the model system. Decellularization and recellularization were optimized, and a stable isotope labeling strategy was developed to differentiate remnant proteins constituting the original scaffold from proteins newly synthesized by reseeded cells. Turnover of matrix and cellular proteins and the effects of cell-scaffold interaction were elucidated. This technique sheds new light on in vitro tissue remodeling and the process of tissue regeneration, and is readily applicable to other tissue and organ systems. PMID:26724458

  10. Development of rheological characterization and twin-screw extrusion/spiral winding processing methods for functionally-graded tissue engineering scaffolds and characterization of cell/biomaterial interactions

    Ozkan, Seher

    Tissue engineering involves the fabrication of biodegradable scaffolds, on which various types of cells are grown, to provide tissue constructs for tissue repair/regeneration. Native tissues have complex structures, with functions and properties changing spatially and temporally, and require special tailoring of tissue engineering scaffolds to allow mimicking of their complex elegance. The understanding of the rheological behavior of the biodegradable polymer and the thermo-mechanical history that the polymer experiences during processing is critical in fabricating scaffolds with appropriate microstructural distributions. This study has first focused on the rheological material functions of various gel-like fluids including biofluids and hydrogels, which can emulate the viscoelastic behavior of biofluids. Viscoplasticity and wall slip were recognized as key attributes of such systems. Furthermore, a new technology base involving twin-screw extrusion/spiral winding (TSESW) process was developed for the shaping of functionally-graded scaffolds. This novel scaffold fabrication technology was applied to the development of polycaprolactone (PCL) scaffolds, incorporated with tricalcium phosphate nanoparticles and various porogens in graded fashion. The protein encapsulation and controlled release capabilities of the TSESW process was also demonstrated by dispersing bovine serum albumin (BSA) protein into the PCL matrix. Effects of processing conditions and porosity distributions on compressive properties, surface topography, encapsulation efficiency, release profiles and the secondary structure of BSA were investigated. The PCL scaffolds were determined to be biocompatible, with the proliferation rates of human fetal osteoblast cells (hFOB) increasing with increasing porosity and decreasing concentration of TCP. BSA proteins were determined to be denatured to a greater extent with melt extrusion in the 80-100°C range (in comparison to wet extrusion using organic

  11. Artificial Affinity Proteins as Ligands of Immunoglobulins

    Barbara Mouratou

    2015-01-01

    Full Text Available A number of natural proteins are known to have affinity and specificity for immunoglobulins. Some of them are widely used as reagents for detection or capture applications, such as Protein G and Protein A. However, these natural proteins have a defined spectrum of recognition that may not fit specific needs. With the development of combinatorial protein engineering and selection techniques, it has become possible to design artificial affinity proteins with the desired properties. These proteins, termed alternative scaffold proteins, are most often chosen for their stability, ease of engineering and cost-efficient recombinant production in bacteria. In this review, we focus on alternative scaffold proteins for which immunoglobulin binders have been identified and characterized.

  12. 12S-lipoxygenase protein associates with α-actin fibers in human umbilical artery vascular smooth muscle cells

    The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to α-actin, a component of the cytoplasmic myofilaments. 12-LO/α-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to α-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein α-actin

  13. Compression of Multilayered Composite Electrospun Scaffolds: A Novel Strategy to Rapidly Enhance Mechanical Properties and Three Dimensionality of Bone Scaffolds

    Parthasarathy A. Madurantakam

    2013-01-01

    Full Text Available One major limitation of electrospun scaffolds intended for bone tissue engineering is their inferior mechanical properties. The present study introduces a novel strategy to engineer stiffer scaffolds by stacking multiple layers and cold welding them under high pressure. Electrospun polydioxanone (PDO and PDO:nanohydroxyapatite (PDO:nHA scaffolds (1, 2, or 4 layered stacks were compressed either before or after mineralizing treatment with simulated body fluid (SBF. After two weeks in SBF, scaffolds were analyzed for total mineral content and stiffness by Alizarin red S and uniaxial tensile testing, respectively. Scaffolds were also analyzed for permeability, pore size, and fiber diameter. Results indicated that compression of multiple layers significantly increased the stiffness of scaffolds while reducing mineralization and permeability. This phenomenon was attributed to increased density of fibers and loss of surface area due to fiber welding. Statistics revealed, the 4-layered PDO:nHA scaffold compressed first followed by mineralization in revised SBF had maximal stiffness, low permeability and pore size, and mineralization second only to noncompressed scaffolds. Within the limitations of permeability and pore size, this scaffold configuration represents an optimal midway for desired stiffness and mineral content for bone tissue engineering.

  14. Taperin (c9orf75, a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1α docking protein

    Tony Ferrar

    2012-02-01

    The promiscuous activity of protein phosphatase one (PP1 is controlled in the cell by associated proteins termed regulatory or targeting subunits. Using biochemical and proteomic approaches we demonstrate that the autosomal recessive nonsyndromic hearing loss gene, taperin (C9orf75, encodes a protein that preferentially docks the alpha isoform of PP1. Taperin associates with PP1 through a classic ‘RVxF’ motif and suppresses the general phosphatase activity of the enzyme. The steady-state localization of taperin is predominantly nuclear, however we demonstrate here that the protein can shuttle between the nucleus and cytoplasm and that it is found complexed to PP1 in both of these cellular compartments. Although originally identified as a hearing loss gene, Western blot analyses with taperin-specific antibodies revealed that the protein is widely expressed across mammalian tissues as multiple splice variants. Taperin is a recent proteome addition appearing during the vertebrate lineage with the PP1 binding site embedded within the most conserved region of the protein. Taperin also shares an ancestral relationship with the cytosolic actin binding protein phostensin, another PP1 interacting partner. Quantitative Stable Isotope Labeling by Amino acids in Culture (SILAC-based mass spectrometry was employed to uncover additional taperin binding partners, and revealed an interaction with the DNA damage response proteins Ku70, Ku80, PARP and topoisomerases I and IIα. Consistent with this, we demonstrate the active recruitment of taperin to sites of DNA damage. This makes taperin a new addition to the family of PP1 targeting subunits involved in the DNA damage repair pathway.

  15. Bio-functionalization of polycaprolactone infiltrated BCP scaffold with silicon and fibronectin enhances osteoblast activity in vitro

    Kwak, Kyung-A.; Kim, Young-Hee; Kim, Minsung; Lee, Byong-Taek; Song, Ho-Yeon

    2013-08-01

    The surface property of a biomaterial plays a fundamental role in cell attachment, proliferation, differentiation, resorption, and biomolecular expression. In this study, the surface of a polycaprolactone-infiltrated biphasic calcium phosphate scaffold was biofunctionalized by silicon (Si) and fibronectin (FN) coating to evaluate the osteoblast-like cells activity in vitro. The surfaces of all scaffolds were characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), whereas the presence of the functional group was determined by Fourier-transform infrared spectroscopy (FT-IR). Coomassie brilliant blue staining was applied to confirm the presence of FN on the scaffold surface. The in vitro bioactivity of the osteoblast-like cells was determined by one cell morphology and proliferation assay at 3, 7, and 14 days by SEM. Cell viability assay by MTT showed higher cell viability rate on coated scaffolds than in those coated with Si only or non-coated surfaces. The mRNA expressions of alkaline phosphatase (ALP) and osteocalcin (OC) were determined using RT-PCR and the expressions of osteopontin (OPN), type I collagen, and osteocalcin (OC) proteins were determined using Western blot. Thus the expression of genes and proteins further confirmed both early and intermediate phases of osteoblast-like cell activity which was found increased by Si-and Fn coating on PCL infiltrated BCP surfaces.

  16. Bio-functionalization of polycaprolactone infiltrated BCP scaffold with silicon and fibronectin enhances osteoblast activity in vitro

    Kwak, Kyung-A.; Kim, Young-Hee [Department of Microbiology and Immunology, School of Medicine, Soonchunhyang University, Cheonan, Chungnum 330-090 (Korea, Republic of); Kim, Minsung; Lee, Byong-Taek [Department of Biomedical Engineering and Materials, School of Medicine, Soonchunhyang University, Cheonan, Chungnum 330-090 (Korea, Republic of); Song, Ho-Yeon, E-mail: songmic@sch.ac.kr [Department of Microbiology and Immunology, School of Medicine, Soonchunhyang University, Cheonan, Chungnum 330-090 (Korea, Republic of)

    2013-08-15

    The surface property of a biomaterial plays a fundamental role in cell attachment, proliferation, differentiation, resorption, and biomolecular expression. In this study, the surface of a polycaprolactone-infiltrated biphasic calcium phosphate scaffold was biofunctionalized by silicon (Si) and fibronectin (FN) coating to evaluate the osteoblast-like cells activity in vitro. The surfaces of all scaffolds were characterized by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), whereas the presence of the functional group was determined by Fourier-transform infrared spectroscopy (FT-IR). Coomassie brilliant blue staining was applied to confirm the presence of FN on the scaffold surface. The in vitro bioactivity of the osteoblast-like cells was determined by one cell morphology and proliferation assay at 3, 7, and 14 days by SEM. Cell viability assay by MTT showed higher cell viability rate on coated scaffolds than in those coated with Si only or non-coated surfaces. The mRNA expressions of alkaline phosphatase (ALP) and osteocalcin (OC) were determined using RT-PCR and the expressions of osteopontin (OPN), type I collagen, and osteocalcin (OC) proteins were determined using Western blot. Thus the expression of genes and proteins further confirmed both early and intermediate phases of osteoblast-like cell activity which was found increased by Si-and Fn coating on PCL infiltrated BCP surfaces.

  17. Silk fibroin porous scaffolds for nucleus pulposus tissue engineering

    Zeng, Chao; Yang, Qiang [Department of Spine Surgery, Tianjin Hospital, Tianjin 300211 (China); Tianjin Medical University, Tianjin 300070 (China); Zhu, Meifeng [The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071 (China); Du, Lilong [Department of Spine Surgery, Tianjin Hospital, Tianjin 300211 (China); Tianjin Medical University, Tianjin 300070 (China); Zhang, Jiamin [The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ma, Xinlong [Department of Spine Surgery, Tianjin Hospital, Tianjin 300211 (China); Xu, Baoshan, E-mail: xubaoshan99@126.com [Department of Spine Surgery, Tianjin Hospital, Tianjin 300211 (China); Wang, Lianyong, E-mail: wly@nankai.edu.cn [The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2014-04-01

    Intervertebral discs (IVDs) are structurally complex tissue that hold the vertebrae together and provide mobility to spine. The nucleus pulposus (NP) degeneration often results in degenerative IVD disease that is one of the most common causes of back and neck pain. Tissue engineered nucleus pulposus offers an alternative approach to regain the function of the degenerative IVD. The aim of this study is to determine the feasibility of porous silk fibroin (SF) scaffolds fabricated by paraffin-sphere-leaching methods with freeze-drying in the application of nucleus pulposus regeneration. The prepared scaffold possessed high porosity of 92.38 ± 5.12% and pore size of 165.00 ± 8.25 μm as well as high pore interconnectivity and appropriate mechanical properties. Rabbit NP cells were seeded and cultured on the SF scaffolds. Scanning electron microscopy, histology, biochemical assays and mechanical tests revealed that the porous scaffolds could provide an appropriate microstructure and environment to support adhesion, proliferation and infiltration of NP cells in vitro as well as the generation of extracellular matrix. The NP cell–scaffold construction could be preliminarily formed after subcutaneously implanted in a nude mice model. In conclusion, The SF porous scaffold offers a potential candidate for tissue engineered NP tissue. - Highlights: • Paraffin microsphere-leaching method is used to fabricate silk fibroin scaffold. • The scaffold has appropriate mechanical property, porosity and pore size • The scaffold supports growth and infiltration of nucleus pulposus cells. • Nucleus pulposus cells can secrete extracellular matrix in the scaffolds. • The scaffold is a potential candidate for tissue engineered nucleus pulposus.

  18. Scaffolds and cells for tissue regeneration: different scaffold pore sizes-different cell effects.

    Bružauskaitė, Ieva; Bironaitė, Daiva; Bagdonas, Edvardas; Bernotienė, Eiva

    2016-05-01

    During the last decade biomaterial sciences and tissue engineering have become new scientific fields supplying rising demand of regenerative therapy. Tissue engineering requires consolidation of a broad knowledge of cell biology and modern biotechnology investigating biocompatibility of materials and their application for the reconstruction of damaged organs and tissues. Stem cell-based tissue regeneration started from the direct cell transplantation into damaged tissues or blood vessels. However, it is difficult to track transplanted cells and keep them in one particular place of diseased organ. Recently, new technologies such as cultivation of stem cell on the scaffolds and subsequently their implantation into injured tissue have been extensively developed. Successful tissue regeneration requires scaffolds with particular mechanical stability or biodegradability, appropriate size, surface roughness and porosity to provide a suitable microenvironment for the sufficient cell-cell interaction, cell migration, proliferation and differentiation. Further functioning of implanted cells highly depends on the scaffold pore sizes that play an essential role in nutrient and oxygen diffusion and waste removal. In addition, pore sizes strongly influence cell adhesion, cell-cell interaction and cell transmigration across the membrane depending on the various purposes of tissue regeneration. Therefore, this review will highlight contemporary tendencies in application of non-degradable scaffolds and stem cells in regenerative medicine with a particular focus on the pore sizes significantly affecting final recover of diseased organs. PMID:26091616

  19. The migration and differentiation of hUC-MSCs(CXCR4/GFP) encapsulated in BDNF/chitosan scaffolds for brain tissue engineering.

    Huang, Chuanjun; Zhao, Longxiang; Gu, Jun; Nie, Dekang; Chen, Yinan; Zuo, Hao; Huan, Wei; Shi, Jinlong; Chen, Jian; Shi, Wei

    2016-01-01

    We previously developed a biomaterial scaffold that could effectively provide seed cells to a lesion cavity resulting from traumatic brain injury. However, we subsequently found that few transplanted human umbilical cord mesenchymal stem cells (hUC-MSCs) are able to migrate from the scaffold to the lesion boundary. Stromal derived-cell factor-1α and its receptor chemokine (C-X-C motif) receptor (CXCR)4 are chemotactic factors that control cell migration and stem cell recruitment to target areas. Given the low expression level of CXCR4 on the hUC-MSC membrane, lentiviral vectors were used to generate hUC-MSCs stably expressing CXCR4 fused to green fluorescent protein (GFP) (hUC-MSCs(CXCR4/GFP)). We constructed a scaffold in which recombinant human brain-derived neurotrophic factor (BDNF) was linked to chitosan scaffolds with the crosslinking agent genipin (CGB scaffold). The scaffold containing hUC-MSCs(CXCR4/GFP) was transplanted into the lesion cavity of a rat brain, providing exogenous hUC-MSCs to both lesion boundary and cavity. These results demonstrate a novel strategy for inducing tissue regeneration after traumatic brain injury. PMID:27147644

  20. Fabrication of Core-Shell PEI/pBMP2-PLGA Electrospun Scaffold for Gene Delivery to Periodontal Ligament Stem Cells

    Qiao Xie

    2016-01-01

    Full Text Available Bone tissue engineering is the most promising technology for enhancing bone regeneration. Scaffolds loaded with osteogenic factors improve the therapeutic effect. In this study, the bioactive PEI (polyethylenimine/pBMP2- (bone morphogenetic protein-2 plasmid- PLGA (poly(D, L-lactic-co-glycolic acid core-shell scaffolds were prepared using coaxial electrospinning for a controlled gene delivery to hPDLSCs (human periodontal ligament stem cells. The pBMP2 was encapsulated in the PEI phase as a core and PLGA was employed to control pBMP2 release as a shell. First, the scaffold characterization and mechanical properties were evaluated. Then the gene release behavior was analyzed. Our results showed that pBMP2 was released at high levels in the first few days, with a continuous release behavior in the next 28 days. At the same time, PEI/pBMP2 showed high transfection efficiency. Moreover, the core-shell electrospun scaffold showed BMP2 expression for a much longer time (more than 28 days compared with the single axial electrospun scaffold, as evaluated by qRT-PCR and western blot after culturing with hPDLSCs. These results suggested that the core-shell PEI/pBMP2-PLGA scaffold fabricated by coaxial electrospinning had a good gene release behavior and showed a prolonged expression time with a high transfection efficiency.