Science.gov

Sample records for 3d extracellular matrix

  1. 3D Extracellular Matrix from Sectioned Human Tissues

    PubMed Central

    Campbell, Catherine B; Cukierman, Edna; Artym, Vira V

    2014-01-01

    Three-dimensional (3D) matrices have significant advantages compared to conventional two-dimensional (2D) matrices for studying cell adhesion, migration, and tissue organization. Cellular behavior is dependent on the surrounding matrix environment for signaling and induction of biological responses (Carletti, et al., 2011; Pampaloni, et al., 2007; Vlodavsky, 1999). 2D cultures induce an artificial polarity in cultured cells between upper and lower surfaces not present normally in the in vivo environment. No longer nonpolar, many aspects of cellular behavior are altered (Beacham, et al., 2007; Grinnell and Petroll, 2010; Yamada and Cukierman, 2007). In addition, 2D models lack the physical properties of 3D matrix, such as topography, stiffness, and dimensionality. To begin to mimic the 3D environment of in vivo connective tissue extracellular matrix (ECM), collagen gels have been used widely (see Unit 10.3). Culture of cells in collagen gels results in a bipolar fibroblast morphology that resembles the in vivo phenotype (Friedl and Brocker, 2000; Even-Ram and Yamada, 2005; Grinnell and Petroll, 2010). Although more physiological, 3D collagen gels lack the complex biochemical and physical microenvironment present in an in vivo ECM that regulates cellular physiological properties (Beacham, et al., 2007). A variety of methods to create a more in vivo-like ECM have been published (Yamada and Cukierman, 2007). Adding critical ECM components to 3D collagen matrices, including fibronectin, hyaluronan, link protein and glycosaminoglycans, can more accurately mimic the structural microenvironment of the native ECM (Friedl and Brocker, 2000). Other ECM models use cultured cell lines, such as fibroblasts, to derive an ECM lattice through secretion of an organized ECM (Beacham, et al., 2007). Different cell lines have been chosen to generate a specific microenvironment for study of particularly types of cellular behavior (Kutys and Yamada, 2013). For example, cultured bovine

  2. Quantitative analysis of 3D extracellular matrix remodelling by pancreatic stellate cells

    PubMed Central

    Robinson, Benjamin K.; Cortes, Ernesto; Rice, Alistair J.; Sarper, Muge

    2016-01-01

    ABSTRACT Extracellular matrix (ECM) remodelling is integral to numerous physiological and pathological processes in biology, such as embryogenesis, wound healing, fibrosis and cancer. Until recently, most cellular studies have been conducted on 2D environments where mechanical cues significantly differ from physiologically relevant 3D environments, impacting cellular behaviour and masking the interpretation of cellular function in health and disease. We present an integrated methodology where cell-ECM interactions can be investigated in 3D environments via ECM remodelling. Monitoring and quantification of collagen-I structure in remodelled matrices, through designated algorithms, show that 3D matrices can be used to correlate remodelling with increased ECM stiffness observed in fibrosis. Pancreatic stellate cells (PSCs) are the key effectors of the stromal fibrosis associated to pancreatic cancer. We use PSCs to implement our methodology and demonstrate that PSC matrix remodelling capabilities depend on their contractile machinery and β1 integrin-mediated cell-ECM attachment. PMID:27170254

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

    PubMed

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

    2015-11-01

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

  4. Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

    PubMed Central

    El-Hamidi, Hamid; Celli, Jonathan P.

    2014-01-01

    The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(ω). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic

  5. Heterogeneous Differentiation of Human Mesenchymal Stem Cells in 3D Extracellular Matrix Composites

    PubMed Central

    Jung, Jangwook P.; Bache-Wiig, Meredith K.; Provenzano, Paolo P.; Ogle, Brenda M.

    2016-01-01

    Abstract Extracellular matrix (ECM) proteins are structural elements of tissue and also potent signaling molecules. Previously, our laboratory showed that ECM of 2D coatings can trigger differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into mesodermal lineages in an ECM-specific manner over 14 days, in some cases comparable to chemical induction. To test whether a similar effect was possible in a 3D, tissue-like environment, we designed a synthetic-natural biomaterial composite. The composite can present whole-molecule ECM proteins to cells, even those that do not spontaneously form hydrogels ex vivo, in 3D. To this end, we entrapped collagen type I, laminin-111, or fibronectin in ECM composites with MSCs and directly compared markers of mesodermal differentiation including cardiomyogenic (ACTC1), osteogenic (SPP1), adipogenic (PPARG), and chondrogenic (SOX9) in 2D versus 3D. We found the 3D condition largely mimicked the 2D condition such that the addition of type I collagen was the most potent inducer of differentiation to all lineages tested. One notable difference between 2D and 3D was pronounced adipogenic differentiation in 3D especially in the presence of exogenous collagen type I. In particular, PPARG gene expression was significantly increased ∼16-fold relative to chemical induction, in 3D and not in 2D. Unexpectedly, 3D engagement of ECM proteins also altered immunomodulatory function of MSCs in that expression of IL-6 gene was elevated relative to basal levels in 2D. In fact, levels of IL-6 gene expression in 3D composites containing exogenously supplied collagen type I or fibronectin were statistically similar to levels attained in 2D with tumor necrosis factor-α (TNF-α) stimulation and these levels were sustained over a 2-week period. Thus, this novel biomaterial platform allowed us to compare the biochemical impact of whole-molecule ECM proteins in 2D versus 3D indicating enhanced adipogenic differentiation and IL-6 expression

  6. Heterogeneous Differentiation of Human Mesenchymal Stem Cells in 3D Extracellular Matrix Composites.

    PubMed

    Jung, Jangwook P; Bache-Wiig, Meredith K; Provenzano, Paolo P; Ogle, Brenda M

    2016-01-01

    Extracellular matrix (ECM) proteins are structural elements of tissue and also potent signaling molecules. Previously, our laboratory showed that ECM of 2D coatings can trigger differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into mesodermal lineages in an ECM-specific manner over 14 days, in some cases comparable to chemical induction. To test whether a similar effect was possible in a 3D, tissue-like environment, we designed a synthetic-natural biomaterial composite. The composite can present whole-molecule ECM proteins to cells, even those that do not spontaneously form hydrogels ex vivo, in 3D. To this end, we entrapped collagen type I, laminin-111, or fibronectin in ECM composites with MSCs and directly compared markers of mesodermal differentiation including cardiomyogenic (ACTC1), osteogenic (SPP1), adipogenic (PPARG), and chondrogenic (SOX9) in 2D versus 3D. We found the 3D condition largely mimicked the 2D condition such that the addition of type I collagen was the most potent inducer of differentiation to all lineages tested. One notable difference between 2D and 3D was pronounced adipogenic differentiation in 3D especially in the presence of exogenous collagen type I. In particular, PPARG gene expression was significantly increased ∼16-fold relative to chemical induction, in 3D and not in 2D. Unexpectedly, 3D engagement of ECM proteins also altered immunomodulatory function of MSCs in that expression of IL-6 gene was elevated relative to basal levels in 2D. In fact, levels of IL-6 gene expression in 3D composites containing exogenously supplied collagen type I or fibronectin were statistically similar to levels attained in 2D with tumor necrosis factor-α (TNF-α) stimulation and these levels were sustained over a 2-week period. Thus, this novel biomaterial platform allowed us to compare the biochemical impact of whole-molecule ECM proteins in 2D versus 3D indicating enhanced adipogenic differentiation and IL-6 expression of MSC in

  7. Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration.

    PubMed

    Pati, Falguni; Song, Tae-Ha; Rijal, Girdhari; Jang, Jinah; Kim, Sung Won; Cho, Dong-Woo

    2015-01-01

    3D printing technique is the most sophisticated technique to produce scaffolds with tailorable physical properties. But, these scaffolds often suffer from limited biological functionality as they are typically made from synthetic materials. Cell-laid mineralized ECM was shown to be potential for improving the cellular responses and drive osteogenesis of stem cells. Here, we intend to improve the biological functionality of 3D-printed synthetic scaffolds by ornamenting them with cell-laid mineralized extracellular matrix (ECM) that mimics a bony microenvironment. We developed bone graft substitutes by using 3D printed scaffolds made from a composite of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP) and mineralized ECM laid by human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs). A rotary flask bioreactor was used to culture hTMSCs on the scaffolds to foster formation of mineralized ECM. A freeze/thaw cycle in hypotonic buffer was used to efficiently decellularize (97% DNA reduction) the ECM-ornamented scaffolds while preserving its main organic and inorganic components. The ECM-ornamented 3D printed scaffolds supported osteoblastic differentiation of newly-seeded hTMSCs by upregulating four typical osteoblastic genes (4-fold higher RUNX2; 3-fold higher ALP; 4-fold higher osteocalcin; and 4-fold higher osteopontin) and increasing calcium deposition compared to bare 3D printed scaffolds. In vivo, in ectopic and orthotopic models in rats, ECM-ornamented scaffolds induced greater bone formation than that of bare scaffolds. These results suggest a valuable method to produce ECM-ornamented 3D printed scaffolds as off-the-shelf bone graft substitutes that combine tunable physical properties with physiological presentation of biological signals.

  8. BIOCOMPATIBILITY OF A SYNTHETIC EXTRACELLULAR MATRIX ON IMMORTALIZED VOCAL FOLD FIBROBLASTS IN 3D CULTURE

    PubMed Central

    Chen, Xia

    2010-01-01

    In order to promote wound repair and induce tissue regeneration, an engineered hyaluronan (HA) hydrogel – Carbylan GSX, which contains di(thiopropionyl) bishydrazide-modified hyaluronic acid (HA-DTPH), di(thiopropionyl) bishydrazide-modified gelatin (Gtn-DTPH) and polyethylene glycol diacrylate (PEGDA), has been developed for extracellular matrix (ECM) defects of the superficial and middle layers of the lamina propria. The purpose of this study was to evaluate the biocompatibility of Carbylan GSX in a previously established immortalized human vocal fold fibroblast (hVFF) cell line prior to human clinical trials. Immortalized hVFF proliferation, viability, apoptosis and transcript analysis for both ECM constituents and inflammatory markers were measured for two-dimensional and three-dimensional culture conditions. There were no significant differences in morphology, cell marker protein expression, proliferation, viability and apoptosis of hVFF cultured with Carbylan GSX compared to Matrigel, a commercial 3D control, after one week. Gene expression levels for fibromodulin, TGF-β1, and TNF-α were similar between Carbylan GSX and Matrigel. Fibronectin, hyaluronidase 1 and COX2 expression levels were induced by Carbylan GSX; whereas IL6, IL8. COL1 and hyaluronic acid synthase 3 expression levels were decreased by Carbylan GSX. This investigation demonstrates that Carbylan GSX may serve as a natural biomaterial for tissue engineering of human vocal folds. PMID:20109588

  9. Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cell response to extracellular matrix.

    PubMed

    Ma, Yufei; Ji, Yuan; Huang, Guoyou; Ling, Kai; Zhang, Xiaohui; Xu, Feng

    2015-01-01

    Periodontitis is an inflammatory disease negatively affecting up to 15% of adults worldwide. Periodontal ligament stem cells (PDLSCs) hold great promises for periodontal tissue regeneration, where it is necessary to find proper extracellular matrix (ECM) materials (e.g., composition, concentration). In this study, we proposed a bioprinting-based approach to generate nano-liter sized three-dimensional (3D) cell-laden hydrogel array with gradient of ECM components, through controlling the volume ratio of two hydrogels, such as gelatin methacrylate (GelMA) and poly(ethylene glycol) (PEG) dimethacrylate. The resulting cell-laden array with a gradient of GelMA/PEG composition was used to screen human PDLSC response to ECM. The behavior (e.g., cell viability, spreading) of human PDLSCs in GelMA/PEG array were found to be depended on the volume ratios of GelMA/PEG, with cell viability and spreading area decreased along with increasing the ratio of PEG. The developed approach would be useful for screening cell-biomaterial interaction in 3D and promoting regeneration of functional tissue.

  10. Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cell response to extracellular matrix.

    PubMed

    Ma, Yufei; Ji, Yuan; Huang, Guoyou; Ling, Kai; Zhang, Xiaohui; Xu, Feng

    2015-01-01

    Periodontitis is an inflammatory disease negatively affecting up to 15% of adults worldwide. Periodontal ligament stem cells (PDLSCs) hold great promises for periodontal tissue regeneration, where it is necessary to find proper extracellular matrix (ECM) materials (e.g., composition, concentration). In this study, we proposed a bioprinting-based approach to generate nano-liter sized three-dimensional (3D) cell-laden hydrogel array with gradient of ECM components, through controlling the volume ratio of two hydrogels, such as gelatin methacrylate (GelMA) and poly(ethylene glycol) (PEG) dimethacrylate. The resulting cell-laden array with a gradient of GelMA/PEG composition was used to screen human PDLSC response to ECM. The behavior (e.g., cell viability, spreading) of human PDLSCs in GelMA/PEG array were found to be depended on the volume ratios of GelMA/PEG, with cell viability and spreading area decreased along with increasing the ratio of PEG. The developed approach would be useful for screening cell-biomaterial interaction in 3D and promoting regeneration of functional tissue. PMID:26696269

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

    PubMed

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

    2016-01-01

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

  12. Extracellular matrix composition and rigidity regulate invasive behavior and response to PDT in 3D pancreatic tumor models

    NASA Astrophysics Data System (ADS)

    Cramer, Gwendolyn; El-Hamidi, Hamid; Jafari, Seyedehrojin; Jones, Dustin P.; Celli, Jonathan P.

    2016-03-01

    The composition and mechanical compliance of the extracellular matrix (ECM) have been shown to serve as regulators of tumor growth and invasive behavior. These effects may be particularly relevant in tumors of the pancreas, noted for a profound desmoplastic reaction and an abundance of stroma rich in ECM. In view of recent progress in the clinical implementation of photodynamic therapy (PDT) for pancreatic tumors, in this report we examine how ECM composition and rheological properties impact upon invasive behavior and response to PDT in 3D multicellular pancreatic tumor spheroids in ECM environments with characterized rheological properties. Tumor spheroids were cultured initially in attachment-free conditions to form millimeter-sized spheroids that were transplanted into reconstituted ECM microenvironments (Matrigel and Type I Collagen) that were characterized using bulk oscillatory shear rheology. Analysis of growth behavior shows that the soft collagen ECM promoted growth and extensive invasion and this microenvironment was used in subsequent assessment of PDT and chemotherapy response. Evaluation of treatment response revealed that primary tumor nodule growth is inhibited more effectively with PDT, while verteporfin PDT response is significantly enhanced in the ECM-infiltrating populations that are non-responsive to oxaliplatin chemotherapy. This finding is potentially significant, suggesting the potential for PDT to target these clinically problematic invasive populations that are associated with aggressive metastatic progression and chemoresistance. Experiments to further validate and identify the mechanistic basis of this observation are ongoing.

  13. Bile canaliculi formation and biliary transport in 3D sandwich-cultured hepatocytes in dependence of the extracellular matrix composition.

    PubMed

    Deharde, Daniela; Schneider, Christin; Hiller, Thomas; Fischer, Nicolas; Kegel, Victoria; Lübberstedt, Marc; Freyer, Nora; Hengstler, Jan G; Andersson, Tommy B; Seehofer, Daniel; Pratschke, Johann; Zeilinger, Katrin; Damm, Georg

    2016-10-01

    Primary human hepatocytes (PHH) are still considered as gold standard for investigation of in vitro metabolism and hepatotoxicity in pharmaceutical research. It has been shown that the three-dimensional (3D) cultivation of PHH in a sandwich configuration between two layers of extracellular matrix (ECM) enables the hepatocytes to adhere three dimensionally leading to formation of in vivo like cell-cell contacts and cell-matrix interactions. The aim of the present study was to investigate the influence of different ECM compositions on morphology, cellular arrangement and bile canaliculi formation as well as bile excretion processes in PHH sandwich cultures systematically. Freshly isolated PHH were cultured for 6 days between two ECM layers made of collagen and/or Matrigel in four different combinations. The cultures were investigated by phase contrast microscopy and immunofluorescence analysis with respect to cell-cell connections, repolarization as well as bile canaliculi formation. The influence of the ECM composition on cell activity and viability was measured using the XTT assay and a fluorescent dead or alive assay. Finally, the bile canalicular transport was analyzed by live cell imaging to monitor the secretion and accumulation of the fluorescent substance CDF in bile canaliculi. Using collagen and Matrigel in different compositions in sandwich cultures of hepatocytes, we observed differences in morphology, cellular arrangement and cell activity of PHH in dependence of the ECM composition. Sandwich-cultured hepatocytes with an underlay of collagen seem to represent the best in vivo tissue architecture in terms of formation of trabecular cell arrangement. Cultures overlaid with collagen were characterized by the formation of abundant bile canaliculi, while the bile canaliculi network in hepatocytes cultured on a layer of Matrigel and overlaid with collagen showed the most branched and stable canalicular network. All cultures showed a time-dependent leakage of

  14. Leishmania amazonensis promastigotes in 3D Collagen I culture: an in vitro physiological environment for the study of extracellular matrix and host cell interactions

    PubMed Central

    Rodrigues, Juliany C.F.; Viana, Nathan B.; Pontes, Bruno; Pereira, Camila F.A.; Silva-Filho, Fernando C.

    2014-01-01

    Leishmania amazonensis is the causative agent of American cutaneous leishmaniasis, an important neglected tropical disease. Once Leishmania amazonensis is inoculated into the human host, promastigotes are exposed to the extracellular matrix (ECM) of the dermis. However, little is known about the interaction between the ECM and Leishmania promastigotes. In this study we established L. amazonensis promastigote culture in a three-dimensional (3D) environment mainly composed of Collagen I (COL I). This 3D culture recreates in vitro some aspects of the human host infection site, enabling the study of the interaction mechanisms of L. amazonensis with the host ECM. Promastigotes exhibited “freeze and run” migration in the 3D COL I matrix, which is completely different from the conventional in vitro swimming mode of migration. Moreover, L. amazonensis promastigotes were able to invade, migrate inside, and remodel the 3D COL I matrix. Promastigote trans-matrix invasion and the freeze and run migration mode were also observed when macrophages were present in the matrix. At least two classes of proteases, metallo- and cysteine proteases, are involved in the 3D COL I matrix degradation caused by Leishmania. Treatment with a mixture of protease inhibitors significantly reduced promastigote invasion and migration through this matrix. Together our results demonstrate that L. amazonensis promastigotes release proteases and actively remodel their 3D environment, facilitating their migration. This raises the possibility that promastigotes actively interact with their 3D environment during the search for their cellular “home”—macrophages. Supporting this hypothesis, promastigotes migrated faster than macrophages in a novel 3D co-culture model. PMID:24765565

  15. Integrating focal adhesion dynamics, cytoskeleton remodeling, and actin motor activity for predicting cell migration on 3D curved surfaces of the extracellular matrix.

    PubMed

    Kim, Min-Cheol; Kim, Choong; Wood, Levi; Neal, Devin; Kamm, Roger D; Asada, H Harry

    2012-11-01

    An integrative cell migration model incorporating focal adhesion (FA) dynamics, cytoskeleton and nucleus remodeling and actin motor activity is developed for predicting cell migration behaviors on 3-dimensional curved surfaces, such as cylindrical lumens in the 3-D extracellular matrix (ECM). The work is motivated by 3-D microfluidic migration experiments suggesting that the migration speed and direction may vary depending on the cross sectional shape of the lumen along which the cell migrates. In this paper, the mechanical structure of the cell is modeled as double elastic membranes of cell and nucleus. The two elastic membranes are connected by stress fibers, which are extended from focal adhesions on the cell surface to the nuclear membrane. The cell deforms and gains traction as transmembrane integrins distributed over the outer cell membrane bind to ligands on the ECM, form focal adhesions, and activate stress fibers. Probabilities at which integrin ligand-receptor bonds are formed as well as ruptures are affected by the surface geometry, resulting in diverse migration behaviors that depend on the curvature of the surface. Monte Carlo simulations of the integrative model reveal that (a) the cell migration speed is dependent on the cross sectional area of the lumen with a maximum speed at a particular diameter or width, (b) as the lumen diameter increases, the cell tends to spread and migrate around the circumference of the lumen, while it moves in the longitudinal direction as the lumen diameter narrows, (c) once the cell moves in one direction, it tends to stay migrating in the same direction despite the stochastic nature of migration. The relationship between the cell migration speed and the lumen width agrees with microfluidic experimental data for cancer cell migration.

  16. Real-time analysis of integrin-mediated chemotactic migration of T lymphocytes within 3-D extracellular matrix-like gels.

    PubMed

    Franitza, S; Alon, R; Lider, O

    1999-05-27

    We have developed a novel 3-D gel reconstituted with major extracellular matrix (ECM) glycoproteins to follow the dynamics of migration of human T cells locomoting, in real-time, on gradients formed by representative chemoattractants: the C-C chemokine RANTES, and the pro-inflammatory cytokine IL-2. In the absence of chemoattractants, none of the T cells migrated directionally and the levels of random migration or cell polarization were low. However, major fractions of T cells placed in IL-2 and RANTES gradients in the gels polarized immediately after exposure to the chemoattractants. Shortly after polarization, 25% of the T cells migrated, in either a random or directional fashion, towards the sources of the chemoattractants; additional 5-10% of the cells remained polarized but stationary. The number of T cells migrating directionally towards RANTES or IL-2 peaked along with the formation of the chemotactic gradients. The directional migration of T cells was increased by a short pre-exposure to low doses of IL-2, which did not alter the level of expression of the beta1 integrins. The directional migration of T cells towards IL-2 and RANTES was mediated by IL-2R and pertussis toxin-sensitive receptors, respectively, and the directional, and to a lesser degree, the random locomotion of T cells induced by both chemoattractants required intact tyrosine kinase signaling and activities of the alpha4, alpha5, and, to a lesser degree, the alpha2 and alpha6 members the beta1 integrins. Our system enables the real-time tracking of individual locomoting lymphocytes and the analysis of their dynamic interactions with ECM components and cytokines. PMID:10365778

  17. 3D cancer cell migration in a confined matrix

    NASA Astrophysics Data System (ADS)

    Alobaidi, Amani; Sun, Bo

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

  18. Extracellular matrix and wound healing.

    PubMed

    Maquart, F X; Monboisse, J C

    2014-04-01

    Extracellular matrix has been known for a long time as an architectural support for the tissues. Many recent data, however, have shown that extracellular matrix macromolecules (collagens, elastin, glycosaminoglycans, proteoglycans and connective tissue glycoproteins) are able to regulate many important cell functions, such as proliferation, migration, protein synthesis or degradation, apoptosis, etc., making them able to play an important role in the wound repair process. Not only the intact macromolecules but some of their specific domains, that we called "Matrikines", are also able to regulate many cell activities. In this article, we will summarize main findings showing the effects of extracellular matrix macromolecules and matrikines on connective tissue and epithelial cells, particularly in skin, and their potential implication in the wound healing process. These examples show that extracellular matrix macromolecules or some of their specific domains may play a major role in wound healing. Better knowledge of these interactions may suggest new therapeutic targets in wound healing defects. PMID:24650524

  19. Extracellular matrix in ovarian follicles.

    PubMed

    Rodgers, R J; Irving-Rodgers, H F; van Wezel, I L

    2000-05-25

    A lot is known about the control of the development of ovarian follicles by growth factors and hormones, but less is known about the roles of extracellular matrix in the control of follicular growth and development. In this review we focus on the specialized extracellular matrix of the basal laminas that are present in ovarian follicles. These include the follicular basal lamina itself, the Call-Exner bodies of the membrana granulosa, the subendothelial and arteriole smooth muscle basal laminas in the theca, and the basal lamina-like material of the thecal matrix. We discuss the evidence that during follicle development the follicular basal lamina changes in composition, that many of its components are produced by the granulosa cells, and that the follicular basal laminas of different follicles have different ultrastructural appearances, linked to the shape of the aligning granulosa cells. All these studies suggest that the follicular basal lamina is extremely dynamic during follicular development. PMID:10963877

  20. Brain Extracellular Matrix in Neurodegeneration

    PubMed Central

    Bonneh-Barkay, Dafna; Wiley, Clayton A.

    2009-01-01

    The role of extracellular matrix (ECM) in neurological development, function and degeneration has evolved from a simplistic physical adhesion to a system of intricate cellular signaling. While most cells require ECM adhesion to survive, it is now clear that differentiated function is intimately dependent upon cellular interaction with the ECM. Therefore, it is not surprising that the ECM is increasingly found to be involved in the enigmatic process of neurodegeneration. Descriptive studies of human neurodegenerative disorders and experimental studies of animal models of neurodegeneration have begun to define potential mechanisms of ECM disruption that can lead to synaptic and neuronal loss. PMID:18662234

  1. The evolution of extracellular matrix.

    PubMed

    Ozbek, Suat; Balasubramanian, Prakash G; Chiquet-Ehrismann, Ruth; Tucker, Richard P; Adams, Josephine C

    2010-12-01

    We present a perspective on the molecular evolution of the extracellular matrix (ECM) in metazoa that draws on research publications and data from sequenced genomes and expressed sequence tag libraries. ECM components do not function in isolation, and the biological ECM system or "adhesome" also depends on posttranslational processing enzymes, cell surface receptors, and extracellular proteases. We focus principally on the adhesome of internal tissues and discuss its origins at the dawn of the metazoa and the expansion of complexity that occurred in the chordate lineage. The analyses demonstrate very high conservation of a core adhesome that apparently evolved in a major wave of innovation in conjunction with the origin of metazoa. Integrin, CD36, and certain domains predate the metazoa, and some ECM-related proteins are identified in choanoflagellates as predicted sequences. Modern deuterostomes and vertebrates have many novelties and elaborations of ECM as a result of domain shuffling, domain innovations and gene family expansions. Knowledge of the evolution of metazoan ECM is important for understanding how it is built as a system, its roles in normal tissues and disease processes, and has relevance for tissue engineering, the development of artificial organs, and the goals of synthetic biology.

  2. The Evolution of Extracellular Matrix

    PubMed Central

    Özbek, Suat; Balasubramanian, Prakash G.; Chiquet-Ehrismann, Ruth; Tucker, Richard P.

    2010-01-01

    We present a perspective on the molecular evolution of the extracellular matrix (ECM) in metazoa that draws on research publications and data from sequenced genomes and expressed sequence tag libraries. ECM components do not function in isolation, and the biological ECM system or “adhesome” also depends on posttranslational processing enzymes, cell surface receptors, and extracellular proteases. We focus principally on the adhesome of internal tissues and discuss its origins at the dawn of the metazoa and the expansion of complexity that occurred in the chordate lineage. The analyses demonstrate very high conservation of a core adhesome that apparently evolved in a major wave of innovation in conjunction with the origin of metazoa. Integrin, CD36, and certain domains predate the metazoa, and some ECM-related proteins are identified in choanoflagellates as predicted sequences. Modern deuterostomes and vertebrates have many novelties and elaborations of ECM as a result of domain shuffling, domain innovations and gene family expansions. Knowledge of the evolution of metazoan ECM is important for understanding how it is built as a system, its roles in normal tissues and disease processes, and has relevance for tissue engineering, the development of artificial organs, and the goals of synthetic biology. PMID:21160071

  3. Extracellular Matrix: Functions in the Nervous System

    PubMed Central

    Barros, Claudia S.; Franco, Santos J.; Müller, Ulrich

    2011-01-01

    An astonishing number of extracellular matrix glycoproteins are expressed in dynamic patterns in the developing and adult nervous system. Neural stem cells, neurons, and glia express receptors that mediate interactions with specific extracellular matrix molecules. Functional studies in vitro and genetic studies in mice have provided evidence that the extracellular matrix affects virtually all aspects of nervous system development and function. Here we will summarize recent findings that have shed light on the specific functions of defined extracellular matrix molecules on such diverse processes as neural stem cell differentiation, neuronal migration, the formation of axonal tracts, and the maturation and function of synapses in the peripheral and central nervous system. PMID:21123393

  4. Extracellular Matrix and Liver Disease

    PubMed Central

    Arriazu, Elena; Ruiz de Galarreta, Marina; Cubero, Francisco Javier; Varela-Rey, Marta; Pérez de Obanos, María Pilar; Leung, Tung Ming; Lopategi, Aritz; Benedicto, Aitor; Abraham-Enachescu, Ioana

    2014-01-01

    Abstract Significance: The extracellular matrix (ECM) is a dynamic microenvironment that undergoes continuous remodeling, particularly during injury and wound healing. Chronic liver injury of many different etiologies such as viral hepatitis, alcohol abuse, drug-induced liver injury, obesity and insulin resistance, metabolic disorders, and autoimmune disease is characterized by excessive deposition of ECM proteins in response to persistent liver damage. Critical Issues: This review describes the main collagenous and noncollagenous components from the ECM that play a significant role in pathological matrix deposition during liver disease. We define how increased myofibroblasts (MF) from different origins are at the forefront of liver fibrosis and how liver cell-specific regulation of the complex scarring process occurs. Recent Advances: Particular attention is paid to the role of cytokines, growth factors, reactive oxygen species, and newly identified matricellular proteins in the regulation of fibrillar type I collagen, a field to which our laboratory has significantly contributed over the years. We compile data from recent literature on the potential mechanisms driving fibrosis resolution such as MF’ apoptosis, senescence, and reversal to quiescence. Future Directions: We conclude with a brief description of how epigenetics, an evolving field, can regulate the behavior of MF and of how new “omics” tools may advance our understanding of the mechanisms by which the fibrogenic response to liver injury occurs. Antioxid. Redox Signal. 21, 1078–1097. PMID:24219114

  5. Extracellular Matrix Stiffness and Architecture Govern Intracellular Rheology in Cancer

    PubMed Central

    Baker, Erin L.; Bonnecaze, Roger T.; Zaman, Muhammad H.

    2009-01-01

    Abstract Little is known about the complex interplay between the extracellular mechanical environment and the mechanical properties that characterize the dynamic intracellular environment. To elucidate this relationship in cancer, we probe the intracellular environment using particle-tracking microrheology. In three-dimensional (3D) matrices, intracellular effective creep compliance of prostate cancer cells is shown to increase with increasing extracellular matrix (ECM) stiffness, whereas modulating ECM stiffness does not significantly affect the intracellular mechanical state when cells are attached to two-dimensional (2D) matrices. Switching from 2D to 3D matrices induces an order-of-magnitude shift in intracellular effective creep compliance and apparent elastic modulus. However, for a given matrix stiffness, partial blocking of β1 integrins mitigates the shift in intracellular mechanical state that is invoked by switching from a 2D to 3D matrix architecture. This finding suggests that the increased cell-matrix engagement inherent to a 3D matrix architecture may contribute to differences observed in viscoelastic properties between cells attached to 2D matrices and cells embedded within 3D matrices. In total, our observations show that ECM stiffness and architecture can strongly influence the intracellular mechanical state of cancer cells. PMID:19686648

  6. Fabrication of 2D and 3D constructs from reconstituted decellularized tissue extracellular matrices.

    PubMed

    Takeda, Yuji S; Xu, Qiaobing

    2014-12-01

    We demonstrated a novel process to reconstitute a decellularized extracellular matrix (Recon-ECM) of heart and liver tissue using a combination of mechanical homogenization and enzymatic digestion. Such Recon-ECM was used as a biomaterial to produce flat or micro-patterned 2D films after crosslinking using replica molding. The mechanical properties of the resulting films were tuned by changing the type of crosslinking reagents. We also demonstrated the fabrication of mechanically robust 3D scaffolds by freeze-drying of the Recon-ECM solution. The porosity of the 3D scaffold was controlled by changing the concentration of the Recon-ECM. HepG2 cells were used to investigate the potential substrate of these engineered 2D patterned and 3D porous structures. The cell attachment, proliferation, and urea synthesis were evaluated, and the results indicate that the scaffold generated from Recon-ECM provides a biologically friendly environment for cells to grow. This method provides a new way to use decellularized ECM as a source of biomaterial to produce novel scaffolds with better controlled micro- and nano-scale structures, tunable physicochemical properties with desired biological functions.

  7. Microrheology and ROCK signaling of human endothelial cells embedded in a 3D matrix.

    PubMed

    Panorchan, Porntula; Lee, Jerry S H; Kole, Thomas P; Tseng, Yiider; Wirtz, Denis

    2006-11-01

    Cell function is profoundly affected by the geometry of the extracellular environment confining the cell. Whether and how cells plated on a two-dimensional matrix or embedded in a three-dimensional (3D) matrix mechanically sense the dimensionality of their environment is mostly unknown, partly because individual cells in an extended matrix are inaccessible to conventional cell-mechanics probes. Here we develop a functional assay based on multiple particle tracking microrheology coupled with ballistic injection of nanoparticles to measure the local intracellular micromechanical properties of individual cells embedded inside a matrix. With our novel assay, we probe the mechanical properties of the cytoplasm of individual human umbilical vein endothelial cells (HUVECs) embedded in a 3D peptide hydrogel in the presence or absence of vascular endothelial growth factor (VEGF). We found that VEGF treatment, which enhances endothelial migration, increases the compliance and reduces the elasticity of the cytoplasm of HUVECs in a matrix. This VEGF-induced softening response of the cytoplasm is abrogated by specific Rho-kinase (ROCK) inhibition. These results establish combined particle-tracking microrheology and ballistic injection as the first method able to probe the micromechanical properties and mechanical response to agonists and/or drug treatments of individual cells inside a matrix. These results suggest that ROCK plays an essential role in the regulation of the intracellular mechanical response to VEGF of endothelial cells in a 3D matrix.

  8. Extracellular environment contribution to astrogliosis—lessons learned from a tissue engineered 3D model of the glial scar

    PubMed Central

    Rocha, Daniela N.; Ferraz-Nogueira, José P.; Barrias, Cristina C.; Relvas, João B.; Pêgo, Ana P.

    2015-01-01

    Glial scars are widely seen as a (bio)mechanical barrier to central nervous system regeneration. Due to the lack of a screening platform, which could allow in-vitro testing of several variables simultaneously, up to now no comprehensive study has addressed and clarified how different lesion microenvironment properties affect astrogliosis. Using astrocytes cultured in alginate gels and meningeal fibroblast conditioned medium, we have built a simple and reproducible 3D culture system of astrogliosis mimicking many features of the glial scar. Cells in this 3D culture model behave similarly to scar astrocytes, showing changes in gene expression (e.g., GFAP) and increased extra-cellular matrix production (chondroitin 4 sulfate and collagen), inhibiting neuronal outgrowth. This behavior being influenced by the hydrogel network properties. Astrocytic reactivity was found to be dependent on RhoA activity, and targeting RhoA using shRNA-mediated lentivirus reduced astrocytic reactivity. Further, we have shown that chemical inhibition of RhoA with ibuprofen or indirectly targeting RhoA by the induction of extracellular matrix composition modification with chondroitinase ABC, can diminish astrogliosis. Besides presenting the extracellular matrix as a key modulator of astrogliosis, this simple, controlled and reproducible 3D culture system constitutes a good scar-like system and offers great potential in future neurodegenerative mechanism studies, as well as in drug screenings envisaging the development of new therapeutic approaches to minimize the effects of the glial scar in the context of central nervous system disease. PMID:26483632

  9. Extracellular environment contribution to astrogliosis-lessons learned from a tissue engineered 3D model of the glial scar.

    PubMed

    Rocha, Daniela N; Ferraz-Nogueira, José P; Barrias, Cristina C; Relvas, João B; Pêgo, Ana P

    2015-01-01

    Glial scars are widely seen as a (bio)mechanical barrier to central nervous system regeneration. Due to the lack of a screening platform, which could allow in-vitro testing of several variables simultaneously, up to now no comprehensive study has addressed and clarified how different lesion microenvironment properties affect astrogliosis. Using astrocytes cultured in alginate gels and meningeal fibroblast conditioned medium, we have built a simple and reproducible 3D culture system of astrogliosis mimicking many features of the glial scar. Cells in this 3D culture model behave similarly to scar astrocytes, showing changes in gene expression (e.g., GFAP) and increased extra-cellular matrix production (chondroitin 4 sulfate and collagen), inhibiting neuronal outgrowth. This behavior being influenced by the hydrogel network properties. Astrocytic reactivity was found to be dependent on RhoA activity, and targeting RhoA using shRNA-mediated lentivirus reduced astrocytic reactivity. Further, we have shown that chemical inhibition of RhoA with ibuprofen or indirectly targeting RhoA by the induction of extracellular matrix composition modification with chondroitinase ABC, can diminish astrogliosis. Besides presenting the extracellular matrix as a key modulator of astrogliosis, this simple, controlled and reproducible 3D culture system constitutes a good scar-like system and offers great potential in future neurodegenerative mechanism studies, as well as in drug screenings envisaging the development of new therapeutic approaches to minimize the effects of the glial scar in the context of central nervous system disease. PMID:26483632

  10. 3D magnetotelluric inversion with full distortion matrix

    NASA Astrophysics Data System (ADS)

    Gribenko, A. V.; Zhdanov, M. S.

    2014-12-01

    Distortion of regional electric fields by local structures represent one of the major problems facing three-dimensional magnetotelluric (MT) interpretation. Effect of 3D local inhomogenities on MT data can be described by a real 2x2 distortion matrix. In this project we develop a method of simultaneous inversion of the full MT impedance data for 3D conductivity distribution and for the distortion matrix. Tikhonov regularization is employed to solve the resulting inverse problem. Integral equations method is used to compute MT responses. Minimization of the cost functional is achieved via conjugate gradient method. The inversion algorithm is tested on the synthetic data from Dublin Secret Model II (DSM 2) for which multiple inversion solutions are available for comparison. Inclusion of the distortion matrix provides faster convergence and allows coarser discretization of the near-surface while achievingsimilar or better data fits as inversion for the conductivity only with finely discretized shallow regions. As a field data example we chose a subset of the EarthScope MT dataset covering Great Basin and adjacent areas of the Western United States. Great Basin data inversion identified several prominent conductive zones which correlate well with areas of tectonic and geothermal activity.

  11. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions

    NASA Astrophysics Data System (ADS)

    Doyle, Andrew D.; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M.

    2015-11-01

    The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils.

  12. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions.

    PubMed

    Doyle, Andrew D; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M

    2015-01-01

    The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils. PMID:26548801

  13. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions

    PubMed Central

    Doyle, Andrew D.; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M.

    2015-01-01

    The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils. PMID:26548801

  14. Techniques for Assessing 3-D Cell-Matrix Mechanical Interactions In Vitro and In Vivo

    PubMed Central

    Miron-Mendoza, Miguel; Koppaka, Vindhya; Zhou, Chengxin; Petroll, W. Matthew

    2013-01-01

    Cellular interactions with extracellular matrices (ECM) through the application of mechanical forces mediate numerous biological processes including developmental morphogenesis, wound healing and cancer metastasis. They also play a key role in the cellular repopulation and/or remodeling of engineered tissues and organs. While 2-D studies can provide important insights into many aspects of cellular mechanobiology, cells reside within 3-D ECMs in vivo, and matrix structure and dimensionality have been shown to impact cell morphology, protein organization and mechanical behavior. Global measurements of cell-induced compaction of 3-D collagen matrices can provide important insights into the regulation of overall cell contractility by various cytokines and signaling pathways. However, to understand how the mechanics of cell spreading, migration, contraction and matrix remodeling are regulated at the molecular level, these processes must also be studied in individual cells. Here we review the evolution and application of techniques for imaging and assessing local cell-matrix mechanical interactions in 3-D culture models, tissue explants and living animals. PMID:23819988

  15. Fibronectin Deposition Participates in Extracellular Matrix Assembly and Vascular Morphogenesis

    PubMed Central

    Hielscher, Abigail; Ellis, Kim; Qiu, Connie; Porterfield, Josh; Gerecht, Sharon

    2016-01-01

    The extracellular matrix (ECM) has been demonstrated to facilitate angiogenesis. In particular, fibronectin has been documented to activate endothelial cells, resulting in their transition from a quiescent state to an active state in which the cells exhibit enhanced migration and proliferation. The goal of this study is to examine the role of polymerized fibronectin during vascular tubulogenesis using a 3 dimensional (3D) cell-derived de-cellularized matrix. A fibronectin-rich 3D de-cellularized ECM was used as a scaffold to study vascular morphogenesis of endothelial cells (ECs). Confocal analyses of several matrix proteins reveal high intra- and extra-cellular deposition of fibronectin in formed vascular structures. Using a small peptide inhibitor of fibronectin polymerization, we demonstrate that inhibition of fibronectin fibrillogenesis in ECs cultured atop de-cellularized ECM resulted in decreased vascular morphogenesis. Further, immunofluorescence and ultrastructural analyses reveal decreased expression of stromal matrix proteins in the absence of polymerized fibronectin with high co-localization of matrix proteins found in association with polymerized fibronectin. Evaluating vascular kinetics, live cell imaging showed that migration, migration velocity, and mean square displacement, are disrupted in structures grown in the absence of polymerized fibronectin. Additionally, vascular organization failed to occur in the absence of a polymerized fibronectin matrix. Consistent with these observations, we tested vascular morphogenesis following the disruption of EC adhesion to polymerized fibronectin, demonstrating that block of integrins α5β1 and αvβ3, abrogated vascular morphogenesis. Overall, fibronectin deposition in a 3D cell-derived de-cellularized ECM appears to be imperative for matrix assembly and vascular morphogenesis. PMID:26811931

  16. Extracellular matrix as a bioactive material for soft tissue reconstruction.

    PubMed

    Hodde, Jason

    2006-12-01

    The extracellular matrix (ECM) directs all phases of healing following trauma or disease and is therefore a natural source of prosthetic mesh material that can be used strategically to induce the repair and restoration of soft tissues following surgery. Biomaterials such as Surgisis (Cook Biotech Incorporated, West Lafayette, IN, USA), which are derived from natural ECM, provide the extracellular components necessary to direct the healing response, allow for the proliferation of new, healthy tissue and restore tissue integrity to the damaged site. The 3-D organization of these extracellular components distinguishes the Surgisis mesh from synthetic materials and is associated with constructive tissue remodelling instead of scar tissue. Common features of this ECM-assisted tissue remodelling include angiogenesis, recruitment of circulating progenitor cells and constructive remodelling of damaged tissue structures. The tissue response to this biologic mesh is discussed in the context of recent reports on clinical hernia repair.

  17. The Extracellular Matrix of Fungal Biofilms.

    PubMed

    Mitchell, Kaitlin F; Zarnowski, Robert; Andes, David R

    2016-01-01

    A key feature of biofilms is their production of an extracellular matrix. This material covers the biofilm cells, providing a protective barrier to the surrounding environment. During an infection setting, this can include such offenses as host cells and products of the immune system as well as drugs used for treatment. Studies over the past two decades have revealed the matrix from different biofilm species to be as diverse as the microbes themselves. This chapter will review the composition and roles of matrix from fungal biofilms, with primary focus on Candida species, Saccharomyces cerevisiae, Aspergillus fumigatus, and Cryptococcus neoformans. Additional coverage will be provided on the antifungal resistance proffered by the Candida albicans matrix, which has been studied in the most depth. A brief section on the matrix produced by bacterial biofilms will be provided for comparison. Current tools for studying the matrix will also be discussed, as well as suggestions for areas of future study in this field. PMID:27271680

  18. Extracellular matrix component signaling in cancer.

    PubMed

    Multhaupt, Hinke A B; Leitinger, Birgit; Gullberg, Donald; Couchman, John R

    2016-02-01

    Cell responses to the extracellular matrix depend on specific signaling events. These are important from early development, through differentiation and tissue homeostasis, immune surveillance, and disease pathogenesis. Signaling not only regulates cell adhesion cytoskeletal organization and motility but also provides survival and proliferation cues. The major classes of cell surface receptors for matrix macromolecules are the integrins, discoidin domain receptors, and transmembrane proteoglycans such as syndecans and CD44. Cells respond not only to specific ligands, such as collagen, fibronectin, or basement membrane glycoproteins, but also in terms of matrix rigidity. This can regulate the release and subsequent biological activity of matrix-bound growth factors, for example, transforming growth factor-β. In the environment of tumors, there may be changes in cell populations and their receptor profiles as well as matrix constitution and protein cross-linking. Here we summarize roles of the three major matrix receptor types, with emphasis on how they function in tumor progression.

  19. Extracellular matrix component signaling in cancer.

    PubMed

    Multhaupt, Hinke A B; Leitinger, Birgit; Gullberg, Donald; Couchman, John R

    2016-02-01

    Cell responses to the extracellular matrix depend on specific signaling events. These are important from early development, through differentiation and tissue homeostasis, immune surveillance, and disease pathogenesis. Signaling not only regulates cell adhesion cytoskeletal organization and motility but also provides survival and proliferation cues. The major classes of cell surface receptors for matrix macromolecules are the integrins, discoidin domain receptors, and transmembrane proteoglycans such as syndecans and CD44. Cells respond not only to specific ligands, such as collagen, fibronectin, or basement membrane glycoproteins, but also in terms of matrix rigidity. This can regulate the release and subsequent biological activity of matrix-bound growth factors, for example, transforming growth factor-β. In the environment of tumors, there may be changes in cell populations and their receptor profiles as well as matrix constitution and protein cross-linking. Here we summarize roles of the three major matrix receptor types, with emphasis on how they function in tumor progression. PMID:26519775

  20. Microtubules regulate GEF-H1 in response to extracellular matrix stiffness

    PubMed Central

    Heck, Jessica N.; Ponik, Suzanne M.; Garcia-Mendoza, Maria G.; Pehlke, Carolyn A.; Inman, David R.; Eliceiri, Kevin W.; Keely, Patricia J.

    2012-01-01

    Breast epithelial cells sense the stiffness of the extracellular matrix through Rho-mediated contractility. In turn, matrix stiffness regulates RhoA activity. However, the upstream signaling mechanisms are poorly defined. Here we demonstrate that the Rho exchange factor GEF-H1 mediates RhoA activation in response to extracellular matrix stiffness. We demonstrate the novel finding that microtubule stability is diminished by a stiff three-dimensional (3D) extracellular matrix, which leads to the activation of GEF-H1. Surprisingly, activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway did not contribute to stiffness-induced GEF-H1 activation. Loss of GEF-H1 decreases cell contraction of and invasion through 3D matrices. These data support a model in which matrix stiffness regulates RhoA through microtubule destabilization and the subsequent release and activation of GEF-H1. PMID:22593214

  1. Nanomechanics of the Cartilage Extracellular Matrix

    NASA Astrophysics Data System (ADS)

    Han, Lin; Grodzinsky, Alan J.; Ortiz, Christine

    2011-08-01

    Cartilage is a hydrated biomacromolecular fiber composite located at the ends of long bones that enables proper joint lubrication, articulation, loading, and energy dissipation. Degradation of extracellular matrix molecular components and changes in their nanoscale structure greatly influence the macroscale behavior of the tissue and result in dysfunction with age, injury, and diseases such as osteoarthritis. Here, the application of the field of nanomechanics to cartilage is reviewed. Nanomechanics involves the measurement and prediction of nanoscale forces and displacements, intra- and intermolecular interactions, spatially varying mechanical properties, and other mechanical phenomena existing at small length scales. Experimental nanomechanics and theoretical nanomechanics have been applied to cartilage at varying levels of material complexity, e.g., nanoscale properties of intact tissue, the matrix associated with single cells, biomimetic molecular assemblies, and individual extracellular matrix biomolecules (such as aggrecan, collagen, and hyaluronan). These studies have contributed to establishing a fundamental mechanism-based understanding of native and engineered cartilage tissue function, quality, and pathology.

  2. Extracellular matrix assembly: a multiscale deconstruction

    PubMed Central

    Mouw, Janna K.; Ou, Guanqing; Weaver, Valerie M.

    2015-01-01

    The biochemical and biophysical properties of the extracellular matrix (ECM) dictate tissue-specific cell behaviour. The molecules that are associated with the ECM of each tissue, including collagens, proteoglycans, laminins and fibronectin, and the manner in which they are assembled determine the structure and the organization of the resultant ECM. The product is a specific ECM signature that is comprised of unique compositional and topographical features that both reflect and facilitate the functional requirements of the tissue. PMID:25370693

  3. Involvement of extracellular matrix constituents in breast cancer

    SciTech Connect

    Lochter, Andre; Bissell, Mina J

    1995-06-01

    It has recently been established that the extracellular matrix is required for normal functional differentiation of mammary epithelia not only in culture, but also in vivo. The mechanisms by which extracellular matrix affects differentiation, as well as the nature of extracellular matrix constituents which have major impacts on mammary gland function, have only now begun to be dissected. The intricate variety of extracellular matrix-mediated events and the remarkable degree of plasticity of extracellular matrix structure and composition at virtually all times during ontogeny, make such studies difficult. Similarly, during carcinogenesis, the extracellular matrix undergoes gross alterations, the consequences of which are not yet precisely understood. Nevertheless, an increasing amount of data suggests that the extracellular matrix and extracellular matrix-receptors might participate in the control of most, if not all, of the successive stages of breast tumors, from appearance to progression and metastasis.

  4. Airway and Extracellular Matrix Mechanics in COPD.

    PubMed

    Bidan, Cécile M; Veldsink, Annemiek C; Meurs, Herman; Gosens, Reinoud

    2015-01-01

    Chronic obstructive pulmonary disease (COPD) is one of the most common lung diseases worldwide, and is characterized by airflow obstruction that is not fully reversible with treatment. Even though airflow obstruction is caused by airway smooth muscle contraction, the extent of airway narrowing depends on a range of other structural and functional determinants that impact on active and passive tissue mechanics. Cells and extracellular matrix in the airway and parenchymal compartments respond both passively and actively to the mechanical stimulation induced by smooth muscle contraction. In this review, we summarize the factors that regulate airway narrowing and provide insight into the relative contributions of different constituents of the extracellular matrix and their biomechanical impact on airway obstruction. We then review the changes in extracellular matrix composition in the airway and parenchymal compartments at different stages of COPD, and finally discuss how these changes impact airway narrowing and the development of airway hyperresponsiveness. Finally, we position these data in the context of therapeutic research focused on defective tissue repair. As a conclusion, we propose that future works should primarily target mild or early COPD, prior to the widespread structural changes in the alveolar compartment that are more characteristic of severe COPD.

  5. Extracellular matrix proteins involved in pseudoislets formation.

    PubMed

    Maillard, Elisa; Sencier, Marie-Christine; Langlois, A; Bietiger, William; Krafft, Mp; Pinget, Michel; Sigrist, Séverine

    2009-01-01

    Extracellular matrix proteins are known to mediate, through integrins, cell adhesion and are involved in a number of cellular processes, including insulin expression and secretion in pancreatic islets. We investigated whether expression of some extracellular matrix proteins were implied in islets-like structure formation, named pseudoislets. For this purpose, we cultured the β-cell line, RINm5F, during 1, 3, 5 and 7 days of culture on treated or untreated culture plate to form adherent cells or pseudoislets and analysed insulin, collagen IV, fibronectin, laminin 5 and β1-integrin expression. We observed that insulin expression and secretion were increased during pseudoislets formation. Moreover, we showed by immunohistochemistry an aggregation of insulin secreting cells in the centre of the pseudoislets. Peripheral β-cells of pseudoislets did not express insulin after 7 days of culture. RT-PCR and immunohistochemistry studies showed a transient expression of type IV collagen in pseudoislets for the first 3 days of culture. Study of fibronectin expression indicated that adherent cells expressed more fibronectin than pseudoislets. In contrast, laminin 5 was more expressed in pseudoislets than in adherent cells. Finally, expression of β1-integrin was increased in pseudoislets as compared to adherent cells. In conclusion, laminin 5 and collagen IV might be implicated in pseudoislets formation whereas fibronectin might be involved in cell adhesion. These data suggested that extracellular matrix proteins may enhance the function of pseudoislets.

  6. Airway and Extracellular Matrix Mechanics in COPD

    PubMed Central

    Bidan, Cécile M.; Veldsink, Annemiek C.; Meurs, Herman; Gosens, Reinoud

    2015-01-01

    Chronic obstructive pulmonary disease (COPD) is one of the most common lung diseases worldwide, and is characterized by airflow obstruction that is not fully reversible with treatment. Even though airflow obstruction is caused by airway smooth muscle contraction, the extent of airway narrowing depends on a range of other structural and functional determinants that impact on active and passive tissue mechanics. Cells and extracellular matrix in the airway and parenchymal compartments respond both passively and actively to the mechanical stimulation induced by smooth muscle contraction. In this review, we summarize the factors that regulate airway narrowing and provide insight into the relative contributions of different constituents of the extracellular matrix and their biomechanical impact on airway obstruction. We then review the changes in extracellular matrix composition in the airway and parenchymal compartments at different stages of COPD, and finally discuss how these changes impact airway narrowing and the development of airway hyperresponsiveness. Finally, we position these data in the context of therapeutic research focused on defective tissue repair. As a conclusion, we propose that future works should primarily target mild or early COPD, prior to the widespread structural changes in the alveolar compartment that are more characteristic of severe COPD. PMID:26696894

  7. Extracellular matrix of the developing ovarian follicle.

    PubMed

    Irving-Rodgers, Helen F; Rodgers, Raymond J

    2006-09-01

    There are many different types of extracellular matrices in the different follicle compartments. These have different roles in follicle development and atresia, and they change in composition during these processes. This review focuses on basal lamina matrix in particular, and considers follicular fluid, the newly identified focimatrix, and thecal matrices. When follicles commence growing, the follicular basal lamina changes in its composition from containing all six alpha chains of type IV collagen to only alpha1 and alpha2. Perlecan and nidogen-1 and -2 subsequently become components of the follicular basal lamina, and there is an increase in the amount of laminin chains alpha1, beta2, and gamma1, in the bovine at least. Late in follicular development and on atresia some follicles contain laminin alpha2. On atresia the follicular basal lamina is not degraded, as occurs in ovulation, but can be breached by cells from the thecal layer when it is not aligned by granulosa cells. A novel type of basal lamina-like matrix, called focimatrix (abbreviated from focal intraepithelial matrix), develops between the cells of the membrana granulosa as aggregates of basal lamina material. It does not envelop cells and so cannot perform functions of basal lamina as currently understood. It is hypothesized that focimatrix assists or initiates depolarization of the membrana granulosa necessary for the transformation into luteal cells. The largest osmotically active molecules in follicular fluid are hyaluronan and chondroitin sulfate proteoglycans, including versican and inter-alpha trypsin inhibitor. It has been suggested that these might be responsible for the formation of follicular fluid by creating an osmotic gradient across the follicular wall. The formation, development, and then either ovulation or regression of follicles requires considerable tissue remodeling, cellular replication, and specialization. The expectation of researchers is that extracellular matrix will be

  8. Regulation of Corneal Stroma Extracellular Matrix Assembly

    PubMed Central

    Chen, Shoujun; Mienaltowski, Michael J.; Birk, David E.

    2014-01-01

    The transparent cornea is the major refractive element of the eye. A finely controlled assembly of the stromal extracellular matrix is critical to corneal function, as well as in establishing the appropriate mechanical stability required to maintain corneal shape and curvature. In the stroma, homogeneous, small diameter collagen fibrils, regularly packed with a highly ordered hierarchical organization, are essential for function. This review focuses on corneal stroma assembly and the regulation of collagen fibrillogenesis. Corneal collagen fibrillogenesis involves multiple molecules interacting in sequential steps, as well as interactions between keratocytes and stroma matrix components. The stroma has the highest collagen V:I ratio in the body. Collagen V regulates the nucleation of protofibril assembly, thus controlling the number of fibrils and assembly of smaller diameter fibrils in the stroma. The corneal stroma is also enriched in small leucine-rich proteoglycans (SLRPs) that cooperate in a temporal and spatial manner to regulate linear and lateral collagen fibril growth. In addition, the fibril-associated collagens (FACITs) such as collagen XII and collagen XIV have roles in the regulation of fibril packing and inter-lamellar interactions. A communicating keratocyte network contributes to the overall and long-range regulation of stromal extracellular matrix assembly, by creating micro-domains where the sequential steps in stromal matrix assembly are controlled. Keratocytes control the synthesis of extracellular matrix components, which interact with the keratocytes dynamically to coordinate the regulatory steps into a cohesive process. Mutations or deficiencies in stromal regulatory molecules result in altered interactions and deficiencies in both transparency and refraction, leading to corneal stroma pathobiology such as stromal dystrophies, cornea plana and keratoconus. PMID:25819456

  9. Achondrogenesis type II, abnormalities of extracellular matrix.

    PubMed

    Horton, W A; Machado, M A; Chou, J W; Campbell, D

    1987-09-01

    Immune and lectin histochemical and microchemical methods were employed to study growth cartilage from seven cases of achondrogenesis type II (Langer-Saldino). The normal architecture of the epiphyseal and growth plate cartilage was replaced by a morphologically heterogeneous tissue. Some areas were comprised of vascular canals surrounded by extensive fibrous tissue and enlarged cells that had the appearance and histochemical characteristics of hypertrophic chondrocytes. Other areas contained a mixture of cells ranging from small to the enlarged chondrocytes. The extracellular matrix in the latter areas was more abundant and had characteristics of both precartilage mesenchymal matrix and typical cartilage matrix; it contained types I and II collagen, cartilage proteoglycan, fibronectin, and peanut agglutinin binding glycoconjugate(s). Peptide mapping of cyanogen bromide cartilage collagen peptides revealed the presence of types I and II collagen. These observations could be explained by a defect in the biosynthesis of type II collagen or in chondrocyte differentiation. PMID:3309860

  10. Individual versus Collective Fibroblast Spreading and Migration: Regulation by Matrix Composition in 3-D Culture

    PubMed Central

    Miron-Mendoza, Miguel; Lin, Xihui; Ma, Lisha; Ririe, Peter; Petroll, W. Matthew

    2012-01-01

    Extracellular matrix (ECM) supplies both physical and chemical signals to cells and provides a substrate through which fibroblasts migrate during wound repair. To directly assess how ECM composition regulates this process, we used a nested 3D matrix model in which cell-populated collagen buttons were embedded in cell-free collagen or fibrin matrices. Time-lapse microscopy was used to record the dynamic pattern of cell migration into the outer matrices, and 3-D confocal imaging was used to assess cell connectivity and cytoskeletal organization. Corneal fibroblasts stimulated with PDGF migrated more rapidly into collagen as compared to fibrin. In addition, the pattern of fibroblast migration into fibrin and collagen ECMs was strikingly different. Corneal fibroblasts migrating into collagen matrices developed dendritic processes and moved independently, whereas cells migrating into fibrin matrices had a more fusiform morphology and formed an interconnected meshwork. A similar pattern was observed when using dermal fibroblasts, suggesting that this response in not unique to corneal cells. We next cultured corneal fibroblasts within and on top of standard collagen and fibrin matrices to assess the impact of ECM composition on the cell spreading response. Similar differences in cell morphology and connectivity were observed – cells remained separated on collagen but coalesced into clusters on fibrin. Cadherin was localized to junctions between interconnected cells, whereas fibronectin was present both between cells and at the tips of extending cell processes. Cells on fibrin matrices also developed more prominent stress fibers than those on collagen matrices. Importantly, these spreading and migration patterns were consistently observed on both rigid and compliant substrates, thus differences in ECM mechanical stiffness were not the underlying cause. Overall, these results demonstrate for the first time that ECM protein composition alone (collagen vs. fibrin) can

  11. Thermoreversible copolymer gels for extracellular matrix.

    PubMed

    Vernon, B; Kim, S W; Bae, Y H

    2000-07-01

    To improve the properties of a reversible synthetic extracellular matrix based on a thermally reversible polymer, copolymers of N-isopropylacrylamide and acrylic acid were prepared in benzene with varying contents of acrylic acid (0 to 3%) and the thermal properties were evaluated. The poly(N-isopropylacrylamide) and copolymers made with acrylic acid had molecular weights from 0.8 to 1.7 x10(6) D. Differential scanning calorimetry (DSC) showed the high-molecular-weight acrylic acid copolymers had similar onset temperatures to the homopolymers, but the peak width was considerably increased with increasing acrylic acid content. DSC and cloud point measurements showed that polymers with 0 to 3% acrylic acid exhibit a lower critical solution temperature (LCST) transition between 30 degrees and 37 degrees C. In swelling studies, the homopolymer showed significant syneresis at temperatures above 31 degrees C. Copolymers with 1 and 1.5% showed syneresis beginning at 32 degrees and 37 degrees C, respectively. At 37 degrees C the copolymers with 1.5-3% acrylic acid showed little or no syneresis. Due to the high water content and a transition near physiologic conditions (below 37 degrees C), the polymers with 1.5-2.0% acrylic acid exhibited properties that would be useful in the development of a refillable synthetic extracellular matrix. Such a matrix could be applied to several cell types, including islets of Langerhans, for a biohybrid artificial pancreas.

  12. Nanomechanics of the Cartilage Extracellular Matrix

    PubMed Central

    Han, Lin; Grodzinsky, Alan J.; Ortiz, Christine

    2012-01-01

    Cartilage is a hydrated biomacromolecular fiber composite located at the ends of long bones that enables proper joint lubrication, articulation, loading, and energy dissipation. Degradation of extracellular matrix molecular components and changes in their nanoscale structure greatly influence the macroscale behavior of the tissue and result in dysfunction with age, injury, and diseases such as osteoarthritis. Here, the application of the field of nanomechanics to cartilage is reviewed. Nanomechanics involves the measurement and prediction of nanoscale forces and displacements, intra- and intermolecular interactions, spatially varying mechanical properties, and other mechanical phenomena existing at small length scales. Experimental nanomechanics and theoretical nanomechanics have been applied to cartilage at varying levels of material complexity, e.g., nanoscale properties of intact tissue, the matrix associated with single cells, biomimetic molecular assemblies, and individual extracellular matrix biomolecules (such as aggrecan, collagen, and hyaluronan). These studies have contributed to establishing a fundamental mechanism-based understanding of native and engineered cartilage tissue function, quality, and pathology. PMID:22792042

  13. Three-dimensional extracellular matrix engineering in the nervous system.

    PubMed

    Borkenhagen, M; Clémence, J F; Sigrist, H; Aebischer, P

    1998-06-01

    Growing neurites are guided through their environment during development and regeneration via different cellular and extracellular matrix (ECM) molecular cues. To mimic cell-matrix interactions, a three-dimensional (3D) hydrogel-based ECM equivalent containing a covalently immobilized laminin oligopeptide sequence was designed to facilitate nerve regeneration. This study illustrates that the oligopeptide domain CDPGYIGSR covalently linked to an agarose gel as a bioartificial 3D substrate successfully supports neurite outgrowth from dorsal root ganglia (DRG) in vitro. The specificity of the neurite promoting activity was illustrated through the inhibition of neurite outgrowth from DRG in a CDPGYIGSR-derivatized gel in the presence of solubilized CDPGYIGSR peptide. Gels derivatized with CDPGYIGSK and CDPGRGSYI peptides stimulated a smaller increase of neurite outgrowth. In vivo experiments revealed the capability of a CDPGYIGSR-derivatized gel to enhance nerve regeneration in a transected rat dorsal root model compared to an underivatized gel, a CDPGRGSYI gel, and saline-filled nerve guidance channels. These data suggest the feasibility of a 3D hydrogel-based ECM equivalent capable of enhancing neurite outgrowth in vitro and in vivo.

  14. Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis

    NASA Astrophysics Data System (ADS)

    Zaman, Muhammad H.; Trapani, Linda M.; Sieminski, Alisha L.; MacKellar, Drew; Gong, Haiyan; Kamm, Roger D.; Wells, Alan; Lauffenburger, Douglas A.; Matsudaira, Paul

    2006-07-01

    Cell migration on 2D surfaces is governed by a balance between counteracting tractile and adhesion forces. Although biochemical factors such as adhesion receptor and ligand concentration and binding, signaling through cell adhesion complexes, and cytoskeletal structure assembly/disassembly have been studied in detail in a 2D context, the critical biochemical and biophysical parameters that affect cell migration in 3D matrices have not been quantitatively investigated. We demonstrate that, in addition to adhesion and tractile forces, matrix stiffness is a key factor that influences cell movement in 3D. Cell migration assays in which Matrigel density, fibronectin concentration, and 1 integrin binding are systematically varied show that at a specific Matrigel density the migration speed of DU-145 human prostate carcinoma cells is a balance between tractile and adhesion forces. However, when biochemical parameters such as matrix ligand and cell integrin receptor levels are held constant, maximal cell movement shifts to matrices exhibiting lesser stiffness. This behavior contradicts current 2D models but is predicted by a recent force-based computational model of cell movement in a 3D matrix. As expected, this 3D motility through an extracellular environment of pore size much smaller than cellular dimensions does depend on proteolytic activity as broad-spectrum matrix metalloproteinase (MMP) inhibitors limit the migration of DU-145 cells and also HT-1080 fibrosarcoma cells. Our experimental findings here represent, to our knowledge, discovery of a previously undescribed set of balances of cell and matrix properties that govern the ability of tumor cells to migration in 3D environments. cell motility | EGF receptor | extracellular matrix | matrix metalloproteinase

  15. Bidirectional extracellular matrix signaling during tissue morphogenesis

    PubMed Central

    Gjorevski, Nikolce; Nelson, Celeste M.

    2009-01-01

    Normal tissue development and function are regulated by the interplay between cells and their surrounding extracellular matrix (ECM). The ECM provides biochemical and mechanical contextual information that is conveyed from the cell membrane through the cytoskeleton to the nucleus to direct cell phenotype. Cells, in turn, remodel the ECM and thereby sculpt their local microenvironment. Here we review the mechanisms by which cells interact with, respond to, and influence the ECM, with particular emphasis placed on the role of this bidirectional communication during tissue morphogenesis. We also discuss the implications for successful engineering of functional tissues ex vivo. PMID:19896886

  16. Rapid fabrication of complex 3D extracellular microenvironments by dynamic optical projection stereolithography.

    PubMed

    Zhang, A Ping; Qu, Xin; Soman, Pranav; Hribar, Kolin C; Lee, Jin W; Chen, Shaochen; He, Sailing

    2012-08-16

    The topographic features of the extracelluar matrix (ECM) lay the foundation for cellular behavior. A novel biofabrication method using a digital-mirror device (DMD), called dynamic optical projection stereolithography (DOPsL) is demonstrated. This robust and versatile platform can generate complex biomimetic scaffolds within seconds. Such 3D scaffolds have promising potentials for studying cell interactions with microenvironments in vitro and in vivo.

  17. Effects of Matrix Alignment and Mechanical Constraints on Cellular Behavior in 3D Engineered Microtissues

    NASA Astrophysics Data System (ADS)

    Bose, Prasenjit; Eyckmans, Jeroen; Chen, Christopher; Reich, Daniel

    The adhesion of cells to the extracellular matrix (ECM) plays a crucial role in a variety of cellular functions. The main building blocks of the ECM are 3D networks of fibrous proteins whose structure and alignments varies with tissue type. However, the impact of ECM alignment on cellular behaviors such as cell adhesion, spreading, extension and mechanics remains poorly understood. We present results on the development of a microtissue-based system that enables control of the structure, orientation, and degree of fibrillar alignment in 3D fibroblast-populated collagen gels. The tissues self-assemble from cell-laden collagen gels placed in micro-fabricated wells containing sets of elastic pillars. The contractile action of the cells leads to controlled alignment of the fibrous collagen, depending on the number and location of the pillars in each well. The pillars are elastic, and are utilized to measure the contractile forces of the microtissues, and by incorporating magnetic material in selected pillars, time-varying forces can be applied to the tissues for dynamic stimulation and measurement of mechanical properties. Results on the effects of varying pillar shape, spacing, location, and stiffness on microtissue organization and contractility will be presented. This work is supported by NSF CMMI-1463011.

  18. Modeling of 3-D Woven Ceramic Matrix Composites

    NASA Technical Reports Server (NTRS)

    Murthy, Pappu L. N.; Sullivan, Roy M.; Mital, Subodh K.

    2003-01-01

    Three different approaches are being pursued at the NASA Glenn Research Center to predict the nanostructural behavior of three-dimensional woven ceramic matrix composites. These are: a micromechanics-based approach using W-CEMCAN (Woven Ceramic Matrix Composite Analyzer), a laminate analogy method and a structural frame approach (based on the finite element method). All three techniques are applied to predict the thermomechanical properties of a three-dimensional woven angle interlock C/SiC composite. The properties are predicted for room temperature and 1100 C and the predicted properties are compared to measurements. General observations regarding the three approaches for three-dimensional composite modeling are discussed.

  19. Matrix-driven formation of mesenchymal stem cell-extracellular matrix microtissues on soft alginate hydrogels.

    PubMed

    Maia, F Raquel; Fonseca, Keila B; Rodrigues, Gabriela; Granja, Pedro L; Barrias, Cristina C

    2014-07-01

    Mesenchymal stem cells (MSCs) can be made to rearrange into microtissues in response to specific matrix cues, a process that depends on a balance between cell-matrix and cell-cell interactions. The effect of such cues, and especially their interplay, is still not fully understood, particularly in three-dimensional (3-D) systems. Here, the behaviour of human MSCs cultured within hydrogel matrices with tailored stiffness and composition was evaluated. MSC aggregation occurred only in more compliant matrices (G'≤ 120 Pa), when compared to stiffer ones, both in the presence and in the absence of matrix-bound arginine-glycine-aspartic acid cell-adhesion ligands (RGD; 0, 100 and 200 μM). Fibronectin assembly stabilized cell-cell contacts within aggregates, even in non-adhesive matrices. However, MSCs were able to substantially contract the artificial matrix only when RGD was present. Moreover, compliant matrices facilitated cell proliferation and provided an environment conducive for MSC osteogenic differentiation, even without RGD. Cell interactions with the original matrix became less important as time progressed, while the de novo-produced extracellular matrix became a more critical determinant of cell fate. These data provide further insights into the mechanisms by which MSCs sense their microenvironment to organize into tissues, and provide new clues to the design of cell-instructive 3-D matrices.

  20. Matrix-driven formation of mesenchymal stem cell-extracellular matrix microtissues on soft alginate hydrogels.

    PubMed

    Maia, F Raquel; Fonseca, Keila B; Rodrigues, Gabriela; Granja, Pedro L; Barrias, Cristina C

    2014-07-01

    Mesenchymal stem cells (MSCs) can be made to rearrange into microtissues in response to specific matrix cues, a process that depends on a balance between cell-matrix and cell-cell interactions. The effect of such cues, and especially their interplay, is still not fully understood, particularly in three-dimensional (3-D) systems. Here, the behaviour of human MSCs cultured within hydrogel matrices with tailored stiffness and composition was evaluated. MSC aggregation occurred only in more compliant matrices (G'≤ 120 Pa), when compared to stiffer ones, both in the presence and in the absence of matrix-bound arginine-glycine-aspartic acid cell-adhesion ligands (RGD; 0, 100 and 200 μM). Fibronectin assembly stabilized cell-cell contacts within aggregates, even in non-adhesive matrices. However, MSCs were able to substantially contract the artificial matrix only when RGD was present. Moreover, compliant matrices facilitated cell proliferation and provided an environment conducive for MSC osteogenic differentiation, even without RGD. Cell interactions with the original matrix became less important as time progressed, while the de novo-produced extracellular matrix became a more critical determinant of cell fate. These data provide further insights into the mechanisms by which MSCs sense their microenvironment to organize into tissues, and provide new clues to the design of cell-instructive 3-D matrices. PMID:24607421

  1. Extracellular matrix motion and early morphogenesis.

    PubMed

    Loganathan, Rajprasad; Rongish, Brenda J; Smith, Christopher M; Filla, Michael B; Czirok, Andras; Bénazéraf, Bertrand; Little, Charles D

    2016-06-15

    For over a century, embryologists who studied cellular motion in early amniotes generally assumed that morphogenetic movement reflected migration relative to a static extracellular matrix (ECM) scaffold. However, as we discuss in this Review, recent investigations reveal that the ECM is also moving during morphogenesis. Time-lapse studies show how convective tissue displacement patterns, as visualized by ECM markers, contribute to morphogenesis and organogenesis. Computational image analysis distinguishes between cell-autonomous (active) displacements and convection caused by large-scale (composite) tissue movements. Modern quantification of large-scale 'total' cellular motion and the accompanying ECM motion in the embryo demonstrates that a dynamic ECM is required for generation of the emergent motion patterns that drive amniote morphogenesis. PMID:27302396

  2. Extracellular Matrix Revisited: Roles in Tissue Engineering

    PubMed Central

    2016-01-01

    The extracellular matrix (ECM) is a heterogeneous, connective network composed of fibrous glycoproteins that coordinate in vivo to provide the physical scaffolding, mechanical stability, and biochemical cues necessary for tissue morphogenesis and homeostasis. This review highlights some of the recently raised aspects of the roles of the ECM as related to the fields of biophysics and biomedical engineering. Fundamental aspects of focus include the role of the ECM as a basic cellular structure, for novel spontaneous network formation, as an ideal scaffold in tissue engineering, and its essential contribution to cell sheet technology. As these technologies move from the laboratory to clinical practice, they are bound to shape the vast field of tissue engineering for medical transplantations. PMID:27230457

  3. Extracellular matrix motion and early morphogenesis.

    PubMed

    Loganathan, Rajprasad; Rongish, Brenda J; Smith, Christopher M; Filla, Michael B; Czirok, Andras; Bénazéraf, Bertrand; Little, Charles D

    2016-06-15

    For over a century, embryologists who studied cellular motion in early amniotes generally assumed that morphogenetic movement reflected migration relative to a static extracellular matrix (ECM) scaffold. However, as we discuss in this Review, recent investigations reveal that the ECM is also moving during morphogenesis. Time-lapse studies show how convective tissue displacement patterns, as visualized by ECM markers, contribute to morphogenesis and organogenesis. Computational image analysis distinguishes between cell-autonomous (active) displacements and convection caused by large-scale (composite) tissue movements. Modern quantification of large-scale 'total' cellular motion and the accompanying ECM motion in the embryo demonstrates that a dynamic ECM is required for generation of the emergent motion patterns that drive amniote morphogenesis.

  4. Defining the extracellular matrix using proteomics

    PubMed Central

    Byron, Adam; Humphries, Jonathan D; Humphries, Martin J

    2013-01-01

    The cell microenvironment has a profound influence on the behaviour, growth and survival of cells. The extracellular matrix (ECM) provides not only mechanical and structural support to cells and tissues but also binds soluble ligands and transmembrane receptors to provide spatial coordination of signalling processes. The ability of cells to sense the chemical, mechanical and topographical features of the ECM enables them to integrate complex, multiparametric information into a coherent response to the surrounding microenvironment. Consequently, dysregulation or mutation of ECM components results in a broad range of pathological conditions. Characterization of the composition of ECM derived from various cells has begun to reveal insights into ECM structure and function, and mechanisms of disease. Proteomic methodologies permit the global analysis of subcellular systems, but extracellular and transmembrane proteins present analytical difficulties to proteomic strategies owing to the particular biochemical properties of these molecules. Here, we review advances in proteomic approaches that have been applied to furthering our understanding of the ECM microenvironment. We survey recent studies that have addressed challenges in the analysis of ECM and discuss major outcomes in the context of health and disease. In addition, we summarize efforts to progress towards a systems-level understanding of ECM biology. PMID:23419153

  5. Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix.

    PubMed

    Piotrowski-Daspit, Alexandra S; Nelson, Celeste M

    2016-01-01

    The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

  6. An integrated statistical model for enhanced murine cardiomyocyte differentiation via optimized engagement of 3D extracellular matrices

    PubMed Central

    Jung, Jangwook P.; Hu, Dongjian; Domian, Ibrahim J.; Ogle, Brenda M.

    2015-01-01

    The extracellular matrix (ECM) impacts stem cell differentiation, but identifying formulations supportive of differentiation is challenging in 3D models. Prior efforts involving combinatorial ECM arrays seemed intuitively advantageous. We propose an alternative that suggests reducing sample size and technological burden can be beneficial and accessible when coupled to design of experiments approaches. We predict optimized ECM formulations could augment differentiation of cardiomyocytes derived in vitro. We employed native chemical ligation to polymerize 3D poly (ethylene glycol) hydrogels under mild conditions while entrapping various combinations of ECM and murine induced pluripotent stem cells. Systematic optimization for cardiomyocyte differentiation yielded a predicted solution of 61%, 24%, and 15% of collagen type I, laminin-111, and fibronectin, respectively. This solution was confirmed by increased numbers of cardiac troponin T, α-myosin heavy chain and α-sarcomeric actinin-expressing cells relative to suboptimum solutions. Cardiomyocytes of composites exhibited connexin43 expression, appropriate contractile kinetics and intracellular calcium handling. Further, adding a modulator of adhesion, thrombospondin-1, abrogated cardiomyocyte differentiation. Thus, the integrated biomaterial platform statistically identified an ECM formulation best supportive of cardiomyocyte differentiation. In future, this formulation could be coupled with biochemical stimulation to improve functional maturation of cardiomyocytes derived in vitro or transplanted in vivo. PMID:26687770

  7. Molecular Adhesion between Cartilage Extracellular Matrix Macromolecules

    PubMed Central

    2015-01-01

    In this study, we investigated the molecular adhesion between the major constituents of cartilage extracellular matrix, namely, the highly negatively charged proteoglycan aggrecan and the type II/IX/XI fibrillar collagen network, in simulated physiological conditions. Colloidal force spectroscopy was applied to measure the maximum adhesion force and total adhesion energy between aggrecan end-attached spherical tips (end radius R ≈ 2.5 μm) and trypsin-treated cartilage disks with undamaged collagen networks. Studies were carried out in various aqueous solutions to reveal the physical factors that govern aggrecan–collagen adhesion. Increasing both ionic strength and [Ca2+] significantly increased adhesion, highlighting the importance of electrostatic repulsion and Ca2+-mediated ion bridging effects. In addition, we probed how partial enzymatic degradation of the collagen network, which simulates osteoarthritic conditions, affects the aggrecan–collagen interactions. Interestingly, we found a significant increase in aggrecan–collagen adhesion even when there were no detectable changes at the macro- or microscales. It is hypothesized that the aggrecan–collagen adhesion, together with aggrecan–aggrecan self-adhesion, works synergistically to determine the local molecular deformability and energy dissipation of the cartilage matrix, in turn, affecting its macroscopic tissue properties. PMID:24491174

  8. Extracellular Matrix Dynamics and Fetal Membrane Rupture

    PubMed Central

    Strauss,, Jerome F.

    2013-01-01

    The extracellular matrix (ECM) plays an important role in determining cell and organ function: (1) it is an organizing substrate that provides tissue tensile strength; (2) it anchors cells and influences cell morphology and function via interaction with cell surface receptors; and (3) it is a reservoir for growth factors. Alterations in the content and the composition of the ECM determine its physical and biological properties, including strength and susceptibility to degradation. The ECM components themselves also harbor cryptic matrikines, which when exposed by conformational change or proteolysis have potent effects on cell function, including stimulating the production of cytokines and matrix metalloproteinases (MMPs). Collectively, these properties of the ECM reflect a dynamic tissue component that influences both tissue form and function. This review illustrates how defects in ECM synthesis and metabolism and the physiological process of ECM turnover contribute to changes in the fetal membranes that precede normal parturition and contribute to the pathological events leading to preterm premature rupture of membranes (PPROM). PMID:22267536

  9. Extracellular Matrix Proteins in Hemostasis and Thrombosis

    PubMed Central

    Bergmeier, Wolfgang; Hynes, Richard O.

    2012-01-01

    The adhesion and aggregation of platelets during hemostasis and thrombosis represents one of the best-understood examples of cell–matrix adhesion. Platelets are exposed to a wide variety of extracellular matrix (ECM) proteins once blood vessels are damaged and basement membranes and interstitial ECM are exposed. Platelet adhesion to these ECM proteins involves ECM receptors familiar in other contexts, such as integrins. The major platelet-specific integrin, αIIbβ3, is the best-understood ECM receptor and exhibits the most tightly regulated switch between inactive and active states. Once activated, αIIbβ3 binds many different ECM proteins, including fibrinogen, its major ligand. In addition to αIIbβ3, there are other integrins expressed at lower levels on platelets and responsible for adhesion to additional ECM proteins. There are also some important nonintegrin ECM receptors, GPIb-V-IX and GPVI, which are specific to platelets. These receptors play major roles in platelet adhesion and in the activation of the integrins and of other platelet responses, such as cytoskeletal organization and exocytosis of additional ECM ligands and autoactivators of the platelets. PMID:21937733

  10. Roles of extracellular matrix in follicular development.

    PubMed

    Rodgers, R J; van Wezel, I L; Irving-Rodgers, H F; Lavranos, T C; Irvine, C M; Krupa, M

    1999-01-01

    The cellular biology and changes in the extracellular matrix of ovarian follicles during their development are reviewed. During growth of the bovine ovarian follicle the follicular basal lamina doubles 19 times in surface area. It changes in composition, having collagen IV alpha 1-26 and laminin alpha 1, beta 2 and gamma 1 at the primordial stage, and collagen IV alpha 1 and alpha 2, reduced amounts of alpha 3-alpha 5, and a higher content of laminin alpha 1, beta 2 and gamma 1 at the antral stage. In atretic antral follicles laminin alpha 2 was also detected. The follicular epithelium also changes from one layer to many layers during follicular growth. It is clear that not all granulosal cells have equal potential to divide, and we have evidence that the granulosal cells arise from a population of stem cells. This finding has important ramifications and supports the concept that different follicular growth factors can act on different subsets of granulosal cells. In antral follicles, the replication of cells occurs in the middle layers of the membrana granulosa, with older granulosal cells towards the antrum and towards the basal lamina. The basal cells in the membrana granulosa have also been observed to vary in shape between follicies. In smaller antral follicles, they were either columnar or rounded, and in follicles > 5 mm the cells were all rounded. The reasons for these changes in matrix and cell shapes are discussed in relation to follicular development. PMID:10692866

  11. 3D Linear Transformations in the Form of Matrix and Vector

    NASA Astrophysics Data System (ADS)

    Zhang, Hua

    2008-11-01

    In this article, results of four 3D linear transformations (translation, rotation, scale and shear) in the form of matrix and vector are simplified into a same 3D physical coordinates system. Comparing the simplified results of those four linear transformations, the results obtained from matrix form are exactly the same as what obtained from vector algebra in final expressions, although they are different from original expressions.

  12. Thrombospondin-2 and extracellular matrix assembly

    PubMed Central

    Calabro, Nicole E.; Kristofik, Nina J.; Kyriakides, Themis R.

    2014-01-01

    Background Numerous proteins and small leucine-rich proteoglycans (SLRPs) make up the composition of the extracellular matrix (ECM). Assembly of individual fibrillar components in the ECM, such as collagen, elastin, and fibronectin is understood at the molecular level. In contrast, the incorporation of non-fibrillar components and their functions in the ECM are not fully understood. Scope of review This review will focus on the role of the matricellular protein thrombospondin (TSP) 2 in ECM assembly. Based on findings in TSP2-null mice and in vitro studies, we describe the participation of TSP2 in ECM assembly, cell-ECM interactions, and modulation of the levels of matrix metalloproteinases (MMPs). Major conclusions Evidence summarized in this review suggests that TSP2 can influence collagen fibrillogenesis without being an integral component of fibrils. Altered ECM assembly and excessive breakdown of ECM can have both positive and negative consequences including increased angiogenesis during tissue repair and compromised cardiac tissue integrity, respectively. General significance Proper ECM assembly is critical for maintaining cell functions and providing structural support. Lack of TSP2 is associated with increased angiogenesis, in part, due to altered endothelial cell-ECM interactions. Therefore, minor changes in ECM composition can have profound effects on cell and tissue function. This article is part of a special issue, “Matrix-Mediated Cell Behavior and Properties.” Highlights TSP2 functions primarily as a modulator of cell-ECM interactions and can influence the assembly of ECM. More importantly, TSP2-null ECM enhances angiogenic responses. Therefore, strategies can be pursued to reduce TSP2 and generate novel ECM via decellularization techniques. PMID:24440155

  13. 3D matrix-based cell cultures: Automated analysis of tumor cell survival and proliferation

    PubMed Central

    EKE, IRIS; HEHLGANS, STEPHANIE; SANDFORT, VEIT; CORDES, NILS

    2016-01-01

    Three-dimensional ex vivo cell cultures mimic physiological in vivo growth conditions thereby significantly contributing to our understanding of tumor cell growth and survival, therapy resistance and identification of novel potent cancer targets. In the present study, we describe advanced three-dimensional cell culture methodology for investigating cellular survival and proliferation in human carcinoma cells after cancer therapy including molecular therapeutics. Single cells are embedded into laminin-rich extracellular matrix and can be treated with cytotoxic drugs, ionizing or UV radiation or any other substance of interest when consolidated and approximating in vivo morphology. Subsequently, cells are allowed to grow for automated determination of clonogenic survival (colony number) or proliferation (colony size). The entire protocol of 3D cell plating takes ~1 h working time and pursues for ~7 days before evaluation. This newly developed method broadens the spectrum of exploration of malignant tumors and other diseases and enables the obtainment of more reliable data on cancer treatment efficacy. PMID:26549537

  14. Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres.

    PubMed

    Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

    2016-01-01

    Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering. PMID:26760956

  15. Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres

    PubMed Central

    Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

    2016-01-01

    Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering. PMID:26760956

  16. Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres.

    PubMed

    Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

    2016-01-01

    Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.

  17. The exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixed-species oral biofilm.

    PubMed

    Xiao, Jin; Klein, Marlise I; Falsetta, Megan L; Lu, Bingwen; Delahunty, Claire M; Yates, John R; Heydorn, Arne; Koo, Hyun

    2012-01-01

    Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and p

  18. Protective Properties of Neural Extracellular Matrix.

    PubMed

    Suttkus, Anne; Morawski, Markus; Arendt, Thomas

    2016-01-01

    The extracellular matrix (ECM) of the central nervous system (CNS) occupies a large part of the neural tissue. It serves a variety of functions ranging from support of cell migration and regulating synaptic transmission and plasticity to the active modulation of the neural tissue after injury. In addition, evidence for neuroprotective properties of ECM components has accumulated more recently. In contrast to other connective tissues, the central nervous ECM is mainly composed of glycosaminoglycans, which can be present unbound in the form of hyaluronan or bound to proteins, thus forming proteoglycans. A subtype of this molecular family are the chondroitin sulphate proteoglycans (CSPGs), which are composed of a core protein that carries at least one covalently bound glycosaminoglycan side chain with a certain degree of sulphation. Several studies could show neuroprotective features of CSPGs against excitotoxicity, amyloid-ß toxicity, or oxidative stress. Recently, we could provide evidence for a neuroprotective function of a specialized form of ECM, the so-called perineuronal net ensheathing a subtype of neurons. Here, we will give an overview on recently emerging aspects of neuroprotective properties of CSPGs and perineuronal nets that might be relevant for our understanding on the distribution and progression of brain pathology and future perspectives toward modifying neurodegenerative diseases.

  19. Extracellular matrix, supramolecular organisation and shape.

    PubMed Central

    Scott, J E

    1995-01-01

    Connective tissue function is defined as the formation and maintenance of shape, without which centralised physiologies (circulatory, digestive or nervous) could not have evolved. Two elements, inextensible (collagenous) fibrils and compression-resistant interfibrillar soluble polymers (proteoglycans), cope with all usual stresses. Relationships between the two are highly specific, as demonstrated by electron histochemistry based on Cupromeronic blue and critical electrolyte concentration (CEC) methodologies. Recent ideas on (1) the protofibrillar or modular structure of collagen fibrils, (2) the nature of specific binding sites for proteoglycans on fibrils, and (3) fundamental similarities in secondary and tertiary structures of the glycosaminoglycans (hyaluronan, chondroitin, keratan and dermatan sulphates) are described. They have greatly illuminated the study of extracellular matrix structure and function in normal, pathological (osteogenesis imperfecta) and ageing tissues. The small proteoglycans are proposed to be tissue organisers, orienting and ordering the collagen fibrils--thus shaping the tissue at a molecular and ultimately macro level. These interfibrillar structures are based on their bifunctional character, the protein parts binding to collagen fibrils at specific sites and the glycosaminoglycans duplexing and aggregating to hold the proteins and hence the collagen fibrils at defined distances from each other, rather like yardsticks. Examples of the way these functions work in specific tissues are drawn from the cornea and vitreous humour of the eye and developing tendon. Images Fig. 3 (cont.) Fig. 3 PMID:7591990

  20. Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

    PubMed

    DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

    2015-05-01

    Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases.

  1. Vascular Extracellular Matrix and Arterial Mechanics

    PubMed Central

    WAGENSEIL, JESSICA E.; MECHAM, ROBERT P.

    2009-01-01

    An important factor in the transition from an open to a closed circulatory system was a change in vessel wall structure and composition that enabled the large arteries to store and release energy during the cardiac cycle. The component of the arterial wall in vertebrates that accounts for these properties is the elastic fiber network organized by medial smooth muscle. Beginning with the onset of pulsatile blood flow in the developing aorta, smooth muscle cells in the vessel wall produce a complex extracellular matrix (ECM) that will ultimately define the mechanical properties that are critical for proper function of the adult vascular system. This review discusses the structural ECM proteins in the vertebrate aortic wall and will explore how the choice of ECM components has changed through evolution as the cardiovascular system became more advanced and pulse pressure increased. By correlating vessel mechanics with physiological blood pressure across animal species and in mice with altered vessel compliance, we show that cardiac and vascular development are physiologically coupled, and we provide evidence for a universal elastic modulus that controls the parameters of ECM deposition in vessel wall development. We also discuss mechanical models that can be used to design better tissue-engineered vessels and to test the efficacy of clinical treatments. PMID:19584318

  2. Extracellular matrix composition of the cricopharyngeus muscle.

    PubMed

    Tavares, Raquel Aguiar; Sennes, Luiz Ubirajara; Mauad, Thais; Imamura, Rui; da Silva, Luiz Fernando Ferraz; Carrau, Ricardo Luis

    2012-06-01

    The aim of this study was to analyze the presence and distribution of total collagen, type I and type III collagen, elastic fibers, fibronectin, and versican in the endomysium of cricopharyngeus muscles from adults of various ages. The study was a cross-sectional analysis of human cricopharyngeus muscles. Twenty-seven muscles obtained from autopsies of men and women ranging in age from 28 to 92 years were analyzed with the Picrosirius method, oxidized Weigert resorcin-fuchsin, immunohistochemistry, and image analysis. Collagen had the highest density among the analyzed components. Elastic fibers surrounded each muscle cell; they were aligned longitudinally by their long axis and associated with traversing fibers, thereby forming a fiber network with embedded muscle cells. The fibronectin and versican contents varied widely among the specimens. We found no statistically significant differences between the proportion of extracellular matrix (ECM) components and factors such as gender and race. We conclude that the higher proportion of type I and type III collagen is compatible with the cricopharyngeus muscle's sphincteric behavior, and the arrangement of the elastic fibers may also contribute to the muscle's elasticity. We found no statistically significant correlation between the ECM components and age. PMID:21874509

  3. Micro- and Macrorheology of Jellyfish Extracellular Matrix

    PubMed Central

    Gambini, Camille; Abou, Bérengère; Ponton, Alain; Cornelissen, Annemiek J.M.

    2012-01-01

    Mechanical properties of the extracellular matrix (ECM) play a key role in tissue organization and morphogenesis. Rheological properties of jellyfish ECM (mesoglea) were measured in vivo at the cellular scale by passive microrheology techniques: microbeads were injected in jellyfish ECM and their Brownian motion was recorded to determine the mechanical properties of the surrounding medium. Microrheology results were compared with macrorheological measurements performed with a shear rheometer on slices of jellyfish mesoglea. We found that the ECM behaved as a viscoelastic gel at the macroscopic scale and as a much softer and heterogeneous viscoelastic structure at the microscopic scale. The fibrous architecture of the mesoglea, as observed by differential interference contrast and scanning electron microscopy, was in accord with these scale-dependent mechanical properties. Furthermore, the evolution of the mechanical properties of the ECM during aging was investigated by measuring microrheological properties at different jellyfish sizes. We measured that the ECM in adult jellyfish was locally stiffer than in juvenile ones. We argue that this stiffening is a consequence of local aggregations of fibers occurring gradually during aging of the jellyfish mesoglea and is enhanced by repetitive muscular contractions of the jellyfish. PMID:22225792

  4. Engineering hydrogels as extracellular matrix mimics

    PubMed Central

    Geckil, Hikmet; Xu, Feng; Zhang, Xiaohui; Moon, SangJun

    2010-01-01

    Extracellular matrix (ECM) is a complex cellular environment consisting of proteins, proteoglycans, and other soluble molecules. ECM provides structural support to mammalian cells and a regulatory milieu with a variety of important cell functions, including assembling cells into various tissues and organs, regulating growth and cell–cell communication. Developing a tailored in vitro cell culture environment that mimics the intricate and organized nanoscale meshwork of native ECM is desirable. Recent studies have shown the potential of hydrogels to mimic native ECM. Such an engineered native-like ECM is more likely to provide cells with rational cues for diagnostic and therapeutic studies. The research for novel biomaterials has led to an extension of the scope and techniques used to fabricate biomimetic hydrogel scaffolds for tissue engineering and regenerative medicine applications. In this article, we detail the progress of the current state-of-the-art engineering methods to create cell-encapsulating hydrogel tissue constructs as well as their applications in in vitro models in biomedicine. PMID:20394538

  5. Enhanced Invasion of Metastatic Cancer Cells via Extracellular Matrix Interface

    PubMed Central

    Zhu, Jiangrui; Liang, Long; Jiao, Yang; Liu, Liyu

    2015-01-01

    Cancer cell invasion is a major component of metastasis and is responsible for extensive cell diffusion into and major destruction of tissues. Cells exhibit complex invasion modes, including a variety of collective behaviors. This phenomenon results in the structural heterogeneity of the extracellular matrix (ECM) in tissues. Here, we systematically investigated the environmental heterogeneity facilitating tumor cell invasion via a combination of in vitro cell migration experiments and computer simulations. Specifically, we constructed an ECM microenvironment in a microfabricated biochip and successfully created a three-dimensional (3D) funnel-like matrigel interface inside. Scanning electron microscopy demonstrated that the interface was at the interior defects of the nano-scale molecular anisotropic orientation and the localized structural density variations in the matrigel. Our results, particularly the correlation of the collective migration pattern with the geometric features of the funnel-like interface, indicate that this heterogeneous in vitro ECM structure strongly guides and promotes aggressive cell invasion in the rigid matrigel space. A cellular automaton model was proposed based on our experimental observations, and the associated quantitative analysis indicated that cell invasion was initiated and controlled by several mechanisms, including microenvironment heterogeneity, long-range cell-cell homotype and gradient-driven directional cellular migration. Our work shows the feasibility of constructing a complex and heterogeneous in vitro 3D ECM microenvironment that mimics the in vivo environment. Moreover, our results indicate that ECM heterogeneity is essential in controlling collective cell invasive behaviors and therefore determining metastasis efficiency. PMID:25706718

  6. Responses of fibroblasts to anchorage of dorsal extracellular matrix receptors.

    PubMed

    Beningo, Karen A; Dembo, Micah; Wang, Yu-li

    2004-12-28

    Fibroblasts in 2D cultures differ dramatically in behavior from those in the 3D environment of a multicellular organism. However, the basis of this disparity is unknown. A key difference is the spatial arrangement of anchored extracellular matrix (ECM) receptors to the ventral surface in 2D cultures and throughout the entire surface in 3D cultures. Therefore, we asked whether changing the topography of ECM receptor anchorage alone could invoke a morphological response. By using polyacrylamide-based substrates to present anchored fibronectin or collagen on dorsal cell surfaces, we found that well spread fibroblasts in 2D cultures quickly changed into a bipolar or stellate morphology similar to fibroblasts in vivo. Cells in this environment lacked lamellipodia and large actin bundles and formed small focal adhesions only near focused sites of protrusion. These responses depend on substrate rigidity, calcium ion, and, likely, the calcium-dependent protease calpain. We suggest that fibroblasts respond to both spatial distribution and mechanical input of anchored ECM receptors. Changes in cell shape may in turn affect diverse cellular activities, including gene expression, growth, and differentiation, as shown in numerous previous studies.

  7. Responses of fibroblasts to anchorage of dorsal extracellular matrix receptors.

    PubMed

    Beningo, Karen A; Dembo, Micah; Wang, Yu-li

    2004-12-28

    Fibroblasts in 2D cultures differ dramatically in behavior from those in the 3D environment of a multicellular organism. However, the basis of this disparity is unknown. A key difference is the spatial arrangement of anchored extracellular matrix (ECM) receptors to the ventral surface in 2D cultures and throughout the entire surface in 3D cultures. Therefore, we asked whether changing the topography of ECM receptor anchorage alone could invoke a morphological response. By using polyacrylamide-based substrates to present anchored fibronectin or collagen on dorsal cell surfaces, we found that well spread fibroblasts in 2D cultures quickly changed into a bipolar or stellate morphology similar to fibroblasts in vivo. Cells in this environment lacked lamellipodia and large actin bundles and formed small focal adhesions only near focused sites of protrusion. These responses depend on substrate rigidity, calcium ion, and, likely, the calcium-dependent protease calpain. We suggest that fibroblasts respond to both spatial distribution and mechanical input of anchored ECM receptors. Changes in cell shape may in turn affect diverse cellular activities, including gene expression, growth, and differentiation, as shown in numerous previous studies. PMID:15601776

  8. Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis

    PubMed Central

    Zaman, Muhammad H.; Trapani, Linda M.; Sieminski, Alisha; MacKellar, Drew; Gong, Haiyan; Kamm, Roger D.; Wells, Alan; Lauffenburger, Douglas A.; Matsudaira, Paul

    2006-01-01

    Cell migration on 2D surfaces is governed by a balance between counteracting tractile and adhesion forces. Although biochemical factors such as adhesion receptor and ligand concentration and binding, signaling through cell adhesion complexes, and cytoskeletal structure assembly/disassembly have been studied in detail in a 2D context, the critical biochemical and biophysical parameters that affect cell migration in 3D matrices have not been quantitatively investigated. We demonstrate that, in addition to adhesion and tractile forces, matrix stiffness is a key factor that influences cell movement in 3D. Cell migration assays in which Matrigel density, fibronectin concentration, and β1 integrin binding are systematically varied show that at a specific Matrigel density the migration speed of DU-145 human prostate carcinoma cells is a balance between tractile and adhesion forces. However, when biochemical parameters such as matrix ligand and cell integrin receptor levels are held constant, maximal cell movement shifts to matrices exhibiting lesser stiffness. This behavior contradicts current 2D models but is predicted by a recent force-based computational model of cell movement in a 3D matrix. As expected, this 3D motility through an extracellular environment of pore size much smaller than cellular dimensions does depend on proteolytic activity as broad-spectrum matrix metalloproteinase (MMP) inhibitors limit the migration of DU-145 cells and also HT-1080 fibrosarcoma cells. Our experimental findings here represent, to our knowledge, discovery of a previously undescribed set of balances of cell and matrix properties that govern the ability of tumor cells to migration in 3D environments. PMID:16832052

  9. Local 3D matrix confinement determines division axis through cell shape.

    PubMed

    He, Lijuan; Chen, Weitong; Wu, Pei-Hsun; Jimenez, Angela; Wong, Bin Sheng; San, Angela; Konstantopoulos, Konstantinos; Wirtz, Denis

    2016-02-01

    How the division axis is determined in mammalian cells embedded in three-dimensional (3D) matrices remains elusive, despite that many types of cells divide in 3D environments. Cells on two-dimensional (2D) substrates typically round up completely to divide. Here, we show that in 3D collagen matrices, mammalian cells such as HT1080 human fibrosarcoma and MDA-MB-231 breast cancer cells exhibit division modes distinct from their Counterparts on 2D substrates, with a markedly higher fraction of cells remaining highly elongated through mitosis in 3D matrices. The long axis of elongated mitotic cells accurately predicts the division axis, independently of matrix density and cell-matrix interactions. This 3D-specific elongated division mode is determined by the local confinement produced by the matrix and the ability of cells to protrude and locally remodel the matrix via β1 integrin. Elongated division is readily recapitulated using collagen-coated microfabricated channels. Cells depleted of β1 integrin still divide in the elongated mode in microchannels, suggesting that 3D confinement is sufficient to induce the elongated cell-division phenotype.

  10. Local 3D matrix confinement determines division axis through cell shape

    PubMed Central

    He, Lijuan; Chen, Weitong; Wu, Pei-Hsun; Jimenez, Angela; Wong, Bin Sheng; San, Angela; Konstantopoulos, Konstantinos; Wirtz, Denis

    2016-01-01

    How the division axis is determined in mammalian cells embedded in three-dimensional (3D) matrices remains elusive, despite that many types of cells divide in 3D environments. Cells on two-dimensional (2D) substrates typically round up completely to divide. Here, we show that in 3D collagen matrices, mammalian cells such as HT1080 human fibrosarcoma and MDA-MB-231 breast cancer cells exhibit division modes distinct from their Counterparts on 2D substrates, with a markedly higher fraction of cells remaining highly elongated through mitosis in 3D matrices. The long axis of elongated mitotic cells accurately predicts the division axis, independently of matrix density and cell-matrix interactions. This 3D-specific elongated division mode is determined by the local confinement produced by the matrix and the ability of cells to protrude and locally remodel the matrix via β1 integrin. Elongated division is readily recapitulated using collagen-coated microfabricated channels. Cells depleted of β1 integrin still divide in the elongated mode in microchannels, suggesting that 3D confinement is sufficient to induce the elongated cell-division phenotype. PMID:26515603

  11. The extracellular matrix of plants: Molecular, cellular and developmental biology

    SciTech Connect

    1996-12-31

    A symposium entitled ``The Extracellular Matrix of Plants: Molecular, Cellular and Developmental Biology was held in Tamarron, Colorado, March 15--21, 1996. The following topics were explored in addresses by 43 speakers: structure and biochemistry of cell walls; biochemistry, molecular biology and biosynthesis of lignin; secretory pathway and synthesis of glycoproteins; biosynthesis of matrix polysaccharides, callose and cellulose; role of the extracellular matrix in plant growth and development; plant cell walls in symbiosis and pathogenesis.

  12. Lung extracellular matrix and redox regulation

    PubMed Central

    Watson, Walter H.; Ritzenthaler, Jeffrey D.; Roman, Jesse

    2016-01-01

    Pulmonary fibrosis affects millions worldwide and, even though there has been a significant investment in understanding the processes involved in wound healing and maladaptive repair, a complete understanding of the mechanisms responsible for lung fibrogenesis eludes us, and interventions capable of reversing or halting disease progression are not available. Pulmonary fibrosis is characterized by the excessive expression and uncontrolled deposition of extracellular matrix (ECM) proteins resulting in erosion of the tissue structure. Initially considered an ‘end-stage’ process elicited after injury, these events are now considered pathogenic and are believed to contribute to the course of the disease. By interacting with integrins capable of signal transduction and by influencing tissue mechanics, ECM proteins modulate processes ranging from cell adhesion and migration to differentiation and growth factor expression. In doing so, ECM proteins help orchestrate complex developmental processes and maintain tissue homeostasis. However, poorly controlled deposition of ECM proteins promotes inflammation, fibroproliferation, and aberrant differentiation of cells, and has been implicated in the pathogenesis of pulmonary fibrosis, atherosclerosis and cancer. Considering their vital functions, ECM proteins are the target of investigation, and oxidation–reduction (redox) reactions have emerged as important regulators of the ECM. Oxidative stress invariably accompanies lung disease and promotes ECM expression directly or through the overproduction of pro-fibrotic growth factors, while affecting integrin binding and activation. In vitro and in vivo investigations point to redox reactions as targets for intervention in pulmonary fibrosis and related disorders, but studies in humans have been disappointing probably due to the narrow impact of the interventions tested, and our poor understanding of the factors that regulate these complex reactions. This review is not meant to

  13. Lung extracellular matrix and redox regulation.

    PubMed

    Watson, Walter H; Ritzenthaler, Jeffrey D; Roman, Jesse

    2016-08-01

    Pulmonary fibrosis affects millions worldwide and, even though there has been a significant investment in understanding the processes involved in wound healing and maladaptive repair, a complete understanding of the mechanisms responsible for lung fibrogenesis eludes us, and interventions capable of reversing or halting disease progression are not available. Pulmonary fibrosis is characterized by the excessive expression and uncontrolled deposition of extracellular matrix (ECM) proteins resulting in erosion of the tissue structure. Initially considered an 'end-stage' process elicited after injury, these events are now considered pathogenic and are believed to contribute to the course of the disease. By interacting with integrins capable of signal transduction and by influencing tissue mechanics, ECM proteins modulate processes ranging from cell adhesion and migration to differentiation and growth factor expression. In doing so, ECM proteins help orchestrate complex developmental processes and maintain tissue homeostasis. However, poorly controlled deposition of ECM proteins promotes inflammation, fibroproliferation, and aberrant differentiation of cells, and has been implicated in the pathogenesis of pulmonary fibrosis, atherosclerosis and cancer. Considering their vital functions, ECM proteins are the target of investigation, and oxidation-reduction (redox) reactions have emerged as important regulators of the ECM. Oxidative stress invariably accompanies lung disease and promotes ECM expression directly or through the overproduction of pro-fibrotic growth factors, while affecting integrin binding and activation. In vitro and in vivo investigations point to redox reactions as targets for intervention in pulmonary fibrosis and related disorders, but studies in humans have been disappointing probably due to the narrow impact of the interventions tested, and our poor understanding of the factors that regulate these complex reactions. This review is not meant to

  14. 3D printed nanocomposite matrix for the study of breast cancer bone metastasis.

    PubMed

    Zhu, Wei; Holmes, Benjamin; Glazer, Robert I; Zhang, Lijie Grace

    2016-01-01

    Bone is one of the most common metastatic sites of breast cancer, but the underlying mechanisms remain unclear, in part due to an absence of advanced platforms for cancer culture and study that mimic the bone microenvironment. In the present study, we integrated a novel stereolithography-based 3D printer and a unique 3D printed nano-ink consisting of hydroxyapatite nanoparticles suspended in hydrogel to create a biomimetic bone-specific environment for evaluating breast cancer bone invasion. Breast cancer cells cultured in a geometrically optimized matrix exhibited spheroid morphology and migratory characteristics. Co-culture of tumor cells with bone marrow mesenchymal stem cells increased the formation of spheroid clusters. The 3D matrix also allowed for higher drug resistance of breast cancer cells than 2D culture. These results validate that our 3D bone matrix can mimic tumor bone microenvironments, suggesting that it can serve as a tool for studying metastasis and assessing drug sensitivity. From the Clinical Editor: Cancer remains a major cause of mortality for patients in the clinical setting. For breast cancer, bone is one of the most common metastatic sites. In this intriguing article, the authors developed a bone-like environment using 3D printing technology to investigate the underlying biology of bone metastasis. Their results would also allow a new model for other researchers who work on cancer to use.

  15. Microfluidic vascularized bone tissue model with hydroxyapatite-incorporated extracellular matrix.

    PubMed

    Jusoh, Norhana; Oh, Soojung; Kim, Sudong; Kim, Jangho; Jeon, Noo Li

    2015-10-21

    Current in vitro systems mimicking bone tissues fail to fully integrate the three-dimensional (3D) microvasculature and bone tissue microenvironments, decreasing their similarity to in vivo conditions. Here, we propose 3D microvascular networks in a hydroxyapatite (HA)-incorporated extracellular matrix (ECM) for designing and manipulating a vascularized bone tissue model in a microfluidic device. Incorporation of HA of various concentrations resulted in ECM with varying mechanical properties. Sprouting angiogenesis was affected by mechanically modulated HA-extracellular matrix interactions, generating a model of vascularized bone microenvironment. Using this platform, we observed that hydroxyapatite enhanced angiogenic properties such as sprout length, sprouting speed, sprout number, and lumen diameter. This new platform integrates fibrin ECM with the synthetic bone mineral HA to provide in vivo-like microenvironments for bone vessel sprouting.

  16. Tuning 3D Collagen Matrix Stiffness Independently of Collagen Concentration Modulates Endothelial Cell Behavior

    PubMed Central

    Mason, Brooke N.; Starchenko, Alina; Williams, Rebecca M.; Bonassar, Lawrence J.; Reinhart-King, Cynthia A.

    2012-01-01

    Numerous studies have described the effects of matrix stiffening on cell behavior using two dimensional (2D) synthetic surfaces; however less is known about the effects of matrix stiffening on cells embedded in three dimensional (3D) in vivo-like matrices. A primary limitation in investigating the effects of matrix stiffness in 3D is the lack of materials that can be tuned to control stiffness independently of matrix density. Here, we use collagen-based scaffolds where the mechanical properties are tuned using non-enzymatic glycation of the collagen in solution, prior to polymerization. Collagen solutions glycated prior to polymerization result in collagen gels with a 3-fold increase in compressive modulus without significant changes to the collagen architecture. Using these scaffolds, we show that endothelial cell spreading increases with matrix stiffness, as does the number and length of angiogenic sprouts and the overall spheroid outgrowth. Differences in sprout length are maintained even when the receptor for advanced glycation endproducts is inhibited. Our results demonstrate the ability to de-couple matrix stiffness from matrix density and structure in collagen gels, and that increased matrix stiffness results in increased sprouting and outgrowth. PMID:22902816

  17. A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis

    PubMed Central

    Reticker-Flynn, Nathan E.; Braga Malta, David F.; Winslow, Monte M.; Lamar, John M.; Xu, Mary J.; Underhill, Gregory H.; Hynes, Richard O.; Jacks, Tyler E.; Bhatia, Sangeeta N.

    2013-01-01

    Extracellular matrix interactions play essential roles in normal physiology and many pathological processes. While the importance of ECM interactions in metastasis is well documented, systematic approaches to identify their roles in distinct stages of tumorigenesis have not been described. Here we report a novel screening platform capable of measuring phenotypic responses to combinations of ECM molecules. Using a genetic mouse model of lung adenocarcinoma, we measure the ECM-dependent adhesion of tumor-derived cells. Hierarchical clustering of the adhesion profiles differentiates metastatic cell lines from primary tumor lines. Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8, or laminin. We show that these molecules correlate with human disease and that their interactions are mediated in part by α3β1 integrin. Thus, our platform allowed us to interrogate interactions between metastatic cells and their microenvironments, and identified ECM and integrin interactions that could serve as therapeutic targets. PMID:23047680

  18. Using a decellularized splenic matrix as a 3D scaffold for hepatocyte cultivation in vitro: a preliminary trial.

    PubMed

    Zheng, Xing-Long; Xiang, Jun-Xi; Wu, Wan-Quan; Wang, Bo; Liu, Wen-Yan; Gao, Rui; Dong, Ding-Hui; Lv, Yi

    2015-08-01

    Using a decellularized liver matrix (DLM) to reengineer liver tissue is a promising therapy for end-stage liver disease. However, the limited supply of donor organs still hampers its potential clinical application, while a xenogenic decellularized matrix may bring a risk of zoonosis and immunological rejection. Therefore, an appropriate alternative scaffold is needed. In this research, we established a decellularized splenic matrix (DSM) in a rodent model, which preserved the 3D ultrastructure, the components of the extracellular matrix (ECM) and the native vascular network. The DSM and DLM had similar components of ECM, and similar mechanical properties. Hepatocytes were seeded to the DSM and DLM for dynamic culturing up to 6 d, and distributed both in decellularized sinusoidal spaces and around the vessels. The TUNEL-positive cell percentage in a dynamic culturing decellularized splenic matrix (dDSM) was 10.7%  ±  3.6% at 3d and 25.8%  ±  5.6% at 5d, although 14.2%  ±  4.5% and 24.8%  ±  2.9%, respectively, in a dynamic culturing decellularized liver matrix (dDLM) at the same time point (p  >  0.05). Primary hepatocytes in the dDSM and dDLM expressed albumin, G6pc and Ugt1a1. The gene expression of Cyp2b1, Cyp1a2 and HNF1α in the gene transcription level revealed hepatocytes had lower gene expression levels in the dDSM compared with the dDLM at 3d, but better than those in a sandwich culture. The cumulative albumin production at 6 d of culture was 80.7   ±   9.6 μg per million cells in the dDSM and 89.6   ±   4.6 μg per million cells in the dDLM (p  >  0.05). In summary, the DSM is a promising 3D scaffold for hepatocyte cultivation in vitro. PMID:26290516

  19. Three-dimensional migration of macrophages requires Hck for podosome organization and extracellular matrix proteolysis

    PubMed Central

    Cougoule, Céline; Le Cabec, Véronique; Poincloux, Renaud; Al Saati, Talal; Mège, Jean-Louis; Tabouret, Guillaume; Lowell, Clifford A.; Laviolette-Malirat, Nathalie

    2010-01-01

    Tissue infiltration of phagocytes exacerbates several human pathologies including chronic inflammations or cancers. However, the mechanisms involved in macrophage migration through interstitial tissues are poorly understood. We investigated the role of Hck, a Src-family kinase involved in the organization of matrix adhesion and degradation structures called podosomes. In Hck−/− mice submitted to peritonitis, we found that macrophages accumulated in interstitial tissues and barely reached the peritoneal cavity. In vitro, 3-dimensional (3D) migration and matrix degradation abilities, 2 protease-dependent properties of bone marrow–derived macrophages (BMDMs), were affected in Hck−/− BMDMs. These macrophages formed few and undersized podosome rosettes and, consequently, had reduced matrix proteolysis operating underneath despite normal expression and activity of matrix metalloproteases. Finally, in fibroblasts unable to infiltrate matrix, ectopic expression of Hck provided the gain–of–3D migration function, which correlated positively with formation of podosome rosettes. In conclusion, spatial organization of podosomes as large rosettes, proteolytic degradation of extracellular matrix, and 3D migration appeared to be functionally linked and regulated by Hck in macrophages. Hck, as the first protein combining a phagocyte-limited expression with a role in 3D migration, could be a target for new anti-inflammatory and antitumor molecules. PMID:19897576

  20. From mechanotransduction to extracellular matrix gene expression in fibroblasts.

    PubMed

    Chiquet, Matthias; Gelman, Laurent; Lutz, Roman; Maier, Silke

    2009-05-01

    Tissue mechanics provide an important context for tissue growth, maintenance and function. On the level of organs, external mechanical forces largely influence the control of tissue homeostasis by endo- and paracrine factors. On the cellular level, it is well known that most normal cell types depend on physical interactions with their extracellular matrix in order to respond efficiently to growth factors. Fibroblasts and other adherent cells sense changes in physical parameters in their extracellular matrix environment, transduce mechanical into chemical information, and integrate these signals with growth factor derived stimuli to achieve specific changes in gene expression. For connective tissue cells, production of the extracellular matrix is a prominent response to changes in mechanical load. We will review the evidence that integrin-containing cell-matrix adhesion contacts are essential for force transmission from the extracellular matrix to the cytoskeleton, and describe novel experiments indicating that mechanotransduction in fibroblasts depends on focal adhesion adaptor proteins that might function as molecular springs. We will stress the importance of the contractile actin cytoskeleton in balancing external with internal forces, and describe new results linking force-controlled actin dynamics directly to the expression of specific genes, among them the extracellular matrix protein tenascin-C. As assembly lines for diverse signaling pathways, matrix adhesion contacts are now recognized as the major sites of crosstalk between mechanical and chemical stimuli, with important consequences for cell growth and differentiation.

  1. Elucidating the Role of Matrix Stiffness in 3D Cell Migration and Remodeling

    PubMed Central

    Ehrbar, M.; Sala, A.; Lienemann, P.; Ranga, A.; Mosiewicz, K.; Bittermann, A.; Rizzi, S.C.; Weber, F.E.; Lutolf, M.P.

    2011-01-01

    Reductionist in vitro model systems which mimic specific extracellular matrix functions in a highly controlled manner, termed artificial extracellular matrices (aECM), have increasingly been used to elucidate the role of cell-ECM interactions in regulating cell fate. To better understand the interplay of biophysical and biochemical effectors in controlling three-dimensional cell migration, a poly(ethylene glycol)-based aECM platform was used in this study to explore the influence of matrix cross-linking density, represented here by stiffness, on cell migration in vitro and in vivo. In vitro, the migration behavior of single preosteoblastic cells within hydrogels of varying stiffness and susceptibilities to degradation by matrix metalloproteases was assessed by time-lapse microscopy. Migration behavior was seen to be strongly dependent on matrix stiffness, with two regimes identified: a nonproteolytic migration mode dominating at relatively low matrix stiffness and proteolytic migration at higher stiffness. Subsequent in vivo experiments revealed a similar stiffness dependence of matrix remodeling, albeit less sensitive to the matrix metalloprotease sensitivity. Therefore, our aECM model system is well suited to unveil the role of biophysical and biochemical determinants of physiologically relevant cell migration phenomena. PMID:21244824

  2. Bioengineered 3D brain tumor model to elucidate the effects of matrix stiffness on glioblastoma cell behavior using PEG-based hydrogels.

    PubMed

    Wang, Christine; Tong, Xinming; Yang, Fan

    2014-07-01

    Glioblastoma (GBM) is the most common and aggressive form of primary brain tumor with a median survival of 12-15 months, and the mechanisms underlying GBM tumor progression remain largely elusive. Given the importance of tumor niche signaling in driving GBM progression, there is a strong need to develop in vitro models to facilitate analysis of brain tumor cell-niche interactions in a physiologically relevant and controllable manner. Here we report the development of a bioengineered 3D brain tumor model to help elucidate the effects of matrix stiffness on GBM cell fate using poly(ethylene-glycol) (PEG)-based hydrogels with brain-mimicking biochemical and mechanical properties. We have chosen PEG given its bioinert nature and tunable physical property, and the resulting hydrogels allow tunable matrix stiffness without changing the biochemical contents. To facilitate cell proliferation and migration, CRGDS and a MMP-cleavable peptide were chemically incorporated. Hyaluronic acid (HA) was also incorporated to mimic the concentration in the brain extracellular matrix. Using U87 cells as a model GBM cell line, we demonstrate that such biomimetic hydrogels support U87 cell growth, spreading, and migration in 3D over the course of 3 weeks in culture. Gene expression analyses showed U87 cells actively deposited extracellular matrix and continued to upregulate matrix remodeling genes. To examine the effects of matrix stiffness on GBM cell fate in 3D, we encapsulated U87 cells in soft (1 kPa) or stiff (26 kPa) hydrogels, which respectively mimics the matrix stiffness of normal brain or GBM tumor tissues. Our results suggest that changes in matrix stiffness induce differential GBM cell proliferation, morphology, and migration modes in 3D. Increasing matrix stiffness led to delayed U87 cell proliferation inside hydrogels, but cells formed denser spheroids with extended cell protrusions. Cells cultured in stiff hydrogels also showed upregulation of HA synthase 1 and matrix

  3. Monitoring of Extracellular Matrix Formation using Nanosecond Pulsed Laser

    NASA Astrophysics Data System (ADS)

    Ishihara, Miya; Sato, Masato; Mitani, Genya; Nagai, Toshihiro; Kutsuna, Toshiharu; Mochida, Joji; Kikuchi, Makoto

    There is a new demand in the field of tissue engineering for evaluation technology of extracellular matrix because the extracellular matrix plays an important role in the function of skeletal tissue such as articular cartilage. We previously proposed a noninvasive method of viscoelastic characterization of tissue phantom, based on the photoacoustic measurement. The purpose of this study was to verify the applicability of the photoacoustic measurement method for monitoring of the development of extracellular matrix using tissue engineering technology. The decay times measured by the photoacoustic method were varied with culture periods when tissue-engineered articular cartilages with various culture periods (-12 weeks) were used as samples. Tissue-engineered cartilage cultured for a long period showed shorter decay times, indicating that the samples approached an elastic solid from a rheological viewpoint. By comparison between biochemical analyses and biomechanical studies, we proved that the photoacoustic signal was a good indicator for evaluating extracellular matrix formation because the change of the photoacoustic decay times would reflect the production of an extracellular matrix.

  4. Implementation of parallel matrix decomposition for NIKE3D on the KSR1 system

    SciTech Connect

    Su, Philip S.; Fulton, R.E.; Zacharia, T.

    1995-06-01

    New massively parallel computer architecture has revolutionized the design of computer algorithms and promises to have significant influence on algorithms for engineering computations. Realistic engineering problems using finite element analysis typically imply excessively large computational requirements. Parallel supercomputers that have the potential for significantly increasing calculation speeds can meet these computational requirements. This report explores the potential for the parallel Cholesky (U{sup T}DU) matrix decomposition algorithm on NIKE3D through actual computations. The examples of two- and three-dimensional nonlinear dynamic finite element problems are presented on the Kendall Square Research (KSR1) multiprocessor system, with 64 processors, at Oak Ridge National Laboratory. The numerical results indicate that the parallel Cholesky (U{sup T}DU) matrix decomposition algorithm is attractive for NIKE3D under multi-processor system environments.

  5. Measurement Matrix Optimization and Mismatch Problem Compensation for DLSLA 3-D SAR Cross-Track Reconstruction

    PubMed Central

    Bao, Qian; Jiang, Chenglong; Lin, Yun; Tan, Weixian; Wang, Zhirui; Hong, Wen

    2016-01-01

    With a short linear array configured in the cross-track direction, downward looking sparse linear array three-dimensional synthetic aperture radar (DLSLA 3-D SAR) can obtain the 3-D image of an imaging scene. To improve the cross-track resolution, sparse recovery methods have been investigated in recent years. In the compressive sensing (CS) framework, the reconstruction performance depends on the property of measurement matrix. This paper concerns the technique to optimize the measurement matrix and deal with the mismatch problem of measurement matrix caused by the off-grid scatterers. In the model of cross-track reconstruction, the measurement matrix is mainly affected by the configuration of antenna phase centers (APC), thus, two mutual coherence based criteria are proposed to optimize the configuration of APCs. On the other hand, to compensate the mismatch problem of the measurement matrix, the sparse Bayesian inference based method is introduced into the cross-track reconstruction by jointly estimate the scatterers and the off-grid error. Experiments demonstrate the performance of the proposed APCs’ configuration schemes and the proposed cross-track reconstruction method. PMID:27556471

  6. Measurement Matrix Optimization and Mismatch Problem Compensation for DLSLA 3-D SAR Cross-Track Reconstruction.

    PubMed

    Bao, Qian; Jiang, Chenglong; Lin, Yun; Tan, Weixian; Wang, Zhirui; Hong, Wen

    2016-01-01

    With a short linear array configured in the cross-track direction, downward looking sparse linear array three-dimensional synthetic aperture radar (DLSLA 3-D SAR) can obtain the 3-D image of an imaging scene. To improve the cross-track resolution, sparse recovery methods have been investigated in recent years. In the compressive sensing (CS) framework, the reconstruction performance depends on the property of measurement matrix. This paper concerns the technique to optimize the measurement matrix and deal with the mismatch problem of measurement matrix caused by the off-grid scatterers. In the model of cross-track reconstruction, the measurement matrix is mainly affected by the configuration of antenna phase centers (APC), thus, two mutual coherence based criteria are proposed to optimize the configuration of APCs. On the other hand, to compensate the mismatch problem of the measurement matrix, the sparse Bayesian inference based method is introduced into the cross-track reconstruction by jointly estimate the scatterers and the off-grid error. Experiments demonstrate the performance of the proposed APCs' configuration schemes and the proposed cross-track reconstruction method. PMID:27556471

  7. Measurement Matrix Optimization and Mismatch Problem Compensation for DLSLA 3-D SAR Cross-Track Reconstruction.

    PubMed

    Bao, Qian; Jiang, Chenglong; Lin, Yun; Tan, Weixian; Wang, Zhirui; Hong, Wen

    2016-08-22

    With a short linear array configured in the cross-track direction, downward looking sparse linear array three-dimensional synthetic aperture radar (DLSLA 3-D SAR) can obtain the 3-D image of an imaging scene. To improve the cross-track resolution, sparse recovery methods have been investigated in recent years. In the compressive sensing (CS) framework, the reconstruction performance depends on the property of measurement matrix. This paper concerns the technique to optimize the measurement matrix and deal with the mismatch problem of measurement matrix caused by the off-grid scatterers. In the model of cross-track reconstruction, the measurement matrix is mainly affected by the configuration of antenna phase centers (APC), thus, two mutual coherence based criteria are proposed to optimize the configuration of APCs. On the other hand, to compensate the mismatch problem of the measurement matrix, the sparse Bayesian inference based method is introduced into the cross-track reconstruction by jointly estimate the scatterers and the off-grid error. Experiments demonstrate the performance of the proposed APCs' configuration schemes and the proposed cross-track reconstruction method.

  8. A 3D printed nano bone matrix for characterization of breast cancer cell and osteoblast interactions

    NASA Astrophysics Data System (ADS)

    Zhu, Wei; Castro, Nathan J.; Cui, Haitao; Zhou, Xuan; Boualam, Benchaa; McGrane, Robert; Glazer, Robert I.; Zhang, Lijie Grace

    2016-08-01

    Bone metastasis is one of the most prevalent complications of late-stage breast cancer, in which the native bone matrix components, including osteoblasts, are intimately involved in tumor progression. The development of a successful in vitro model would greatly facilitate understanding the underlying mechanism of breast cancer bone invasion as well as provide a tool for effective discovery of novel therapeutic strategies. In the current study, we fabricated a series of in vitro bone matrices composed of a polyethylene glycol hydrogel and nanocrystalline hydroxyapatite of varying concentrations to mimic the native bone microenvironment for the investigation of breast cancer bone metastasis. A stereolithography-based three-dimensional (3D) printer was used to fabricate the bone matrices with precisely controlled architecture. The interaction between breast cancer cells and osteoblasts was investigated in the optimized bone matrix. Using a Transwell® system to separate the two cell lines, breast cancer cells inhibited osteoblast proliferation, while osteoblasts stimulated breast cancer cell growth, whereas, both cell lines increased IL-8 secretion. Breast cancer cells co-cultured with osteoblasts within the 3D bone matrix formed multi-cellular spheroids in comparison to two-dimensional monolayers. These findings validate the use of our 3D printed bone matrices as an in vitro metastasis model, and highlights their potential for investigating breast cancer bone metastasis.

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

    PubMed

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

    2013-02-15

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

  10. A novel culture device for the evaluation of three-dimensional extracellular matrix materials.

    PubMed

    Akhyari, Payam; Ziegler, Heiko; Gwanmesia, Patricia; Barth, Mareike; Schilp, Soeren; Huelsmann, Joern; Hoffmann, Stefanie; Bosch, Julia; Kögler, Gesine; Lichtenberg, Artur

    2014-09-01

    Cell-matrix interactions in a three-dimensional (3D) extracellular matrix (ECM) are of fundamental importance in living tissue, and their in vitro reconstruction in bioartificial structures represents a core target of contemporary tissue engineering concepts. For a detailed analysis of cell-matrix interaction under highly controlled conditions, we developed a novel ECM evaluation culture device (EECD) that allows for a precisely defined surface-seeding of 3D ECM scaffolds, irrespective of their natural geometry. The effectiveness of EECD was evaluated in the context of heart valve tissue engineering. Detergent decellularized pulmonary cusps were mounted in EECD and seeded with endothelial cells (ECs) to study EC adhesion, morphology and function on a 3D ECM after 3, 24, 48 and 96 h. Standard EC monolayers served as controls. Exclusive top-surface-seeding of 3D ECM by viable ECs was demonstrated by laser scanning microscopy (LSM), resulting in a confluent re-endothelialization of the ECM after 96 h. Cell viability and protein expression, as demonstrated by MTS assay and western blot analysis (endothelial nitric oxide synthase, von Willebrand factor), were preserved at maintained levels over time. In conclusion, EECD proves as a highly effective system for a controlled repopulation and in vitro analysis of cell-ECM interactions in 3D ECM. PMID:22761130

  11. The Effects of Matrix Stiffness and RhoA on the Phenotypic Plasticity of Smooth Muscle Cells in a 3-D Biosynthetic Hydrogel System

    PubMed Central

    Peyton, Shelly R.; Kim, Peter D.; Ghajar, Cyrus M.; Seliktar, Dror; Putnam, Andrew J.

    2008-01-01

    Studies using 2-D cultures have shown that the mechanical properties of the extracellular matrix (ECM) influence cell migration, spreading, proliferation, and differentiation; however, cellular mechanosensing in 3-D remains under-explored. To investigate this topic, a unique biomaterial system based on poly(ethylene glycol)-conjugated fibrinogen was adapted to study phenotypic plasticity in smooth muscle cells (SMCs) as a function of ECM mechanics in 3-D. Tuning compressive modulus between 448–5804 Pa modestly regulated SMC cytoskeletal assembly in 3-D, with spread cells in stiff matrices having a slightly higher degree of F-actin bundling after prolonged culture. However, vinculin expression in all 3-D conditions was qualitatively low and was not assembled into the classic focal adhesions typically seen in 2-D cultures. Given the evidence that RhoA-mediated cytoskeletal contractility represents a critical node in mechanosensing, we molecularly upregulated contractility by inducing SMCs to express constitutively active RhoA. In these cells, F-actin bundling and total vinculin expression increased, and focal adhesion-like structures began to emerge, consistent with RhoA’s mechanism of action cells cultured on 2-D substrates. Furthermore, SMC proliferation in 3-D did not depend significantly on matrix stiffness, and was reduced by constitutive activation of RhoA irrespective of ECM mechanical properties. Conversely, the expression of contractile markers globally increased with constitutive RhoA activation and depended on 3-D matrix stiffness only in cells with heightened RhoA activity. Combined, these data suggest the synergistic effects of ECM mechanics and RhoA activity on SMC phenotype in 3-D are distinct from those in 2-D, and highlight the importance of studying the mechanical role of cell-matrix interactions in tunable 3-D environments. PMID:18342366

  12. Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix.

    PubMed

    Xie, Daniel; Ju, Donghong; Speyer, Cecilia; Gorski, David; Kosir, Mary A

    2016-08-14

    Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis(1). Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

  13. Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix.

    PubMed

    Xie, Daniel; Ju, Donghong; Speyer, Cecilia; Gorski, David; Kosir, Mary A

    2016-01-01

    Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis(1). Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor. PMID:27585062

  14. Vitamin A Deficiency and Alterations in the Extracellular Matrix

    PubMed Central

    Barber, Teresa; Esteban-Pretel, Guillermo; Marín, María Pilar; Timoneda, Joaquín

    2014-01-01

    Vitamin A or retinol which is the natural precursor of several biologically active metabolites can be considered the most multifunctional vitamin in mammals. Its deficiency is currently, along with protein malnutrition, the most serious and common nutritional disorder worldwide. It is necessary for normal embryonic development and postnatal tissue homeostasis, and exerts important effects on cell proliferation, differentiation and apoptosis. These actions are produced mainly by regulating the expression of a variety of proteins through transcriptional and non-transcriptional mechanisms. Extracellular matrix proteins are among those whose synthesis is known to be modulated by vitamin A. Retinoic acid, the main biologically active form of vitamin A, influences the expression of collagens, laminins, entactin, fibronectin, elastin and proteoglycans, which are the major components of the extracellular matrix. Consequently, the structure and macromolecular composition of this extracellular compartment is profoundly altered as a result of vitamin A deficiency. As cell behavior, differentiation and apoptosis, and tissue mechanics are influenced by the extracellular matrix, its modifications potentially compromise organ function and may lead to disease. This review focuses on the effects of lack of vitamin A in the extracellular matrix of several organs and discusses possible molecular mechanisms and pathologic implications. PMID:25389900

  15. Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms.

    PubMed

    Klein, Marlise I; Hwang, Geelsu; Santos, Paulo H S; Campanella, Osvaldo H; Koo, Hyun

    2015-01-01

    Biofilms are highly structured microbial communities that are enmeshed in a self-produced extracellular matrix. Within the complex oral microbiome, Streptococcus mutans is a major producer of extracellular polymeric substances including exopolysaccharides (EPS), eDNA, and lipoteichoic acid (LTA). EPS produced by S. mutans-derived exoenzymes promote local accumulation of microbes on the teeth, while forming a spatially heterogeneous and diffusion-limiting matrix that protects embedded bacteria. The EPS-rich matrix provides mechanical stability/cohesiveness and facilitates the creation of highly acidic microenvironments, which are critical for the pathogenesis of dental caries. In parallel, S. mutans also releases eDNA and LTA, which can contribute with matrix development. eDNA enhances EPS (glucan) synthesis locally, increasing the adhesion of S. mutans to saliva-coated apatitic surfaces and the assembly of highly cohesive biofilms. eDNA and other extracellular substances, acting in concert with EPS, may impact the functional properties of the matrix and the virulence of cariogenic biofilms. Enhanced understanding about the assembly principles of the matrix may lead to efficacious approaches to control biofilm-related diseases.

  16. Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms

    PubMed Central

    Klein, Marlise I.; Hwang, Geelsu; Santos, Paulo H. S.; Campanella, Osvaldo H.; Koo, Hyun

    2015-01-01

    Biofilms are highly structured microbial communities that are enmeshed in a self-produced extracellular matrix. Within the complex oral microbiome, Streptococcus mutans is a major producer of extracellular polymeric substances including exopolysaccharides (EPS), eDNA, and lipoteichoic acid (LTA). EPS produced by S. mutans-derived exoenzymes promote local accumulation of microbes on the teeth, while forming a spatially heterogeneous and diffusion-limiting matrix that protects embedded bacteria. The EPS-rich matrix provides mechanical stability/cohesiveness and facilitates the creation of highly acidic microenvironments, which are critical for the pathogenesis of dental caries. In parallel, S. mutans also releases eDNA and LTA, which can contribute with matrix development. eDNA enhances EPS (glucan) synthesis locally, increasing the adhesion of S. mutans to saliva-coated apatitic surfaces and the assembly of highly cohesive biofilms. eDNA and other extracellular substances, acting in concert with EPS, may impact the functional properties of the matrix and the virulence of cariogenic biofilms. Enhanced understanding about the assembly principles of the matrix may lead to efficacious approaches to control biofilm-related diseases. PMID:25763359

  17. A Look inside the Listeria monocytogenes Biofilms Extracellular Matrix.

    PubMed

    Colagiorgi, Angelo; Di Ciccio, Pierluigi; Zanardi, Emanuela; Ghidini, Sergio; Ianieri, Adriana

    2016-01-01

    Listeria monocytogenes is a foodborne pathogen able to persist in food industry and is responsible for a severe illness called listeriosis. The ability of L. monocytogenes to persist in environments is due to its capacity to form biofilms that are a sessile community of microorganisms embedded in a self-produced matrix of extracellular polymeric substances (EPS's). In this review, we summarized recent efforts performed in order to better characterize the polymeric substances that compose the extracellular matrix (ECM) of L. monocytogenes biofilms. EPS extraction and analysis led to the identification of polysaccharides, proteins, extracellular DNA, and other molecules within the listerial ECM. All this knowledge will be useful for increasing food protection, suggesting effective strategies for the minimization of persistence of L. monocytogenes in food industry environments. PMID:27681916

  18. A Look inside the Listeria monocytogenes Biofilms Extracellular Matrix

    PubMed Central

    Colagiorgi, Angelo; Di Ciccio, Pierluigi; Zanardi, Emanuela; Ghidini, Sergio; Ianieri, Adriana

    2016-01-01

    Listeria monocytogenes is a foodborne pathogen able to persist in food industry and is responsible for a severe illness called listeriosis. The ability of L. monocytogenes to persist in environments is due to its capacity to form biofilms that are a sessile community of microorganisms embedded in a self-produced matrix of extracellular polymeric substances (EPS’s). In this review, we summarized recent efforts performed in order to better characterize the polymeric substances that compose the extracellular matrix (ECM) of L. monocytogenes biofilms. EPS extraction and analysis led to the identification of polysaccharides, proteins, extracellular DNA, and other molecules within the listerial ECM. All this knowledge will be useful for increasing food protection, suggesting effective strategies for the minimization of persistence of L. monocytogenes in food industry environments.

  19. A Look inside the Listeria monocytogenes Biofilms Extracellular Matrix

    PubMed Central

    Colagiorgi, Angelo; Di Ciccio, Pierluigi; Zanardi, Emanuela; Ghidini, Sergio; Ianieri, Adriana

    2016-01-01

    Listeria monocytogenes is a foodborne pathogen able to persist in food industry and is responsible for a severe illness called listeriosis. The ability of L. monocytogenes to persist in environments is due to its capacity to form biofilms that are a sessile community of microorganisms embedded in a self-produced matrix of extracellular polymeric substances (EPS’s). In this review, we summarized recent efforts performed in order to better characterize the polymeric substances that compose the extracellular matrix (ECM) of L. monocytogenes biofilms. EPS extraction and analysis led to the identification of polysaccharides, proteins, extracellular DNA, and other molecules within the listerial ECM. All this knowledge will be useful for increasing food protection, suggesting effective strategies for the minimization of persistence of L. monocytogenes in food industry environments. PMID:27681916

  20. Regulation of Osteoblast Survival by the Extracellular Matrix and Gravity

    NASA Technical Reports Server (NTRS)

    Globus. Ruth K.; Almeida, Eduardo A. C.; Searby, Nancy D.; Bowley, Susan M. (Technical Monitor)

    2000-01-01

    Spaceflight adversely affects the skeleton, posing a substantial risk to astronaut's health during long duration missions. The reduced bone mass observed in growing animals following spaceflight is due at least in part to inadequate bone formation by osteoblasts. Thus, it is of central importance to identify basic cellular mechanisms underlying normal bone formation. The fundamental ideas underlying our research are that interactions between extracellular matrix proteins, integrin adhesion receptors, cytoplasmic signaling and cytoskeletal proteins are key ingredients for the proper functioning of osteoblasts, and that gravity impacts these interactions. As an in vitro model system we used primary fetal rat calvarial cells which faithfully recapitulate osteoblast differentiation characteristically observed in vivo. We showed that specific integrin receptors ((alpha)3(beta)1), ((alpha)5(beta)1), ((alpha)8(betal)1) and extracellular matrix proteins (fibronectin, laminin) were needed for the differentiation of immature osteoblasts. In the course of maturation, cultured osteoblasts switched from depending on fibronectin and laminin for differentiation to depending on these proteins for their very survival. Furthermore, we found that manipulating the gravity vector using ground-based models resulted in activation of key intracellular survival signals generated by integrin/extracellular matrix interactions. We are currently testing the in vivo relevance of some of these observations using targeted transgenic technology. In conclusion, mechanical factors including gravity may participate in regulating survival via cellular interactions with the extracellular matrix. This leads us to speculate that microgravity adversely affects the survival of osteoblasts and contributes to spaceflight-induced osteoporosis.

  1. Functional Ultrasound Imaging for Assessment of Extracellular Matrix Scaffolds Used for Liver Organoid Formation

    PubMed Central

    Gessner, Ryan C.; Hanson, Ariel D.; Feingold, Steven; Cashion, Avery T.; Corcimaru, Ana; Wu, Bryant T.; Mullins, Christopher R.; Aylward, Stephen R.; Reid, Lola M.; Dayton, Paul A.

    2015-01-01

    A method of 3D functional ultrasound imaging has been developed to enable non-destructive assessment of extracellular matrix scaffolds that have been prepared by decellularization protocols and are intended for recellularization to create organoids. A major challenge in organ decellularization is retaining patent micro-vascular structures crucial for nutrient access and functionality of organoids. The imaging method described here provides statistical distributions of flow rates throughout the tissue volumes, 3D vessel network architecture visualization, characterization of microvessel volumes and sizes, and delineation of matrix from vascular circuits. The imaging protocol was tested on matrix scaffolds that are tissue-specific, but not species-specific, matrix extracts, prepared by a process that preserved >98% of the collagens, collagen-associated matrix components, and matrix-bound growth factors and cytokines. Image-derived data are discussed with respect to assessment of scaffolds followed by proof-of-concept studies in organoid establishment using Hep3B, human hepatoblast-like cells. Histology showed that the cells attached to scaffolds with patent vasculature within minutes, achieved engraftment at near 100%, expressed liver-specific functions within 24h, and yielded evidence of proliferation and increasing differentiation of cells throughout the two weeks of culture studies. This imaging method should prove valuable in analyses of such matrix scaffolds. PMID:24011714

  2. Modeling the tumor extracellular matrix: Tissue engineering tools repurposed towards new frontiers in cancer biology.

    PubMed

    Gill, Bartley J; West, Jennifer L

    2014-06-27

    Cancer progression is mediated by complex epigenetic, protein and structural influences. Critical among them are the biochemical, mechanical and architectural properties of the extracellular matrix (ECM). In recognition of the ECM's important role, cancer biologists have repurposed matrix mimetic culture systems first widely used by tissue engineers as new tools for in vitro study of tumor models. In this review we discuss the pathological changes in tumor ECM, the limitations of 2D culture on both traditional and polyacrylamide hydrogel surfaces in modeling these characteristics and advances in both naturally derived and synthetic scaffolds to facilitate more complex and controllable 3D cancer cell culture. Studies using naturally derived matrix materials like Matrigel and collagen have produced significant findings related to tumor morphogenesis and matrix invasion in a 3D environment and the mechanotransductive signaling that mediates key tumor-matrix interaction. However, lack of precise experimental control over important matrix factors in these matrices have increasingly led investigators to synthetic and semi-synthetic scaffolds that offer the engineering of specific ECM cues and the potential for more advanced experimental manipulations. Synthetic scaffolds composed of poly(ethylene glycol) (PEG), for example, facilitate highly biocompatible 3D culture, modular bioactive features like cell-mediated matrix degradation and complete independent control over matrix bioactivity and mechanics. Future work in PEG or similar reductionist synthetic matrix systems should enable the study of increasingly complex and dynamic tumor-ECM relationships in the hopes that accurate modeling of these relationships may reveal new cancer therapeutics targeting tumor progression and metastasis.

  3. 3-D FEM Modeling of fiber/matrix interface debonding in UD composites including surface effects

    NASA Astrophysics Data System (ADS)

    Pupurs, A.; Varna, J.

    2012-02-01

    Fiber/matrix interface debond growth is one of the main mechanisms of damage evolution in unidirectional (UD) polymer composites. Because for polymer composites the fiber strain to failure is smaller than for the matrix multiple fiber breaks occur at random positions when high mechanical stress is applied to the composite. The energy released due to each fiber break is usually larger than necessary for the creation of a fiber break therefore a partial debonding of fiber/matrix interface is typically observed. Thus the stiffness reduction of UD composite is contributed both from the fiber breaks and from the interface debonds. The aim of this paper is to analyze the debond growth in carbon fiber/epoxy and glass fiber/epoxy UD composites using fracture mechanics principles by calculation of energy release rate GII. A 3-D FEM model is developed for calculation of energy release rate for fiber/matrix interface debonds at different locations in the composite including the composite surface region where the stress state differs from the one in the bulk composite. In the model individual partially debonded fiber is surrounded by matrix region and embedded in a homogenized composite.

  4. The effects of extracellular sugar extraction on the 3D-structure of biological soil crusts from different ecosystems

    NASA Astrophysics Data System (ADS)

    Felde, Vincent; Rossi, Federico; Colesie, Claudia; Uteau-Puschmann, Daniel; Felix-Henningsen, Peter; Peth, Stephan; De Philippis, Roberto

    2015-04-01

    Biological soil crusts (BSCs) play important roles in the hydrological cycles of many different ecosystems around the world. In arid and semi-arid regions, they alter the availability and redistribution of water. Especially in early successional stage BSCs, this feature can be attributed to the presence and characteristics of extracellular polymeric substances (EPS) that are excreted by the crusts' organisms. In a previous study, the extraction of EPS from BSCs of the SW United States lead to a significant change in their hydrological behavior, namely the sorptivity of water (Rossi et al. 2012). This was concluded to be the effect of a change in the pore structure of these crusts, which is why in this work we investigated the effect of the EPS-extraction on soil structure using 3D-computed micro-tomography (µCT). We studied different types of BSCs from Svalbard, Germany, Israel and South Africa with varying grain sizes and species compositions (from green algae to light and dark cyanobacterial crusts with and without lichens and/or mosses). Unlike other EPS-extraction methods, the one utilized here is aimed at removing the extracellular matrix from crust samples whilst acting non-destructively (Rossi et al. 2012). For every crust sample, we physically cut out a small piece (1cm) from a larger sample contained in Petri dish, and scanned it in a CT at a high resolution (voxel edge length: 7µm). After putting it back in the dish, approximately in the same former position, it was treated for EPS-extraction and then removed and scanned again in order to check for a possible effect of the EPS-extraction. Our results show that the utilized EPS-extraction method had varying extraction efficiencies: while in some cases the amount removed was barely significant, in other cases up to 50% of the total content was recovered. Notwithstanding, no difference in soil micro-structure could be detected, neither in total porosity, nor in the distribution of pore sizes, the

  5. Specialisation of extracellular matrix for function in tendons and ligaments

    PubMed Central

    Birch, Helen L.; Thorpe, Chavaunne T.; Rumian, Adam P.

    2013-01-01

    Summary Tendons and ligaments are similar structures in terms of their composition, organisation and mechanical properties. The distinction between them stems from their anatomical location; tendons form a link between muscle and bone while ligaments link bones to bones. A range of overlapping functions can be assigned to tendon and ligaments and each structure has specific mechanical properties which appear to be suited for particular in vivo function. The extracellular matrix in tendon and ligament varies in accordance with function, providing appropriate mechanical properties. The most useful framework in which to consider extracellular matrix differences therefore is that of function rather than anatomical location. In this review we discuss what is known about the relationship between functional requirements, structural properties from molecular to gross level, cellular gene expression and matrix turnover. The relevance of this information is considered by reviewing clinical aspects of tendon and ligament repair and reconstructive procedures. PMID:23885341

  6. Mean deformation metrics for quantifying 3D cell–matrix interactions without requiring information about matrix material properties

    PubMed Central

    Stout, David A.; Bar-Kochba, Eyal; Estrada, Jonathan B.; Toyjanova, Jennet; Kesari, Haneesh; Reichner, Jonathan S.; Franck, Christian

    2016-01-01

    Mechanobiology relates cellular processes to mechanical signals, such as determining the effect of variations in matrix stiffness with cell tractions. Cell traction recorded via traction force microscopy (TFM) commonly takes place on materials such as polyacrylamide- and polyethylene glycol-based gels. Such experiments remain limited in physiological relevance because cells natively migrate within complex tissue microenvironments that are spatially heterogeneous and hierarchical. Yet, TFM requires determination of the matrix constitutive law (stress–strain relationship), which is not always readily available. In addition, the currently achievable displacement resolution limits the accuracy of TFM for relatively small cells. To overcome these limitations, and increase the physiological relevance of in vitro experimental design, we present a new approach and a set of associated biomechanical signatures that are based purely on measurements of the matrix's displacements without requiring any knowledge of its constitutive laws. We show that our mean deformation metrics (MDM) approach can provide significant biophysical information without the need to explicitly determine cell tractions. In the process of demonstrating the use of our MDM approach, we succeeded in expanding the capability of our displacement measurement technique such that it can now measure the 3D deformations around relatively small cells (∼10 micrometers), such as neutrophils. Furthermore, we also report previously unseen deformation patterns generated by motile neutrophils in 3D collagen gels. PMID:26929377

  7. Mean deformation metrics for quantifying 3D cell-matrix interactions without requiring information about matrix material properties.

    PubMed

    Stout, David A; Bar-Kochba, Eyal; Estrada, Jonathan B; Toyjanova, Jennet; Kesari, Haneesh; Reichner, Jonathan S; Franck, Christian

    2016-03-15

    Mechanobiology relates cellular processes to mechanical signals, such as determining the effect of variations in matrix stiffness with cell tractions. Cell traction recorded via traction force microscopy (TFM) commonly takes place on materials such as polyacrylamide- and polyethylene glycol-based gels. Such experiments remain limited in physiological relevance because cells natively migrate within complex tissue microenvironments that are spatially heterogeneous and hierarchical. Yet, TFM requires determination of the matrix constitutive law (stress-strain relationship), which is not always readily available. In addition, the currently achievable displacement resolution limits the accuracy of TFM for relatively small cells. To overcome these limitations, and increase the physiological relevance of in vitro experimental design, we present a new approach and a set of associated biomechanical signatures that are based purely on measurements of the matrix's displacements without requiring any knowledge of its constitutive laws. We show that our mean deformation metrics (MDM) approach can provide significant biophysical information without the need to explicitly determine cell tractions. In the process of demonstrating the use of our MDM approach, we succeeded in expanding the capability of our displacement measurement technique such that it can now measure the 3D deformations around relatively small cells (∼10 micrometers), such as neutrophils. Furthermore, we also report previously unseen deformation patterns generated by motile neutrophils in 3D collagen gels. PMID:26929377

  8. The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps

    PubMed Central

    Cabezas-Olcoz, Jonathan; Wang, Steven X.; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E.

    2016-01-01

    Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix. PMID:27622514

  9. The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

    PubMed

    Johnson, Chad J; Cabezas-Olcoz, Jonathan; Kernien, John F; Wang, Steven X; Beebe, David J; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E

    2016-09-01

    Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix. PMID:27622514

  10. 3-D shear lag model for the analysis of interface damage in ceramic matrix composites

    SciTech Connect

    Dharani, L.R.; Ji, F.

    1995-12-31

    In this paper a micromechanics analytical model is presented for characterizing the behavior of a unidirectional brittle matrix composite containing initial matrix flaws, specifically, as they approach a fiber-matrix interface. It is contemplated that when a matrix crack impinges on the interface it may go around the fiber or go through the fiber by breaking it or debond the fiber/matrix interface. It has been experimentally observed that the crack front does not remain straight, rather it bows once it impinges on a row of fibers. If a unit cell approach is used, the problem is clearly non-axisymmetric and three-dimensional. Since most of the previous analyses dealing with self-similar cracking and interface debonding have considered axisymmetric cracking or two-dimensional planar geometries, the development of an analytical micromechanics model using a 3-D (non-axisymmetric) formulation is needed. The model is based on the consistent shear lag constitutive relations and does account for the large stiffness of the ceramic matrix. Since the present consistent shear lag model is for Cartesian coordinates, we have first derived the consistent shear lag constitutive relations in cylindrical coordinates. The governing equations are obtained by minimizing the potential energy in which the three displacements are represented by means of finite exponential series. Since the full field stresses and displacements are known, the strain energy release rates for self-similar extension of the matrix crack (Gp) and the interface debonding (Gd) are calculated using the Compliance method. The competition between various failure modes will be assessed based on the above strain energy release rates and the corresponding critical (toughness) values. The type of interfaces addressed include fictional, elastic, and gradient with varying properties (interphase). An extensive parametric study will be presented involving different constitutive properties and interface conditions.

  11. Front-end receiver electronics for a matrix transducer for 3-D transesophageal echocardiography.

    PubMed

    Yu, Zili; Blaak, Sandra; Chang, Zu-yao; Yao, Jiajian; Bosch, Johan G; Prins, Christian; Lancée, Charles T; de Jong, Nico; Pertijs, Michiel A P; Meijer, Gerard C M

    2012-07-01

    There is a clear clinical need for creating 3-D images of the heart. One promising technique is the use of transesophageal echocardiography (TEE). To enable 3-D TEE, we are developing a miniature ultrasound probe containing a matrix piezoelectric transducer with more than 2000 elements. Because a gastroscopic tube cannot accommodate the cables needed to connect all transducer elements directly to an imaging system, a major challenge is to locally reduce the number of channels, while maintaining a sufficient signal-to-noise ratio. This can be achieved by using front-end receiver electronics bonded to the transducers to provide appropriate signal conditioning in the tip of the probe. This paper presents the design of such electronics, realizing time-gain compensation (TGC) and micro-beamforming using simple, low-power circuits. Prototypes of TGC amplifiers and micro-beamforming cells have been fabricated in 0.35-μm CMOS technology. These prototype chips have been combined on a printed circuit board (PCB) to form an ultrasound-receiver system capable of reading and combining the signals of three transducer elements. Experimental results show that this design is a suitable candidate for 3-D TEE.

  12. Extracellular matrix components direct porcine muscle stem cell behavior

    SciTech Connect

    Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J.

    2010-02-01

    In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.

  13. The Extracellular Matrix Regulates Granuloma Necrosis in Tuberculosis.

    PubMed

    Al Shammari, Basim; Shiomi, Takayuki; Tezera, Liku; Bielecka, Magdalena K; Workman, Victoria; Sathyamoorthy, Tarangini; Mauri, Francesco; Jayasinghe, Suwan N; Robertson, Brian D; D'Armiento, Jeanine; Friedland, Jon S; Elkington, Paul T

    2015-08-01

    A central tenet of tuberculosis pathogenesis is that caseous necrosis leads to extracellular matrix destruction and bacterial transmission. We reconsider the underlying mechanism of tuberculosis pathology and demonstrate that collagen destruction may be a critical initial event, causing caseous necrosis as opposed to resulting from it. In human tuberculosis granulomas, regions of extracellular matrix destruction map to areas of caseous necrosis. In mice, transgenic expression of human matrix metalloproteinase 1 causes caseous necrosis, the pathological hallmark of human tuberculosis. Collagen destruction is the principal pathological difference between humanised mice and wild-type mice with tuberculosis, whereas the release of proinflammatory cytokines does not differ, demonstrating that collagen breakdown may lead to cell death and caseation. To investigate this hypothesis, we developed a 3-dimensional cell culture model of tuberculosis granuloma formation, using bioelectrospray technology. Collagen improved survival of Mycobacterium tuberculosis-infected cells analyzed on the basis of a lactate dehydrogenase release assay, propidium iodide staining, and measurement of the total number of viable cells. Taken together, these findings suggest that collagen destruction is an initial event in tuberculosis immunopathology, leading to caseous necrosis and compromising the immune response, revealing a previously unappreciated role for the extracellular matrix in regulating the host-pathogen interaction. PMID:25676469

  14. Vascular wall extracellular matrix proteins and vascular diseases

    PubMed Central

    Xu, Junyan; Shi, Guo-Ping

    2014-01-01

    Extracellular matrix proteins form the basic structure of blood vessels. Along with providing basic structural support to blood vessels, matrix proteins interact with different sets of vascular cells via cell surface integrin or non-integrin receptors. Such interactions induce vascular cell de novo synthesis of new matrix proteins during blood vessel development or remodeling. Under pathological conditions, vascular matrix proteins undergo proteolytic processing, yielding bioactive fragments to influence vascular wall matrix remodeling. Vascular cells also produce alternatively spliced variants that induce vascular cell production of different matrix proteins to interrupt matrix homeostasis, leading to increased blood vessel stiffness; vascular cell migration, proliferation, or death; or vascular wall leakage and rupture. Destruction of vascular matrix proteins leads to vascular cell or blood-borne leukocyte accumulation, proliferation, and neointima formation within the vascular wall; blood vessels prone to uncontrolled enlargement during blood flow diastole; tortuous vein development; and neovascularization from existing pathological tissue microvessels. Here we summarize discoveries related to blood vessel matrix proteins within the past decade from basic and clinical studies in humans and animals — from expression to cross-linking, assembly, and degradation under physiological and vascular pathological conditions, including atherosclerosis, aortic aneurysms, varicose veins, and hypertension. PMID:25045854

  15. Gene evolution and functions of extracellular matrix proteins in teeth

    PubMed Central

    Yoshizaki, Keigo; Yamada, Yoshihiko

    2013-01-01

    The extracellular matrix (ECM) not only provides physical support for tissues, but it is also critical for tissue development, homeostasis and disease. Over 300 ECM molecules have been defined as comprising the “core matrisome” in mammals through the analysis of whole genome sequences. During tooth development, the structure and functions of the ECM dynamically change. In the early stages, basement membranes (BMs) separate two cell layers of the dental epithelium and the mesenchyme. Later in the differentiation stages, the BM layer is replaced with the enamel matrix and the dentin matrix, which are secreted by ameloblasts and odontoblasts, respectively. The enamel matrix genes and the dentin matrix genes are each clustered in two closed regions located on human chromosome 4 (mouse chromosome 5), except for the gene coded for amelogenin, the major enamel matrix protein, which is located on the sex chromosomes. These genes for enamel and dentin matrix proteins are derived from a common ancestral gene, but as a result of evolution, they diverged in terms of their specific functions. These matrix proteins play important roles in cell adhesion, polarity, and differentiation and mineralization of enamel and dentin matrices. Mutations of these genes cause diseases such as odontogenesis imperfect (OI) and amelogenesis imperfect (AI). In this review, we discuss the recently defined terms matrisome and matrixome for ECMs, as well as focus on genes and functions of enamel and dentin matrix proteins. PMID:23539364

  16. [The corneal wound healing and the extracellular matrix].

    PubMed

    Varkoly, Gréta; Bencze, János; Hortobágyi, Tibor; Módis, László

    2016-06-19

    The cornea is the first refractive element of the eye. The transparency of the cornea results from the regularly arranged collagen fibrils, forming lamellar structure and the leucin rich proteoglycans, which make interactions between the fibrils. The adult cornea consists mainly of fibril-forming collagens. The cornea has less amount of fibril associated and non-fibrillar collagens. The main proteoglycans of the cornea are keratan-sulfate proteoglycans and it also contains dermatan-sulfate proteoglycans. Disorders of the proteoglycan synthesis lead to the disruption of the unique pattern and result in thicker collagen fibrils. The abnormal structure of the extracellular matrix can generate corneal disorders and the loss of corneal transparency. Furthermore, proteoglycans and collagens have an important role in wound healing. In injury the keratocytes produce higher amounts of collagens and proteoglycans mediated by growth factors. Depending on the ratio of the cells and growth factors the extracellular matrix returns to normal or corneal scar tissue develops. PMID:27287839

  17. Proteomics Analysis of the Zebrafish Skeletal Extracellular Matrix

    PubMed Central

    Kessels, Maurijn Y.; Huitema, Leonie F. A.; Boeren, Sjef; Kranenbarg, Sander; Schulte-Merker, Stefan; van Leeuwen, Johan L.; de Vries, Sacco C.

    2014-01-01

    The extracellular matrix of the immature and mature skeleton is key to the development and function of the skeletal system. Notwithstanding its importance, it has been technically challenging to obtain a comprehensive picture of the changes in skeletal composition throughout the development of bone and cartilage. In this study, we analyzed the extracellular protein composition of the zebrafish skeleton using a mass spectrometry-based approach, resulting in the identification of 262 extracellular proteins, including most of the bone and cartilage specific proteins previously reported in mammalian species. By comparing these extracellular proteins at larval, juvenile, and adult developmental stages, 123 proteins were found that differed significantly in abundance during development. Proteins with a reported function in bone formation increased in abundance during zebrafish development, while analysis of the cartilage matrix revealed major compositional changes during development. The protein list includes ligands and inhibitors of various signaling pathways implicated in skeletogenesis such as the Int/Wingless as well as the insulin-like growth factor signaling pathways. This first proteomic analysis of zebrafish skeletal development reveals that the zebrafish skeleton is comparable with the skeleton of other vertebrate species including mammals. In addition, our study reveals 6 novel proteins that have never been related to vertebrate skeletogenesis and shows a surprisingly large number of differences in the cartilage and bone proteome between the head, axis and caudal fin regions. Our study provides the first systematic assessment of bone and cartilage protein composition in an entire vertebrate at different stages of development. PMID:24608635

  18. Conformal Nanopatterning of Extracellular Matrix Proteins onto Topographically Complex Surfaces

    PubMed Central

    Sun, Yan; Jallerat, Quentin; Szymanski, John M.

    2015-01-01

    We report a method for conformal nanopatterning of extracellular matrix proteins onto engineered surfaces independent of underlying microtopography. This enables fibronectin, laminin, and other proteins to be applied to biomaterial surfaces in complex geometries inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface, used here to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment. PMID:25506720

  19. Tendon Extracellular Matrix Alterations in Ullrich Congenital Muscular Dystrophy

    PubMed Central

    Sardone, Francesca; Traina, Francesco; Bondi, Alice; Merlini, Luciano; Santi, Spartaco; Maraldi, Nadir Mario; Faldini, Cesare; Sabatelli, Patrizia

    2016-01-01

    Collagen VI (COLVI) is a non-fibrillar collagen expressed in skeletal muscle and most connective tissues. Mutations in COLVI genes cause two major clinical forms, Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). In addition to congenital muscle weakness, patients affected by COLVI myopathies show axial and proximal joint contractures and distal joint hypermobility, which suggest the involvement of the tendon function. We examined a peroneal tendon biopsy and tenocyte culture of a 15-year-old patient affected by UCMD with compound heterozygous COL6A2 mutations. In patient’s tendon biopsy, we found striking morphological alterations of tendon fibrils, consisting in irregular profiles and reduced mean diameter. The organization of the pericellular matrix of tenocytes, the primary site of collagen fibril assembly, was severely affected, as determined by immunoelectron microscopy, which showed an abnormal accumulation of COLVI and altered distribution of collagen I (COLI) and fibronectin (FBN). In patient’s tenocyte culture, COLVI web formation and cell surface association were severely impaired; large aggregates of COLVI, which matched with COLI labeling, were frequently detected in the extracellular matrix. In addition, metalloproteinase MMP-2, an extracellular matrix-regulating enzyme, was increased in the conditioned medium of patient’s tenocytes, as determined by gelatin zymography and western blot. Altogether, these data indicate that COLVI deficiency may influence the organization of UCMD tendon matrix, resulting in dysfunctional fibrillogenesis. The alterations of tendon matrix may contribute to the complex pathogenesis of COLVI related myopathies. PMID:27375477

  20. Tendon Extracellular Matrix Alterations in Ullrich Congenital Muscular Dystrophy.

    PubMed

    Sardone, Francesca; Traina, Francesco; Bondi, Alice; Merlini, Luciano; Santi, Spartaco; Maraldi, Nadir Mario; Faldini, Cesare; Sabatelli, Patrizia

    2016-01-01

    Collagen VI (COLVI) is a non-fibrillar collagen expressed in skeletal muscle and most connective tissues. Mutations in COLVI genes cause two major clinical forms, Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). In addition to congenital muscle weakness, patients affected by COLVI myopathies show axial and proximal joint contractures and distal joint hypermobility, which suggest the involvement of the tendon function. We examined a peroneal tendon biopsy and tenocyte culture of a 15-year-old patient affected by UCMD with compound heterozygous COL6A2 mutations. In patient's tendon biopsy, we found striking morphological alterations of tendon fibrils, consisting in irregular profiles and reduced mean diameter. The organization of the pericellular matrix of tenocytes, the primary site of collagen fibril assembly, was severely affected, as determined by immunoelectron microscopy, which showed an abnormal accumulation of COLVI and altered distribution of collagen I (COLI) and fibronectin (FBN). In patient's tenocyte culture, COLVI web formation and cell surface association were severely impaired; large aggregates of COLVI, which matched with COLI labeling, were frequently detected in the extracellular matrix. In addition, metalloproteinase MMP-2, an extracellular matrix-regulating enzyme, was increased in the conditioned medium of patient's tenocytes, as determined by gelatin zymography and western blot. Altogether, these data indicate that COLVI deficiency may influence the organization of UCMD tendon matrix, resulting in dysfunctional fibrillogenesis. The alterations of tendon matrix may contribute to the complex pathogenesis of COLVI related myopathies. PMID:27375477

  1. Degradation of extracellular matrix and its components by hypobromous acid

    PubMed Central

    Rees, Martin D.; McNiven, Tane N.; Davies, Michael J.

    2006-01-01

    EPO (eosinophil peroxidase) and MPO (myeloperoxidase) are highly basic haem enzymes that can catalyse the production of HOBr (hypobromous acid). They are released extracellularly by activated leucocytes and their binding to the polyanionic glycosa-minoglycan components of extracellular matrix (proteoglycans and hyaluronan) may localize the production of HOBr to these materials. It is shown in the present paper that the reaction of HOBr with glycosaminoglycans (heparan sulfate, heparin, chondroitin sulfate and hyaluronan) generates polymer-derived N-bromo derivatives (bromamines, dibromamines, N-bromosulfon-amides and bromamides). Decomposition of these species, which can occur spontaneously and/or via one-electron reduction by low-valent transition metal ions (Cu+ and Fe2+), results in polymer fragmentation and modification. One-electron reduction of the N-bromo derivatives generates radicals that have been detected by EPR spin trapping. The species detected are consistent with metal ion-dependent polymer fragmentation and modification being initiated by the formation of nitrogen-centred (aminyl, N-bromoaminyl, sulfonamidyl and amidyl) radicals. Previous studies have shown that the reaction of HOBr with proteins generates N-bromo derivatives and results in fragmentation of the polypeptide backbone. The reaction of HOBr with extracellular matrix synthesized by smooth muscle cells in vitro induces the release of carbohydrate and protein components in a time-dependent manner, which is consistent with fragmentation of these materials via the formation of N-bromo derivatives. The degradation of extracellular matrix glycosaminoglycans and proteins by HOBr may contribute to tissue damage associated with inflammatory diseases such as asthma. PMID:17014424

  2. The role of extracellular matrix in spinal cord development.

    PubMed

    Wiese, Stefan; Faissner, Andreas

    2015-12-01

    The development of the spinal cord represents one of the most complex structure developments of the central nervous system (CNS) as it has to unfold along the longitudinal axis and within segmental cues. There it has to cope with on the one hand connection to the periphery (skeletal muscle, dermomyotome, smooth muscles) and connect it to the higher midbrain and cortical regions of the CNS. Major studies have been performed to analyze the specific subset of transcription factors of the different types of cells within the different segments of the spinal cord. But transcription factor expression is always a result of cellular positioning as the environment defines the intracellular changes during differentiation and in adulthood. The surrounding composed of mainly extracellular matrix does not only provide a "glue" to attach cells to each other but also provides signals with special domains docking to cell surface receptors and presents soluble molecules such as basic fibroblast growth factors (bFGFs) or Wnt-proteins. The availability of these molecules depends on the matrix composition and influences the transcription factor code of each cell. Recent research has also provided strong evidence that depletion of single matrix molecules like Tenascin C (TnC) can lead to developmental changes within the progenitor pools. Therefore beyond the transcription factor code that defines cellular properties we want to focus on the role of the extracellular matrix in the development of the spinal cord. PMID:26028310

  3. Extracellular matrix structure governs invasion resistance in bacterial biofilms.

    PubMed

    Nadell, Carey D; Drescher, Knut; Wingreen, Ned S; Bassler, Bonnie L

    2015-08-01

    Many bacteria are highly adapted for life in communities, or biofilms. A defining feature of biofilms is the production of extracellular matrix that binds cells together. The biofilm matrix provides numerous fitness benefits, including protection from environmental stresses and enhanced nutrient availability. Here we investigate defense against biofilm invasion using the model bacterium Vibrio cholerae. We demonstrate that immotile cells, including those identical to the biofilm resident strain, are completely excluded from entry into resident biofilms. Motile cells can colonize and grow on the biofilm exterior, but are readily removed by shear forces. Protection from invasion into the biofilm interior is mediated by the secreted protein RbmA, which binds mother-daughter cell pairs to each other and to polysaccharide components of the matrix. RbmA, and the invasion protection it confers, strongly localize to the cell lineages that produce it.

  4. Response of microscale cell/matrix constructs to successive force application in a 3D environment

    NASA Astrophysics Data System (ADS)

    Liu, Alan; Chen, Christopher; Reich, Daniel

    2014-03-01

    Mechanical dilation of arteries by pulsatile blood flow is directly opposed by coordinated contraction of a band of smooth muscle tissue that envelops the vessels. This mechanical adaptation of smooth muscle cells to external loading is a critical feature of normal blood vessel function. While most previous studies on biomechanical systems have focused on single cells or large excised tissue, we utilize a device to apply forces to engineered smooth muscle microtissues. This device consists of arrayed pairs of elastomeric micro-cantilevers capable of magnetic actuation. Tissues are formed through self-assembly following the introduction of cell-infused collagen gel to the array. With this system, we are able to dynamically stretch and relax these sub-millimeter sized tissues. The timing and magnitude of the force application can be precisely controlled and thus can be used to mimic a wide range of physiological behavior. In particular, we will discuss results that show that the interval between successive force applications mediates the both the subsequent mechanical and active dynamics of the cell/matrix composite system. Understanding this process will lead to better understanding of the interplay between cell and extracellular matrix responses to mechanical stimulus at a novel length scale.

  5. Microscale Diffusion Properties of the Cartilage Pericellular Matrix Measured Using 3D Scanning Microphotolysis

    PubMed Central

    Leddy, Holly A.; Christensen, Susan E.; Guilak, Farshid

    2009-01-01

    Chondrocytes (cartilage cells) are enclosed within a pericellular matrix (PCM) whose composition and structure differ from those of the extracellular matrix (ECM). Since the PCM surrounds each cell, molecules that interact with the chondrocyte must pass through the pericellular environment. A quantitative understanding of the diffusional properties of the PCM will help elucidate the PCM’s regulatory role in controlling transport to and from the chondrocyte. The diffusivity of a fluorescently-labeled 70 kDa dextran was quantified within the PCM of porcine articular cartilage using a newly-developed mathematical model of scanning microphotolysis (SCAMP). SCAMP is a rapid, line photobleaching method that accounts for out-of-plane bleaching attributable to high magnification. Data were analyzed by best-fit comparison to simulations generated using a discretization of the diffusion-reaction equation in conjunction with the microscope-specific three-dimensional excitation and detection profiles. The diffusion coefficient of dextran was significantly lower in the PCM than in the ECM in normal cartilage. In early-stage arthritic tissue, however, no significant differences in diffusivity were detectable. These results support the hypothesis that the diffusivity of the PCM is lower than that of the ECM, presumably due to differences in proteoglycan content, and that osteoarthritic changes in tissue affect the transport properties of the PCM. PMID:19045531

  6. The molecular elasticity of the extracellular matrix protein tenascin

    NASA Astrophysics Data System (ADS)

    Oberhauser, Andres F.; Marszalek, Piotr E.; Erickson, Harold P.; Fernandez, Julio M.

    1998-05-01

    Extracellular matrix proteins are thought to provide a rigid mechanical anchor that supports and guides migrating and rolling cells. Here we examine the mechanical properties of the extracellular matrix protein tenascin by using atomic-force-microscopy techniques. Our results indicate that tenascin is an elastic protein. Single molecules of tenascin could be stretched to several times their resting length. Force-extension curves showed a saw-tooth pattern, with peaks of force at 137pN. These peaks were ~25nm apart. Similar results have been obtained by study of titin. We also found similar results by studying recombinant tenascin fragments encompassing the 15 fibronectin type III domains of tenascin. This indicates that the extensibility of tenascin may be due to the stretch-induced unfolding of its fibronectin type III domains. Refolding of tenascin after stretching, observed when the force was reduced to near zero, showed a double-exponential recovery with time constants of 42 domains refolded per second and 0.5 domains per second. The former speed of refolding is more than twice as fast as any previously reported speed of refolding of a fibronectin type III domain,. We suggest that the extensibility of the modular fibronectin type III region may be important in allowing tenascin-ligand bonds to persist over long extensions. These properties of fibronectin type III modules may be of widespread use in extracellular proteins containing such domain,.

  7. Tracking immune-related cell responses to drug delivery microparticles in 3D dense collagen matrix.

    PubMed

    Obarzanek-Fojt, Magdalena; Curdy, Catherine; Loggia, Nicoletta; Di Lena, Fabio; Grieder, Kathrin; Bitar, Malak; Wick, Peter

    2016-10-01

    Beyond the therapeutic purpose, the impact of drug delivery microparticles on the local tissue and inflammatory responses remains to be further elucidated specifically for reactions mediated by the host immune cells. Such immediate and prolonged reactions may adversely influence the release efficacy and intended therapeutic pathway. The lack of suitable in vitro platforms limits our ability to gain insight into the nature of immune responses at a single cell level. In order to establish an in vitro 3D system mimicking the connective host tissue counterpart, we utilized reproducible, compressed, rat-tail collagen polymerized matrices. THP1 cells (human acute monocytic leukaemia cells) differentiated into macrophage-like cells were chosen as cell model and their functionality was retained in the dense rat-tail collagen matrix. Placebo microparticles were later combined in the immune cell seeded system during collagen polymerization and secreted pro-inflammatory factors: TNFα and IL-8 were used as immune response readout (ELISA). Our data showed an elevated TNFα and IL-8 secretion by macrophage THP1 cells indicating that Placebo microparticles trigger certain immune cell responses under 3D in vivo like conditions. Furthermore, we have shown that the system is sensitive to measure the differences in THP1 macrophage pro-inflammatory responses to Active Pharmaceutical Ingredient (API) microparticles with different API release kinetics. We have successfully developed a tissue-like, advanced, in vitro system enabling selective "readouts" of specific responses of immune-related cells. Such system may provide the basis of an advanced toolbox enabling systemic evaluation and prediction of in vivo microparticle reactions on human immune-related cells.

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

    PubMed

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

    2016-03-28

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

  9. Probing Micromechanical Properties of the Extracellular Matrix of Soft Tissues by Atomic Force Microscopy.

    PubMed

    Jorba, Ignasi; Uriarte, Juan J; Campillo, Noelia; Farré, Ramon; Navajas, Daniel

    2017-01-01

    The extracellular matrix (ECM) determines 3D tissue architecture and provides structural support and chemical and mechanical cues to the cells. Atomic force microscopy (AFM) has unique capabilities to measure ECM mechanics at the scale at which cells probe the mechanical features of their microenvironment. Moreover, AFM measurements can be readily combined with bright field and fluorescence microscopy. Performing reliable mechanical measurements with AFM requires accurate calibration of the device and correct computation of the mechanical parameters. A suitable approach to isolate ECM mechanics from cell contribution is removing the cells by means of an effective decellularization process that preserves the composition, structure and mechanical properties of the ECM. AFM measurement of ECM micromechanics provides important insights into organ biofabrication, cell-matrix mechanical crosstalk and disease-induced tissue stiffness alterations. J. Cell. Physiol. 232: 19-26, 2017. © 2016 Wiley Periodicals, Inc. PMID:27163411

  10. Probing Micromechanical Properties of the Extracellular Matrix of Soft Tissues by Atomic Force Microscopy.

    PubMed

    Jorba, Ignasi; Uriarte, Juan J; Campillo, Noelia; Farré, Ramon; Navajas, Daniel

    2017-01-01

    The extracellular matrix (ECM) determines 3D tissue architecture and provides structural support and chemical and mechanical cues to the cells. Atomic force microscopy (AFM) has unique capabilities to measure ECM mechanics at the scale at which cells probe the mechanical features of their microenvironment. Moreover, AFM measurements can be readily combined with bright field and fluorescence microscopy. Performing reliable mechanical measurements with AFM requires accurate calibration of the device and correct computation of the mechanical parameters. A suitable approach to isolate ECM mechanics from cell contribution is removing the cells by means of an effective decellularization process that preserves the composition, structure and mechanical properties of the ECM. AFM measurement of ECM micromechanics provides important insights into organ biofabrication, cell-matrix mechanical crosstalk and disease-induced tissue stiffness alterations. J. Cell. Physiol. 232: 19-26, 2017. © 2016 Wiley Periodicals, Inc.

  11. Cell Invasion Dynamics into a Three Dimensional Extracellular Matrix Fibre Network

    PubMed Central

    Kim, Min-Cheol; Whisler, Jordan; Silberberg, Yaron R.; Kamm, Roger D.; Asada, H. Harry

    2015-01-01

    The dynamics of filopodia interacting with the surrounding extracellular matrix (ECM) play a key role in various cell-ECM interactions, but their mechanisms of interaction with the ECM in 3D environment remain poorly understood. Based on first principles, here we construct an individual-based, force-based computational model integrating four modules of 1) filopodia penetration dynamics; 2) intracellular mechanics of cellular and nuclear membranes, contractile actin stress fibers, and focal adhesion dynamics; 3) structural mechanics of ECM fiber networks; and 4) reaction-diffusion mass transfers of seven biochemical concentrations in related with chemotaxis, proteolysis, haptotaxis, and degradation in ECM to predict dynamic behaviors of filopodia that penetrate into a 3D ECM fiber network. The tip of each filopodium crawls along ECM fibers, tugs the surrounding fibers, and contracts or retracts depending on the strength of the binding and the ECM stiffness and pore size. This filopodium-ECM interaction is modeled as a stochastic process based on binding kinetics between integrins along the filopodial shaft and the ligands on the surrounding ECM fibers. This filopodia stochastic model is integrated into migratory dynamics of a whole cell in order to predict the cell invasion into 3D ECM in response to chemotaxis, haptotaxis, and durotaxis cues. Predicted average filopodia speed and that of the cell membrane advance agreed with experiments of 3D HUVEC migration at r2 > 0.95 for diverse ECMs with different pore sizes and stiffness. PMID:26436883

  12. Streptococcus pyogenes degrades extracellular matrix in chondrocytes via MMP-13

    SciTech Connect

    Sakurai, Atsuo; Okahashi, Nobuo; Maruyama, Fumito; Ooshima, Takashi; Hamada, Shigeyuki; Nakagawa, Ichiro

    2008-08-29

    Group A streptococcus (GAS) causes a wide range of human diseases, including bacterial arthritis. The pathogenesis of arthritis is characterized by synovial proliferation and the destruction of cartilage and subchondral bone in joints. We report here that GAS strain JRS4 invaded a chondrogenic cell line ATDC5 and induced the degradation of the extracellular matrix (ECM), whereas an isogenic mutant of JRS4 lacking a fibronectin-binding protein, SAM1, failed to invade the chondrocytes or degrade the ECM. Reverse transcription-PCR and Western blot analysis revealed that the expression of matrix metalloproteinase (MMP)-13 was strongly elevated during the infection with GAS. A reporter assay revealed that the activation of the AP-1 transcription factor and the phosphorylation of c-Jun terminal kinase participated in MMP-13 expression. These results suggest that MMP-13 plays an important role in the destruction of infected joints during the development of septic arthritis.

  13. The design of reversible hydrogels to capture extracellular matrix dynamics

    NASA Astrophysics Data System (ADS)

    Rosales, Adrianne M.; Anseth, Kristi S.

    2016-02-01

    The extracellular matrix (ECM) is a dynamic environment that constantly provides physical and chemical cues to embedded cells. Much progress has been made in engineering hydrogels that can mimic the ECM, but hydrogel properties are, in general, static. To recapitulate the dynamic nature of the ECM, many reversible chemistries have been incorporated into hydrogels to regulate cell spreading, biochemical ligand presentation and matrix mechanics. For example, emerging trends include the use of molecular photoswitches or biomolecule hybridization to control polymer chain conformation, thereby enabling the modulation of the hydrogel between two states on demand. In addition, many non-covalent, dynamic chemical bonds have found increasing use as hydrogel crosslinkers or tethers for cell signalling molecules. These reversible chemistries will provide greater temporal control of adhered cell behaviour, and they allow for more advanced in vitro models and tissue-engineering scaffolds to direct cell fate.

  14. Interaction of Angiogenic Microvessels with the Extracellular Matrix

    PubMed Central

    Krishnan, Laxminarayanan; Hoying, James B.; Nguyen, Hoa; Song, Helen; Weiss, Jeffrey A.

    2010-01-01

    The extracellular matrix (ECM) plays a critical role in angiogenesis by providing biochemical and positional cues as well as by mechanically influencing microvessel cell behavior. Considerable information is known concerning the biochemical cues relevant to angiogenesis, but less is known about the mechanical dynamics during active angiogenesis. The objective of this study was to characterize changes in the material properties of a simple angiogenic tissue before and during angiogenesis. During sprouting, there was an overall decrease in tissue stiffness followed by an increase during neovessel elongation. The fall in matrix stiffness coincided with peak MMP mRNA expression and elevated proteolytic activity. An elevated expression of genes for ECM componenets and cell-ECM interaction molecules and a subsequent drop in proteolytic activity (although enzyme levels remained elevated) coincided with the subsequent stiffening.. The results of this study show that the mechanical properties of a scaffold tissue may be actively modified during angiogenesis by the growing microvasculature. PMID:17933969

  15. Recent advances in the study of zebrafish extracellular matrix proteins.

    PubMed

    Jessen, Jason R

    2015-05-01

    The zebrafish extracellular matrix (ECM) is a dynamic and pleomorphic structure consisting of numerous proteins that together regulate a variety of cellular and morphogenetic events beginning as early as gastrulation. The zebrafish genome encodes a similar complement of ECM proteins as found in other vertebrate organisms including glycoproteins, fibrous proteins, proteoglycans, glycosaminoglycans, and interacting or modifying proteins such as integrins and matrix metalloproteinases. As a genetic model system combined with its amenability to high-resolution microscopic imaging, the zebrafish allows interrogation of ECM protein structure and function in both the embryo and adult. Accumulating data have identified important roles for zebrafish ECM proteins in processes as diverse as cell polarity, migration, tissue mechanics, organ laterality, muscle contraction, and regeneration. In this review, I highlight recently published data on these topics that demonstrate how the ECM proteins fibronectin, laminin, and collagen contribute to zebrafish development and adult homeostasis.

  16. Extracellular Matrix Modulates Angiogenesis in Physiological and Pathological Conditions

    PubMed Central

    Neve, Anna; Cantatore, Francesco Paolo; Maruotti, Nicola; Corrado, Addolorata; Ribatti, Domenico

    2014-01-01

    Angiogenesis is a multistep process driven by a wide range of positive and negative regulatory factors. Extracellular matrix (ECM) plays a crucial role in the regulation of this process. The degradation of ECM, occurring in response to an angiogenic stimulus, leads to degradation or partial modification of matrix molecules, release of soluble factors, and exposure of cryptic sites with pro- and/or antiangiogenic activity. ECM molecules and fragments, resulting from proteolysis, can also act directly as inflammatory stimuli, and this can explain the exacerbated angiogenesis that drives and maintains several inflammatory diseases. In this review we have summarized some of the more recent literature data concerning the molecular control of ECM in angiogenesis in both physiological and pathological conditions. PMID:24949467

  17. Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions.

    PubMed

    Karkampouna, Sofia; Kloen, Peter; Obdeijn, Miryam C; Riester, Scott M; van Wijnen, Andre J; Kruithof-de Julio, Marianna

    2015-01-01

    Organ fibrosis or "scarring" is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic. To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first "organ" culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

  18. The Extracellular Matrix In Development and Morphogenesis: A Dynamic View

    PubMed Central

    Rozario, Tania; DeSimone, Douglas W.

    2009-01-01

    The extracellular matrix (ECM) is synthesized and secreted by embryonic cells beginning at the earliest stages of development. Our understanding of ECM composition, structure and function has grown considerably in the last several decades and this knowledge has revealed that the extracellular microenvironment is critically important for cell growth, survival, differentiation and morphogenesis. ECM and the cellular receptors that interact with it mediate both physical linkages with the cytoskeleton and the bidirectional flow of information between the extracellular and intracellular compartments. This review considers the range of cell and tissue functions attributed to ECM molecules and summarizes recent findings specific to key developmental processes. The importance of ECM as a dynamic repository for growth factors is highlighted along with more recent studies implicating the 3-dimensional organization and physical properties of the ECM as it relates to cell signaling and the regulation of morphogenetic cell behaviors. Embryonic cell and tissue generated forces and mechanical signals arising from ECM adhesion represent emerging areas of interest in this field. PMID:19854168

  19. Ultrasonic assessment of extracellular matrix content in healing Achilles tendon.

    PubMed

    Ghorayeb, Sleiman R; Shah, Neil V; Edobor-Osula, Folorunsho; Lane, Lewis B; Razzano, Pasquale; Chahine, Nadeen; Grande, Daniel A

    2012-04-01

    Although several imaging modalities have been utilized to observe tendons, assessing injured tendons by tracking the healing response over time with ultrasound is a desirable method which is yet to be realized. This study examines the use of ultrasound for non-invasive monitoring of the healing process of Achilles tendons after surgical transection. The overall extracellular matrix content of the transection site is monitored and quantified as a function of time. B-mode images (built from successive A-scan signatures) of the injury site were obtained and compared to biomechanical properties. A quantitative measure of tendon healing using the extracellular matrix (ECM) content of the injury site was analyzed using linear regression with all biomechanical measures. Contralateral tendons were used as controls. The trend in the degree of ECM regrowth in the 4 weeks following complete transection of excised tendons was found to be most closely paralleled with that of linear stiffness (R(2) = 0.987, p < .05) obtained with post-ultrasound biomechanical tests. Results suggest that ultrasound can be an effective imaging technique in assessing the degree of tendon healing, and can be used to correlate structural properties of Achilles tendons.

  20. Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy.

    PubMed

    Edginton, Ryan S; Mattana, Sara; Caponi, Silvia; Fioretto, Daniele; Green, Ellen; Winlove, C Peter; Palombo, Francesca

    2016-01-01

    Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis. PMID:27684584

  1. Extracellular matrix communication and turnover in cardiac physiology and pathology.

    PubMed

    Takawale, Abhijit; Sakamuri, Siva S V P; Kassiri, Zamaneh

    2015-04-01

    Despite significant advances in treating heart disease, heart failure remains a major cause of morbidity and mortality. Regardless of the initiating cause(s), heart failure is associated with disruptions in the myocardial extracellular matrix (ECM). ECM is a dynamic structure and its physiological turnover is mediated by matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). Research in the past two decades has revealed that the function of ECM extends beyond its role in providing structural support. Similarly, ECM regulatory proteins, MMPs and TIMPs, have been demonstrated to play diverse and ECM-independent roles in tissue remodeling and homeostasis. ECM is a network structure that in addition to providing structural support, serves as an extracellular reservoir for a number of growth factors and cytokines, and plays a central role in interstitial transport of different molecules (hormones, growth factors, drugs, etc.). This is mainly through the action of nonstructural ECM components, proteoglycans and matricellular proteins, which are also critical in cell-ECM interactions and overall ECM remodeling. As such, sustaining the ECM integrity is not only critical in preserving cardiac geometry and function, it is essential in ensuring optimal delivery of different molecules to their site of action. Further, ECM composition and integrity in disease should be considered in designing drugs with a specific site of action. In this review article, we provide an overview of the ECM structure, components, its function in interstitial transport, heart disease-dependent ECM remodeling, and the potential therapeutic approaches in preserving the diseased myocardial ECM and cardiac function.

  2. Highlighting the impact of aging on type I collagen: label-free investigation using confocal reflectance microscopy and diffuse reflectance spectroscopy in 3D matrix model.

    PubMed

    Guilbert, Marie; Roig, Blandine; Terryn, Christine; Garnotel, Roselyne; Jeannesson, Pierre; Sockalingum, Ganesh D; Manfait, Michel; Perraut, François; Dinten, Jean-Marc; Koenig, Anne; Piot, Olivier

    2016-02-23

    During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models.

  3. Highlighting the impact of aging on type I collagen: label-free investigation using confocal reflectance microscopy and diffuse reflectance spectroscopy in 3D matrix model

    PubMed Central

    Terryn, Christine; Garnotel, Roselyne; Jeannesson, Pierre; Sockalingum, Ganesh D.; Manfait, Michel; Perraut, François; Dinten, Jean-Marc; Koenig, Anne; Piot, Olivier

    2016-01-01

    During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models. PMID:26885896

  4. Myelinating glia differentiation is regulated by extracellular matrix elasticity

    PubMed Central

    Urbanski, Mateusz M.; Kingsbury, Lyle; Moussouros, Daniel; Kassim, Imran; Mehjabeen, Saraf; Paknejad, Navid; Melendez-Vasquez, Carmen V.

    2016-01-01

    The mechanical properties of living tissues have a significant impact on cell differentiation, but remain unexplored in the context of myelin formation and repair. In the PNS, the extracellular matrix (ECM) incorporates a basal lamina significantly denser than the loosely organized CNS matrix. Inhibition of non-muscle myosin II (NMII) enhances central but impairs peripheral myelination and NMII has been implicated in cellular responses to changes in the elasticity of the ECM. To directly evaluate whether mechanotransduction plays a role in glial cell differentiation, we cultured Schwann cells (SC) and oligodendrocytes (OL) on matrices of variable elastic modulus, mimicking either their native environment or conditions found in injured tissue. We found that a rigid, lesion-like matrix inhibited branching and differentiation of OL in NMII-dependent manner. By contrast, SC developed normally in both soft and stiffer matrices. Although SC differentiation was not significantly affected by changes in matrix stiffness alone, we found that expression of Krox-20 was potentiated on rigid matrices at high laminin concentration. These findings are relevant to the design of biomaterials to promote healing and regeneration in both CNS and PNS, via transplantation of glial progenitors or the implantation of tissue scaffolds. PMID:27646171

  5. Hydrogels Derived from Central Nervous System Extracellular Matrix

    PubMed Central

    Medberry, Christopher J.; Crapo, Peter M.; Siu, Bernard F.; Carruthers, Christopher A.; Wolf, Matthew T.; Nagarkar, Shailesh P.; Agrawal, Vineet; Jones, Kristen E.; Kelly, Jeremy; Johnson, Scott A.; Velankar, Sachin S.; Watkins, Simon C.; Modo, Michel

    2012-01-01

    Biologic scaffolds composed of extracellular matrix (ECM) are commonly used repair devices in preclinical and clinical settings; however the use of these scaffolds for peripheral and central nervous system (CNS) repair has been limited. Biologic scaffolds developed from brain and spinal cord tissue have recently been described, yet the conformation of the harvested ECM limits therapeutic utility. An injectable CNS-ECM derived hydrogel capable of in vivo polymerization and conformation to irregular lesion geometries may aid in tissue reconstruction efforts following complex neurologic trauma. The objectives of the present study were to develop hydrogel forms of brain and spinal cord ECM and compare the resulting biochemical composition, mechanical properties, and neurotrophic potential of a brain derived cell line to a non-CNS-ECM hydrogel, urinary bladder matrix. Results showed distinct differences between compositions of brain ECM, spinal cord ECM, and urinary bladder matrix. The rheologic modulus of spinal cord ECM hydrogel was greater than that of brain ECM and urinary bladder matrix. All ECMs increased the number of cells expressing neurites, but only brain ECM increased neurite length, suggesting a possible tissue-specific effect. All hydrogels promoted three-dimensional uni- or bi-polar neurite outgrowth following 7 days in culture. These results suggest that CNS-ECM hydrogels may provide supportive scaffolding to promote in vivo axonal repair. PMID:23158935

  6. Myelinating glia differentiation is regulated by extracellular matrix elasticity.

    PubMed

    Urbanski, Mateusz M; Kingsbury, Lyle; Moussouros, Daniel; Kassim, Imran; Mehjabeen, Saraf; Paknejad, Navid; Melendez-Vasquez, Carmen V

    2016-01-01

    The mechanical properties of living tissues have a significant impact on cell differentiation, but remain unexplored in the context of myelin formation and repair. In the PNS, the extracellular matrix (ECM) incorporates a basal lamina significantly denser than the loosely organized CNS matrix. Inhibition of non-muscle myosin II (NMII) enhances central but impairs peripheral myelination and NMII has been implicated in cellular responses to changes in the elasticity of the ECM. To directly evaluate whether mechanotransduction plays a role in glial cell differentiation, we cultured Schwann cells (SC) and oligodendrocytes (OL) on matrices of variable elastic modulus, mimicking either their native environment or conditions found in injured tissue. We found that a rigid, lesion-like matrix inhibited branching and differentiation of OL in NMII-dependent manner. By contrast, SC developed normally in both soft and stiffer matrices. Although SC differentiation was not significantly affected by changes in matrix stiffness alone, we found that expression of Krox-20 was potentiated on rigid matrices at high laminin concentration. These findings are relevant to the design of biomaterials to promote healing and regeneration in both CNS and PNS, via transplantation of glial progenitors or the implantation of tissue scaffolds. PMID:27646171

  7. Extracellular matrix of the bovine ovarian membrana granulosa.

    PubMed

    Rodgers, R J; Irving Rodgers, H F

    2002-05-31

    Much is known about the control of the development of ovarian follicles by growth factors and hormones. The study of extracellular matrix in the ovary, though, is a relatively new area. To date much research has focused on identifying the matrix components present, and more recently, its production and the physiological roles. In this review we focus on the changes that occur in the follicular basal lamina from primordial follicles through to ovulation and formation of the corpus luteum, the changes that occur during follicular atresia, and we discuss our observations of a novel matrix which forms in the membrana granulosa. The follicular basal lamina changes considerably during follicular development in its expression pattern of type IV collagens. Of the laminin chains examined, there appears only to be an increase in amount, except for laminin alpha2. It is expressed only in a small proportion of healthy antral follicles and in the majority of atretic antral follicles. Call-Exner bodies have the same composition as the basal lamina, except they do not contain laminin alpha2, even when the follicular basal lamina does. The novel matrix that develops within the membrana granulosa is similar in composition to Call-Exner bodies which occur predominantly in preantral follicles, except that it is far more common in large antral follicles, does not induce polarization of the surrounding granulosa cells, and does not contain follicular fluid-like material as the Call-Exner bodies of some species do. The expression of this matrix occurs prior to and during the time when granulosa cells express steroidogenic enzymes. It does not exist in corpora lutea. In addition large luteal cells, derived from granulosa cells, do not appear to have a basal lamina. These findings suggest that the maturational changes in the membrana granulosa are accompanied by changes in the matrix. PMID:12044919

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

    PubMed

    Akhmanova, Maria; Osidak, Egor; Domogatsky, Sergey; Rodin, Sergey; Domogatskaya, Anna

    2015-01-01

    Extracellular matrix can influence stem cell choices, such as self-renewal, quiescence, migration, proliferation, phenotype maintenance, differentiation, or apoptosis. Three aspects of extracellular matrix were extensively studied during the last decade: physical properties, spatial presentation of adhesive epitopes, and molecular complexity. Over 15 different parameters have been shown to influence stem cell choices. Physical aspects include stiffness (or elasticity), viscoelasticity, pore size, porosity, amplitude and frequency of static and dynamic deformations applied to the matrix. Spatial aspects include scaffold dimensionality (2D or 3D) and thickness; cell polarity; area, shape, and microscale topography of cell adhesion surface; epitope concentration, epitope clustering characteristics (number of epitopes per cluster, spacing between epitopes within cluster, spacing between separate clusters, cluster patterns, and level of disorder in epitope arrangement), and nanotopography. Biochemical characteristics of natural extracellular matrix molecules regard diversity and structural complexity of matrix molecules, affinity and specificity of epitope interaction with cell receptors, role of non-affinity domains, complexity of supramolecular organization, and co-signaling by growth factors or matrix epitopes. Synergy between several matrix aspects enables stem cells to retain their function in vivo and may be a key to generation of long-term, robust, and effective in vitro stem cell culture systems. PMID:26351461

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

    PubMed Central

    Akhmanova, Maria; Osidak, Egor; Domogatsky, Sergey; Rodin, Sergey; Domogatskaya, Anna

    2015-01-01

    Extracellular matrix can influence stem cell choices, such as self-renewal, quiescence, migration, proliferation, phenotype maintenance, differentiation, or apoptosis. Three aspects of extracellular matrix were extensively studied during the last decade: physical properties, spatial presentation of adhesive epitopes, and molecular complexity. Over 15 different parameters have been shown to influence stem cell choices. Physical aspects include stiffness (or elasticity), viscoelasticity, pore size, porosity, amplitude and frequency of static and dynamic deformations applied to the matrix. Spatial aspects include scaffold dimensionality (2D or 3D) and thickness; cell polarity; area, shape, and microscale topography of cell adhesion surface; epitope concentration, epitope clustering characteristics (number of epitopes per cluster, spacing between epitopes within cluster, spacing between separate clusters, cluster patterns, and level of disorder in epitope arrangement), and nanotopography. Biochemical characteristics of natural extracellular matrix molecules regard diversity and structural complexity of matrix molecules, affinity and specificity of epitope interaction with cell receptors, role of non-affinity domains, complexity of supramolecular organization, and co-signaling by growth factors or matrix epitopes. Synergy between several matrix aspects enables stem cells to retain their function in vivo and may be a key to generation of long-term, robust, and effective in vitro stem cell culture systems. PMID:26351461

  10. The Extracellular Matrix in Photosynthetic Mats: A Cyanobacterial Gingerbread House

    NASA Astrophysics Data System (ADS)

    Stuart, R.; Stannard, W.; Bebout, B.; Pett-Ridge, J.; Mayali, X.; Weber, P. K.; Lipton, M. S.; Lee, J.; Everroad, R. C.; Thelen, M.

    2014-12-01

    Hypersaline laminated cyanobacterial mats are excellent model systems for investigating photoautotrophic contributions to biogeochemical cycling on a millimeter scale. These self-sustaining ecosystems are characterized by steep physiochemical gradients that fluctuate dramatically on hour timescales, providing a dynamic environment to study microbial response. However, elucidating the distribution of energy from light absorption into biomass requires a complete understanding of the various constituents of the mat. Extracellular polymeric substances (EPS), which can be composed of proteins, polysaccharides, lipids and DNA are a major component of these mats and may function in the redistribution of nutrients and metabolites within the community. To test this notion, we established a model mat-building culture for comparison with the phylogenetically diverse natural mat communities. In these two systems we determined how proteins and glycans in the matrix changed as a function of light and tracked nutrient flow from the matrix. Using mass spectrometry metaproteomics analysis, we found homologous proteins in both field and culture extracellular matrix that point to cyanobacterial turnover of amino acids, inorganic nutrients, carbohydrates and nucleic acids from the EPS. Other abundant functions identified included oxidative stress response from both the cyanobacteria and heterotrophs and cyanobacterial structural proteins that may play a role in mat cohesion. Several degradative enzymes also varied in abundance in the EPS in response to light availability, suggesting active secretion. To further test cyanobacterial EPS turnover, we generated isotopically-labeled EPS and used NanoSIMS to trace uptake of this labeled EPS. Our findings suggest Cyanobacteria may facilitate nutrient transfer to other groups, as well as uptake of their own products through degradation of EPS components. This work provides evidence for the essential roles of EPS for storage, structural

  11. [Inhibitory proteins of neuritic regeneration in the extracellular matrix: structure, molecular interactions and their functions. Mechanisms of extracellular balance].

    PubMed

    Vargas, Javier; Uribe-Escamilla, Rebeca; Alfaro-Rodríguez, Alfonso

    2013-01-01

    After injury of the central nervous system (CNS) in higher vertebrates, neurons neither grow nor reconnect with their targets because their axons or dendrites cannot regenerate within the injured site. In the CNS, the signal from the environment regulating neurite regeneration is not exclusively generated by one molecular group. This signal is generated by the interaction of various types of molecules such as extracellular matrix proteins, soluble factors and surface membrane molecules; all these elements interact with one another generating the matrix's biological state: the extracellular balance. Proteins in the balanced extracellular matrix, support and promote cellular physiological states, including neuritic regeneration. We have reviewed three types of proteins of the extracellular matrix possessing an inhibitory effect and that are determinant of neuritic regeneration failure in the CNS: chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and tenascin. We also review some of the mechanisms involved in the balance of extracellular proteins such as isomerization, epimerization, sulfation and glycosylation as well as the assemblage of the extracellular matrix, the interaction between the matrix and soluble factors and its proteolytic degradation. In the final section, we have presented some examples of the matrix's role in development and in tumor propagation.

  12. Synthetic osteogenic extracellular matrix formed by coated silicon dioxide nanosprings

    PubMed Central

    2012-01-01

    Background The design of biomimetic materials that parallel the morphology and biology of extracellular matrixes is key to the ability to grow functional tissues in vitro and to enhance the integration of biomaterial implants into existing tissues in vivo. Special attention has been put into mimicking the nanostructures of the extracellular matrix of bone, as there is a need to find biomaterials that can enhance the bonding between orthopedic devices and this tissue. Methods We have tested the ability of normal human osteoblasts to propagate and differentiate on silicon dioxide nanosprings, which can be easily grown on practically any surface. In addition, we tested different metals and metal alloys as coats for the nanosprings in tissue culture experiments with bone cells. Results Normal human osteoblasts grown on coated nanosprings exhibited an enhanced rate of propagation, differentiation into bone forming cells and mineralization. While osteoblasts did not attach effectively to bare nanowires grown on glass, these cells propagated successfully on nanosprings coated with titanium oxide and gold. We observed a 270 fold increase in the division rate of osteoblasts when grow on titanium/gold coated nanosprings. This effect was shown to be dependent on the nanosprings, as the coating by themselves did not alter the growth rate of osteoblast. We also observed that titanium/zinc/gold coated nanosprings increased the levels of osteoblast production of alkaline phosphatase seven folds. This result indicates that osteoblasts grown on this metal alloy coated nanosprings are differentiating to mature bone making cells. Consistent with this hypothesis, we showed that osteoblasts grown on the same metal alloy coated nanosprings have an enhanced ability to deposit calcium salt. Conclusion We have established that metal/metal alloy coated silicon dioxide nanosprings can be used as a biomimetic material paralleling the morphology and biology of osteogenic extracellular matrix

  13. Extracellular Matrix Modulation: Optimizing Skin Care and Rejuvenation Procedures.

    PubMed

    Widgerow, Alan D; Fabi, Sabrina G; Palestine, Roberta F; Rivkin, Alexander; Ortiz, Arisa; Bucay, Vivian W; Chiu, Annie; Naga, Lina; Emer, Jason; Chasan, Paul E

    2016-04-01

    Normal aging and photoaging of the skin are chronic processes that progress gradually. The extracellular matrix (ECM), constituting over 70% of the skin, is the central hub for repair and regeneration of the skin. As such, the ECM is the area where changes related to photodamage are most evident. Degradation of the ECM with fragmentation of proteins significantly affects cross talk and signaling between cells, the matrix, and its constituents. The accumulation of collagen fragments, amorphous elastin agglutinations, and abnormal cross-linkages between the collagen fragments impedes the ECM from its normal repair and regenerative capacity, which manifests as wrinkled, non-elastic skin. Similar to how the chronic wound healing process requires wound bed preparation before therapeutic intervention, treatment of chronic aging of the skin would likely benefit from a "skin bed preparation" to optimize the outcome of rejuvenation procedures and skin maintenance programs. This involves introducing agents that can combat stress-induced oxidation, proteasome dysfunction, and non-enzymatic cross linkages involved in glycation end products, to collectively modulate this damaged ECM, and upregulate neocollagenesis and elastin production. Agents of particular interest are matrikines, peptides originating from the fragmentation of matrix proteins that exhibit a wide range of biological activities. Peptides of this type (tripeptide and hexapeptide) are incorporated in ALASTIN™ Skin Nectar with TriHex™ technology (ALASTIN Skincare, Inc., Carlsbad, CA), which is designed to target ECM modulation with a goal of optimizing results following invasive and non-invasive dermal rejuvenating procedures. PMID:27050707

  14. Tendon Differentiation on Decellularized Extracellular Matrix Under Cyclic Loading.

    PubMed

    Youngstrom, Daniel W; Barrett, Jennifer G

    2016-01-01

    Tendon bioreactors combine cells, scaffold, and mechanical stimulation to drive tissue neogenesis ex vivo. Faithful recapitulation of the native tendon microenvironment is essential for stimulating graft maturation or modeling tendon biology. As the mediator between cells and mechanical stimulation, the properties of a scaffold constitute perhaps the most essential elements in a bioreactor system. One method of achieving native scaffold properties is to process tendon allograft in a manner that removes cells without modifying structure and function: "decellularization." This chapter describes (1) production of tendon scaffolds derived from native extracellular matrix, (2) preparation of cell-laden scaffolds prior to bioreactor culture, and (3) tissue processing post-harvest for gene expression analysis. These methods may be applied for a variety of applications including graft production, cell priming prior to transplantation and basic investigations of tendon cell biology. PMID:27062597

  15. Remodelling the extracellular matrix in development and disease

    PubMed Central

    Bonnans, Caroline; Chou, Jonathan; Werb, Zena

    2015-01-01

    The extracellular matrix (ECM) is a highly dynamic structure that is present in all tissues and continuously undergoes controlled remodelling. This process involves quantitative and qualitative changes in the ECM, mediated by specific enzymes that are responsible for ECM degradation, such as metalloproteinases. The ECM interacts with cells to regulate diverse functions, including proliferation, migration and differentiation. ECM remodelling is crucial for regulating the morphogenesis of the intestine and lungs, as well as of the mammary and submandibular glands. Dysregulation of ECM composition, structure, stiffness and abundance contributes to several pathological conditions, such as fibrosis and invasive cancer. A better understanding of how the ECM regulates organ structure and function and of how ECM remodelling affects disease progression will contribute to the development of new therapeutics. PMID:25415508

  16. Characteristic adaptations of the extracellular matrix in dilated cardiomyopathy.

    PubMed

    Louzao-Martinez, Laura; Vink, Aryan; Harakalova, Magdalena; Asselbergs, Folkert W; Verhaar, Marianne C; Cheng, Caroline

    2016-10-01

    Dilated cardiomyopathy (DCM) is a relatively common heart muscle disease characterized by the dilation and thinning of the left ventricle accompanied with left ventricular systolic dysfunction. Myocardial fibrosis is a major feature in DCM and therefore it is inevitable that corresponding extracellular matrix (ECM) changes are involved in DCM onset and progression. Increasing our understanding of how ECM adaptations are involved in DCM could be important for the development of future interventions. This review article discusses the molecular adaptations in ECM composition and structure that have been reported in both animal and human studies of DCM. Furthermore, we provide a transcriptome-based catalogue of ECM genes that are associated with DCM, generated by using NCBI Gene Expression Omnibus database sets for DCM. Based on this in silico analysis, many novel ECM components involved in DCM are identified and discussed in this review. With the information gathered, we propose putative pathways of ECM adaptations in onset and progression of DCM.

  17. Cell stiffness, contractile stress and the role of extracellular matrix

    SciTech Connect

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

    2009-05-15

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

  18. Cell stiffness, contractile stress and the role of extracellular matrix

    PubMed Central

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

    2010-01-01

    Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genes in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses. PMID:19327344

  19. Characterization of canine platelet adhesion to extracellular matrix proteins.

    PubMed

    Pelagalli, Alessandra; Pero, Maria Elena; Mastellone, Vincenzo; Cestaro, Anna; Signoriello, Simona; Lombardi, Pietro; Avallone, Luigi

    2011-07-01

    Canine platelets have been extensively studied but little is known about specific aspects such as adhesion. Platelet adhesion is a critical step during haemostasis and thrombosis as well as during inflammatory and immunopathogenic responses. The aim of this study was to evaluate the adhesive properties of canine platelets using fibrinogen and collagen as substrates immobilized on plates. Adhesion was monitored for 120 min and the effect of adenosine 5'-diphosphate (ADP) was assayed. The results showed that canine platelets displayed good adhesion activity that was significantly time-dependent. Moreover, ADP was able to enhance platelet adhesion in a dose-dependent manner. The findings aid knowledge of the adhesion process and suggest a specific role of surface platelet receptors in mediating the interaction with extracellular matrix proteins.

  20. Bioactive extracellular matrix fragments in lung health and disease.

    PubMed

    Gaggar, Amit; Weathington, Nathaniel

    2016-09-01

    The extracellular matrix (ECM) is the noncellular component critical in the maintenance of organ structure and the regulation of tissue development, organ structure, and cellular signaling. The ECM is a dynamic entity that undergoes continuous degradation and resynthesis. In addition to compromising structure, degradation of the ECM can liberate bioactive fragments that cause cellular activation and chemotaxis of a variety of cells. These fragments are termed matrikines, and their cellular activities are sentinel in the development and progression of tissue injury seen in chronic lung disease. Here, we discuss the matrikines that are known to be active in lung biology and their roles in lung disease. We also consider the use of matrikines as disease markers and potential therapeutic targets in lung disease. PMID:27584731

  1. Tendon Differentiation on Decellularized Extracellular Matrix Under Cyclic Loading.

    PubMed

    Youngstrom, Daniel W; Barrett, Jennifer G

    2016-01-01

    Tendon bioreactors combine cells, scaffold, and mechanical stimulation to drive tissue neogenesis ex vivo. Faithful recapitulation of the native tendon microenvironment is essential for stimulating graft maturation or modeling tendon biology. As the mediator between cells and mechanical stimulation, the properties of a scaffold constitute perhaps the most essential elements in a bioreactor system. One method of achieving native scaffold properties is to process tendon allograft in a manner that removes cells without modifying structure and function: "decellularization." This chapter describes (1) production of tendon scaffolds derived from native extracellular matrix, (2) preparation of cell-laden scaffolds prior to bioreactor culture, and (3) tissue processing post-harvest for gene expression analysis. These methods may be applied for a variety of applications including graft production, cell priming prior to transplantation and basic investigations of tendon cell biology.

  2. Insight On Colorectal Carcinoma Infiltration by Studying Perilesional Extracellular Matrix.

    PubMed

    Nebuloni, Manuela; Albarello, Luca; Andolfo, Annapaola; Magagnotti, Cinzia; Genovese, Luca; Locatelli, Irene; Tonon, Giovanni; Longhi, Erika; Zerbi, Pietro; Allevi, Raffaele; Podestà, Alessandro; Puricelli, Luca; Milani, Paolo; Soldarini, Armando; Salonia, Andrea; Alfano, Massimo

    2016-03-04

    The extracellular matrix (ECM) from perilesional and colorectal carcinoma (CRC), but not healthy colon, sustains proliferation and invasion of tumor cells. We investigated the biochemical and physical diversity of ECM in pair-wised comparisons of healthy, perilesional and CRC specimens. Progressive linearization and degree of organization of fibrils was observed from healthy to perilesional and CRC ECM, and was associated with a steady increase of stiffness and collagen crosslinking. In the perilesional ECM these modifications coincided with increased vascularization, whereas in the neoplastic ECM they were associated with altered modulation of matrisome proteins, increased content of hydroxylated lysine and lysyl oxidase. This study identifies the increased stiffness and crosslinking of the perilesional ECM predisposing an environment suitable for CRC invasion as a phenomenon associated with vascularization. The increased stiffness of colon areas may represent a new predictive marker of desmoplastic region predisposing to invasion, thus offering new potential application for monitoring adenoma with invasive potential.

  3. Insight On Colorectal Carcinoma Infiltration by Studying Perilesional Extracellular Matrix

    PubMed Central

    Nebuloni, Manuela; Albarello, Luca; Andolfo, Annapaola; Magagnotti, Cinzia; Genovese, Luca; Locatelli, Irene; Tonon, Giovanni; Longhi, Erika; Zerbi, Pietro; Allevi, Raffaele; Podestà, Alessandro; Puricelli, Luca; Milani, Paolo; Soldarini, Armando; Salonia, Andrea; Alfano, Massimo

    2016-01-01

    The extracellular matrix (ECM) from perilesional and colorectal carcinoma (CRC), but not healthy colon, sustains proliferation and invasion of tumor cells. We investigated the biochemical and physical diversity of ECM in pair-wised comparisons of healthy, perilesional and CRC specimens. Progressive linearization and degree of organization of fibrils was observed from healthy to perilesional and CRC ECM, and was associated with a steady increase of stiffness and collagen crosslinking. In the perilesional ECM these modifications coincided with increased vascularization, whereas in the neoplastic ECM they were associated with altered modulation of matrisome proteins, increased content of hydroxylated lysine and lysyl oxidase. This study identifies the increased stiffness and crosslinking of the perilesional ECM predisposing an environment suitable for CRC invasion as a phenomenon associated with vascularization. The increased stiffness of colon areas may represent a new predictive marker of desmoplastic region predisposing to invasion, thus offering new potential application for monitoring adenoma with invasive potential. PMID:26940881

  4. Insight On Colorectal Carcinoma Infiltration by Studying Perilesional Extracellular Matrix.

    PubMed

    Nebuloni, Manuela; Albarello, Luca; Andolfo, Annapaola; Magagnotti, Cinzia; Genovese, Luca; Locatelli, Irene; Tonon, Giovanni; Longhi, Erika; Zerbi, Pietro; Allevi, Raffaele; Podestà, Alessandro; Puricelli, Luca; Milani, Paolo; Soldarini, Armando; Salonia, Andrea; Alfano, Massimo

    2016-01-01

    The extracellular matrix (ECM) from perilesional and colorectal carcinoma (CRC), but not healthy colon, sustains proliferation and invasion of tumor cells. We investigated the biochemical and physical diversity of ECM in pair-wised comparisons of healthy, perilesional and CRC specimens. Progressive linearization and degree of organization of fibrils was observed from healthy to perilesional and CRC ECM, and was associated with a steady increase of stiffness and collagen crosslinking. In the perilesional ECM these modifications coincided with increased vascularization, whereas in the neoplastic ECM they were associated with altered modulation of matrisome proteins, increased content of hydroxylated lysine and lysyl oxidase. This study identifies the increased stiffness and crosslinking of the perilesional ECM predisposing an environment suitable for CRC invasion as a phenomenon associated with vascularization. The increased stiffness of colon areas may represent a new predictive marker of desmoplastic region predisposing to invasion, thus offering new potential application for monitoring adenoma with invasive potential. PMID:26940881

  5. Constructive remodeling of a synthetic endothelial extracellular matrix

    PubMed Central

    Han, Sewoon; Shin, Yoojin; Jeong, Hyo Eun; Jeon, Jessie S.; Kamm, Roger D.; Huh, Dongeun; Sohn, Lydia L.; Chung, Seok

    2015-01-01

    The construction of well-controllable in vitro models of physiological and pathological vascular endothelium remains a fundamental challenge in tissue engineering and drug development. Here, we present an approach for forming a synthetic endothelial extracellular matrix (ECM) that closely resembles that of the native structure by locally depositing basement membrane materials onto type 1 collagen nanofibers only in a region adjacent to the endothelial cell (EC) monolayer. Culturing the EC monolayer on this synthetic endothelial ECM remarkably enhanced its physiological properties, reducing its vascular permeability, and promoting a stabilized, quiescent phenotype. We demonstrated that the EC monolayer on the synthetic endothelial ECM neither creates non-physiological barriers to cell-cell or cell-ECM interactions, nor hinders molecular diffusion of growth factors and other molecules. The synthetic endothelial ECM and vascular endothelium on it may help us enter in a new phase of research in which various models of the biological barrier behavior can be tested experimentally. PMID:26687334

  6. Extracellular matrix and growth factors in corneal wound healing.

    PubMed

    Nishida, T; Tanaka, T

    1996-08-01

    The crystal clear cornea has been challenged by refractive surgeries. The surgical outcome depends on the healing responses of the cornea. The factors responsible for the corneal wound healing have been characterized. The orchestrated action of extracellular matrix proteins, growth factors, cytokines, and their receptors have been investigated extensively over the past decade. The clinical results with refractive surgeries provide us various important information with regard to the physiology and pathology of the cornea. The role of basement membrane or Bowman's membrane is now challenged for the maintenance and repair of the epithelium. Furthermore, the interactions between epithelium and stroma is another field to be investigated. The regulatory mechanisms of the maintenance of stromal collagen by keratocytes is also studied. This review discusses the current advancement in the healing responses of the cornea to various injuries and refractive surgeries.

  7. Aging-associated changes in renal extracellular matrix.

    PubMed Central

    Abrass, C. K.; Adcox, M. J.; Raugi, G. J.

    1995-01-01

    The composition of renal extracellular matrices was examined in 6-, 12-, 18-, and 24-month-old rats by immunofluorescence microscopy. No change in composition of tubular basement membrane was detected. Increased immunostaining for laminin chains B1 and s-laminin and thrombospondin characterized the thickened glomerular basement membrane. Interstitial collagens I and III were not detected in globally sclerotic glomeruli. The major change noted in the aged rat kidney at 24 months was generalized expansion of the interstitium by thrombospondin and fibronectin. In areas of tubular atrophy there was new expression of extra domain A (EDA)+ fibronectin. Collagens I and III were detected focally in the interstitium adjacent to areas of tubular atrophy, but otherwise collagens I, III, and IV and laminin did not contribute to the interstitial fibrosis. Interstitial fibrosis was detectable at 18 months of age and preceded the development of sclerotic glomeruli, tubular atrophy, or accumulations of interstitial collagen. These changes in extracellular matrix composition distinguish the aging kidney from other sclerotic forms of renal disease. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7887455

  8. Extracellular matrix glycoproteins and diffusion barriers in human astrocytic tumours.

    PubMed

    Zámecník, J; Vargová, L; Homola, A; Kodet, R; Syková, E

    2004-08-01

    The extracellular matrix (ECM) and changes in the size and geometry of the extracellular space (ECS) in tumour tissue are thought to be of critical importance in influencing the migratory abilities of tumour cells as well as the delivery of therapeutic agents into the tumour. In 21 astrocytic neoplasms, the ECM composition was investigated in situ by the immunohistochemical detection of ECM glycoproteins (tenascin, laminin, vitronectin, fibronectin, collagen types I-VI). To explain the changes in ECS size and to detect barriers to diffusion in the tumour tissue, the ECM composition, the cellularity, the density of glial fibrillary acidic protein (GFAP)-positive tumour cell processes and the proliferative activity of the tumours were compared with the size and geometry of the ECS. The ECS volume fraction and the complex of hindrances to diffusion in the ECS (i.e. the tortuosity) were revealed by the real-time iontophoretic tetramethylammonium method. Increased proliferative activity of the tumours correlated with increased ECS volume fraction and tortuosity. The tortuosity of the tumour tissue was not significantly influenced by tumour cell density. Higher tortuosity was found in low-grade astrocytomas associated with the presence of a dense net of GFAP-positive fibrillary processes of the tumour cells. The increase in tortuosity in high-grade tumours correlated with an increased accumulation of ECM molecules, particularly of tenascin. We conclude that the increased malignancy of astrocytic tumours correlates with increases in both ECS volume and ECM deposition.

  9. Binding of the extracellular matrix component entactin to Candida albicans.

    PubMed Central

    López-Ribot, J L; Chaffin, W L

    1994-01-01

    We have investigated the interaction between Candida albicans and entactin, a recently characterized glycoprotein present in the extracellular matrix, especially in the basement membrane. Organisms of both the yeast and the hyphal morphologies of the fungus had the ability to bind recombinant entactin, as detected by an indirect immunofluorescence assay. Material present in the 2-mercaptoethanol cell wall extracts from both C. albicans growth forms was capable of binding to immobilized recombinant entactin in a dose-dependent manner. Binding to entactin was approximately twice that observed for laminin. Binding of an extract component(s) to entactin was partially inhibited by an Arg-Gly-Asp-Ser peptide. A polyclonal antientactin antiserum, as well as a pooled antiserum preparation raised against components present in different C. albicans cell wall extracts, completely or almost completely abolished binding. The existence of morphology-specific receptor-like molecules which bind to different domains of the entactin molecule was ruled out in a competition binding assay. The entactin-binding material(s) in the cell wall also displayed some ability to bind laminin and fibronectin, since preadsorption in the presence of these extracellular matrix components resulted in reduction of binding to entactin. Moieties with a molecular mass of approximately 25, 44, and 65 kDa present in the 2-mercaptoethanol cell wall extracts from both blastoconidia and germ tubes were detected in a ligand affinity blotting experiment as having the ability to bind entactin. Interactions between C. albicans and entactin could be important in mediating adhesion of the fungus to the host tissues and may play a role in the establishment of the disseminated form of the disease. Images PMID:7927722

  10. Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins.

    PubMed Central

    Löhr, M.; Trautmann, B.; Göttler, M.; Peters, S.; Zauner, I.; Maillet, B.; Klöppel, G.

    1994-01-01

    Pancreatic ductal adenocarcinomas are characterised by a dense connective tissue reaction. To test the hypothesis that stroma components are synthesised and produced by the tumour cells themselves, eight cell lines as well as six xenografted tumours from human ductal adenocarcinomas of the pancreas were examined for the expression of extracellular matrix proteins (ECM), using cDNA probes and antibodies to collagen types I, III and IV, vitronectin, fibronectin, undulin and laminin. All tumour cell lines (CAPAN-1, CAPAN-2, AsPC-1, BxPC-3, PANC-1, PaCa-2, PaCa-3, PaCa-44) and xenografted human pancreatic tumours expressed at least one of the examined ECM at the RNA (collagen type IV > laminin = fibronectin = vitronectin > collagen type III > undulin > collagen type I) or protein level (collagen type IV = collagen type III > vitronectin > laminin > collagen type I = fibronectin > undulin). In nude mouse tumours expression of laminin and collagen I was most pronounced in well-differentiated carcinomas. In a few tumours, collagen type III, vitronectin and undulin were expressed on the luminal side of the neoplastic glands, suggesting loss of normal polar differentiation. Incubation with fetal calf serum modulated ECM RNA levels to a varying extent in all but one cell line (AsPC-1). The results suggest that human pancreatic ductal adenocarcinomas cells are capable of synthesising and producing extracellular matrix proteins in vitro and in vivo, but that the extent and pattern of ECM expression differs between the various tumours and conditions tested. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8286197

  11. Cells involved in extracellular matrix remodeling after acute myocardial infarction

    PubMed Central

    Garcia, Larissa Ferraz; Mataveli, Fábio D’Aguiar; Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell; Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva

    2015-01-01

    Objective Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Methods Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. Results There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. Conclusion The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct. PMID:25993074

  12. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation

    PubMed Central

    Hwang, Jun-Ha; Byun, Mi Ran; Kim, A. Rum; Kim, Kyung Min; Cho, Hang Jun; Lee, Yo Han; Kim, Juwon; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2015-01-01

    Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation. PMID:26262877

  13. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    PubMed

    Hwang, Jun-Ha; Byun, Mi Ran; Kim, A Rum; Kim, Kyung Min; Cho, Hang Jun; Lee, Yo Han; Kim, Juwon; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2015-01-01

    Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  14. Hydrogels derived from demineralized and decellularized bone extracellular matrix

    PubMed Central

    Sawkins, M.J.; Bowen, W.; Dhadda, P.; Markides, H.; Sidney, L.E.; Taylor, A.J.; Rose, F.R.A.J.; Badylak, S.F.; Shakesheff, K.M.; White, L.J.

    2013-01-01

    The extracellular matrix (ECM) of mammalian tissues has been isolated, decellularized and utilized as a scaffold to facilitate the repair and reconstruction of numerous tissues. Recent studies have suggested that superior function and complex tissue formation occurred when ECM scaffolds were derived from site-specific homologous tissues compared with heterologous tissues. The objectives of the present study were to apply a stringent decellularization process to demineralized bone matrix (DBM), prepared from bovine bone, and to characterize the structure and composition of the resulting ECM materials and DBM itself. Additionally, we sought to produce a soluble form of DBM and ECM which could be induced to form a hydrogel. Current clinical delivery of DBM particles for treatment of bone defects requires incorporation of the particles within a carrier liquid. Differences in osteogenic activity, inflammation and nephrotoxicity have been reported with various carrier liquids. The use of hydrogel forms of DBM or ECM may reduce the need for carrier liquids. DBM and ECM hydrogels exhibited sigmoidal gelation kinetics consistent with a nucleation and growth mechanism, with ECM hydrogels characterized by lower storage moduli than the DBM hydrogels. Enhanced proliferation of mouse primary calvarial cells was achieved on ECM hydrogels, compared with collagen type I and DBM hydrogels. These results show that DBM and ECM hydrogels have distinct structural, mechanical and biological properties and have the potential for clinical delivery without the need for carrier liquids. PMID:23624219

  15. Domain organizations of modular extracellular matrix proteins and their evolution.

    PubMed

    Engel, J

    1996-11-01

    Multidomain proteins which are composed of modular units are a rather recent invention of evolution. Domains are defined as autonomously folding regions of a protein, and many of them are similar in sequence and structure, indicating common ancestry. Their modular nature is emphasized by frequent repetitions in identical or in different proteins and by a large number of different combinations with other domains. The extracellular matrix is perhaps the largest biological system composed of modular mosaic proteins, and its astonishing complexity and diversity are based on them. A cluster of minireviews on modular proteins is being published in Matrix Biology. These deal with the evolution of modular proteins, the three-dimensional structure of domains and the ways in which these interact in a multidomain protein. They discuss structure-function relationships in calcium binding domains, collagen helices, alpha-helical coiled-coil domains and C-lectins. The present minireview is focused on some general aspects and serves as an introduction to the cluster.

  16. Extracellular Matrix Powder Protects Against Bleomycin-Induced Pulmonary Fibrosis

    PubMed Central

    Manni, Michelle L.; Czajka, Caitlin A.; Oury, Tim D.

    2011-01-01

    Pulmonary fibrosis refers to a group of lung diseases characterized by inflammation, fibroblast proliferation, and excessive collagen deposition. Although the mechanisms underlying pulmonary fibrosis are poorly understood, current evidence suggests that epithelial injury contributes to the development of fibrosis. Regenerative medicine approaches using extracellular matrix (ECM) scaffolds have been shown to promote site-specific tissue remodeling. This led to the hypothesis that particulate ECM would promote normal tissue repair and attenuate bleomycin-induced pulmonary fibrosis. C57BL/6 mice were treated intratracheally with bleomycin or saline with or without a particulate form of ECM scaffold from porcine urinary bladder matrix (UBM-ECM) or enzymatically digested UBM-ECM. Mice were sacrificed 5 and 14 days after exposure. Compared to control mice, bleomycin-exposed mice had similar increases in inflammation in the bronchoalveolar lavage fluid regardless of UBM-ECM treatment. However, 14 days after exposure, lung histology and collagen levels revealed that mice treated with bleomycin and the particulate or digested UBM-ECM had negligible fibrosis, whereas mice given only bleomycin had marked fibrosis. Administration of the particulate UBM-ECM 24 h after bleomycin exposure also significantly protected against pulmonary injury. In vitro epithelial cell migration and wound healing assays revealed that particulate UBM-ECM promoted epithelial cell chemotaxis and migration. This suggests that promotion of epithelial wound repair may be one mechanism in which UBM-ECM limits pulmonary fibrosis. PMID:21797754

  17. In vitro 3D angiogenesis assay in egg white matrix: comparison to Matrigel, compatibility to various species, and suitability for drug testing.

    PubMed

    Mousseau, Yoanne; Mollard, Séverine; Qiu, Hao; Richard, Laurence; Cazal, Raphael; Nizou, Angélique; Vedrenne, Nicolas; Rémi, Séverine; Baaj, Yasser; Fourcade, Laurent; Funalot, Benoit; Sturtz, Franck G

    2014-03-01

    In vitro angiogenesis assays are commonly used to assess pro- or anti-angiogenic drug properties. Extracellular matrix (ECM) substitutes such as Matrigel and collagen gel became very popular in in vitro 3D angiogenesis assays as they enable tubule formation by endothelial cells from culture or aortic rings. However, these assays are usually used with a single cell type, lacking the complex cellular interactions occurring during angiogenesis. Here, we report a novel angiogenesis assay using egg white as ECM substitute. We found that, similar to Matrigel, egg white elicited prevascular network formation by endothelial and/or smooth muscle cell coculture. This matrix was suitable for various cells from human, mouse, and rat origin. It is compatible with aortic ring assay and also enables vascular and tumor cell coculture. Through simple labeling (DAPI, Hoechst 33258), cell location and resulting prevascular network formation can easily be quantified. Cell transfection with green fluorescent protein improved whole cell visualization and 3D structure characterization. Finally, egg-based assay dedicated to angiogenesis studies represents a reliable and cost-effective way to produce and analyze data regarding drug effects on vascular cells. PMID:24395110

  18. [Single mechanism of remodelling extracellular matrix in thymus and pineal gland at aging].

    PubMed

    Lin'kova, N S; Poliakova, V O; Kvetnoĭ, I M

    2011-01-01

    The expression of matrix metalloproteinase 2 and 9 in thymus and pineal gland has been verified. These data demonstrate single mechanism of remodelling extracellular matrix in thymus and pineal gland at aging.

  19. Electrical conduction mechanisms in PbSe and PbS nano crystals 3D matrix layer

    NASA Astrophysics Data System (ADS)

    Arbell, Matan; Hechster, Elad; Sarusi, Gabby

    2016-02-01

    A simulation study and measurements of the electrical conductance in a PbSe and PbS spherical Nano-crystal 3D matrix layer was carried out focusing on its dependences of Nano-crystal size distribution and size gradient along the layer thickness (z-direction). The study suggests a new concept of conductance enhancement by utilizing a size gradient along the layer thickness from mono-layer to the next mono-layer of the Nano-crystals, in order to create a gradient of the energy levels and thus improve directional conductance in this direction. A Monte Carlo simulation of the charge carriers path along the layer thickness of the Nano-crystals 3D matrix using the Miller-Abrahams hopping model was performed. We then compared the conductance characteristics of the gradual size 3D matrix layer to a constant-sized 3D matrix layer that was used as a reference in the simulation. The numerical calculations provided us with insights into the actual conductance mechanism of the PbSe and PbS Nano-crystals 3D matrix and explained the discrepancies in actual conductance and the variability in measured mobilities published in the literature. It is found that the mobility and thus conductance are dependent on a critical electrical field generated between two adjacent nano-crystals. Our model explains the conductance dependents on the: Cathode-Anode distance, the distance between the adjacent nano-crystals in the 3D matrix layer and the size distribution along the current direction. Part of the model (current-voltage dependence) was validated using a current-voltage measurements taken on a constant size normal distribution nano-crystals PbS layer (330nm thick) compared with the predicted I-V curves. It is shown that under a threshold bias, the current is very low, while after above a threshold bias the conductance is significantly increased due to increase of hopping probability. Once reaching the maximum probability the current tend to level-off reaching the maximal conductance

  20. Laser nanostructuring 3-D bioconstruction based on carbon nanotubes in a water matrix of albumin

    NASA Astrophysics Data System (ADS)

    Gerasimenko, Alexander Y.; Ichkitidze, Levan P.; Podgaetsky, Vitaly M.; Savelyev, Mikhail S.; Selishchev, Sergey V.

    2016-04-01

    3-D bioconstructions were created using the evaporation method of the water-albumin solution with carbon nanotubes (CNTs) by the continuous and pulsed femtosecond laser radiation. It is determined that the volume structure of the samples created by the femtosecond radiation has more cavities than the one created by the continuous radiation. The average diameter for multi-walled carbon nanotubes (MWCNTs) samples was almost two times higher (35-40 nm) than for single-walled carbon nanotubes (SWCNTs) samples (20-30 nm). The most homogenous 3-D bioconstruction was formed from MWCNTs by the continuous laser radiation. The hardness of such samples totaled up to 370 MPa at the nanoscale. High strength properties and the resistance of the 3-D bioconstructions produced by the laser irradiation depend on the volume nanotubes scaffold forming inside them. The scaffold was formed by the electric field of the directed laser irradiation. The covalent bond energy between the nanotube carbon molecule and the oxygen of the bovine serum albumin aminoacid residue amounts 580 kJ/mol. The 3-D bioconstructions based on MWCNTs and SWCNTs becomes overgrown with the cells (fibroblasts) over the course of 72 hours. The samples based on the both types of CNTs are not toxic for the cells and don't change its normal composition and structure. Thus the 3-D bioconstructions that are nanostructured by the pulsed and continuous laser radiation can be applied as implant materials for the recovery of the connecting tissues of the living body.

  1. Differential sensitivity of epithelial cells to extracellular matrix in polarity establishment.

    PubMed

    Yonemura, Shigenobu

    2014-01-01

    Establishment of apical-basal polarity is crucial for epithelial sheets that form a compartment in the body, which function to maintain the environment in the compartment. Effects of impaired polarization are easily observed in three-dimensional (3-D) culture systems rather than in two-dimensional (2-D) culture systems. Although the mechanisms for establishing the polarity are not completely understood, signals from the extracellular matrix (ECM) are considered to be essential for determining the basal side and eventually generating polarity in the epithelial cells. To elucidate the common features and differences in polarity establishment among various epithelial cells, we analyzed the formation of epithelial apical-basal polarity using three cell lines of different origin: MDCK II cells (dog renal tubules), EpH4 cells (mouse mammary gland), and R2/7 cells (human colon) expressing wild-type α-catenin (R2/7 α-Cate cells). These cells showed clear apical-basal polarity in 2-D cultures. In 3-D cultures, however, each cell line displayed different responses to the same ECM. In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel. In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens. In EpH4 cells, the spheroids displayed an apical-basal polarity that was opposite to that seen in the other two cell types in both ECM gels, at least during the culture period. On the other hand, the three cell lines showed the same apical-basal polarity both in 2-D cultures and in 3-D cultures using the hanging drop method. The three lines also had similar cellular responses to ECM secreted by the cells themselves. Therefore, appropriate culture conditions should be carefully determined in advance when using various epithelial cells to analyze cell polarity or 3-D morphogenesis.

  2. Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development

    PubMed Central

    Stanic, Karen; Saldivia, Natalia; Förstera, Benjamín; Torrejón, Marcela; Montecinos, Hernán; Caprile, Teresa

    2016-01-01

    Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons. PMID:27733818

  3. Molecular Signaling Pathways Controlling Vascular Tube Morphogenesis and Pericyte-Induced Tube Maturation in 3D Extracellular Matrices.

    PubMed

    Bowers, S L K; Norden, P R; Davis, G E

    2016-01-01

    During capillary network formation, ECs establish interconnecting tubes with defined lumens that reside within vascular guidance tunnels (physical spaces generated during EC tubulogenesis). Pericytes are recruited to EC tubes within these tunnels and capillary basement membrane deposition occurs to facilitate tube maturation. Here, we discuss molecular mechanisms controlling EC tubulogenesis demonstrating the involvement of integrins, MT1-MMP, extracellular matrix, Cdc42, Rac1, Rac2, k-Ras, Rap1b, and key downstream effectors including Pak2, Pak4, IQGAP1, MRCKβ, and Rasip1. These molecules activate kinase cascades controlling EC tube formation, in conjunction with growth factor receptor signaling, which involve PKCɛ, Src family, Raf, Mek, and Erk kinases. These molecules and signaling cascades stimulate EC lumen and tube formation by: regulating MT-MMP-dependent lumen expansion and vascular guidance tunnel formation; generation of intracellular vacuoles/vesicles to create EC apical membranes; and establishing cytoskeletal polarity with acetylated tubulin distributed subapically (and F-actin basally) to facilitate vacuole trafficking/fusion in a polarized, perinuclear region. Using defined serum-free models, we have demonstrated that human EC tubulogenesis and EC-pericyte tube coassembly requires five exogenously applied growth factors which are SCF, IL-3, SDF-1α, FGF-2, and insulin (Factors). Also, we have demonstrated that EC-derived PDGF-BB and HB-EGF are necessary for pericytes to proliferate, recruit to tubes, and induce basement membrane assembly. Finally, we have shown that VEGF fails to directly stimulate EC tubulogenesis. In contrast, it acts as an upstream EC primer of downstream "Factor"-induced tubulogenic and EC-pericyte tube coassembly by upregulating c-Kit, IL-3Rα, and CXCR4 as well as PDGF-BB and HB-EGF expression. PMID:27451100

  4. Monte Carlo - Metropolis Investigations of Shape and Matrix Effects in 2D and 3D Spin-Crossover Nanoparticles

    NASA Astrophysics Data System (ADS)

    Guerroudj, Salim; Caballero, Rafael; De Zela, Francisco; Jureschi, Catalin; Linares, Jorge; Boukheddaden, Kamel

    2016-08-01

    The Ising like model, taking into account short-, long-range interaction as well as surface effects is used to investigate size and shape effects on the thermal behaviour of 2D and 3D spin crossover (SCO) nanoparticles embedded in a matrix. We analyze the role of the parametert, representing the ratio between the number of surface and volume molecules, on the unusual thermal hysteresis behaviour (appearance of the hysteresis and a re-entrance phase transition) at small scales.

  5. Neoplastic extracellular matrix environment promotes cancer invasion in vitro.

    PubMed

    Sundquist, Elias; Renko, Outi; Salo, Sirpa; Magga, Johanna; Cervigne, Nilva K; Nyberg, Pia; Risteli, Juha; Sormunen, Raija; Vuolteenaho, Olli; Zandonadi, Flávia; Paes Leme, Adriana F; Coletta, Ricardo D; Ruskoaho, Heikki; Salo, Tuula

    2016-06-10

    The invasion of carcinoma cells is a crucial feature in carcinogenesis. The penetration efficiency not only depends on the cancer cells, but also on the composition of the tumor microenvironment. Our group has developed a 3D invasion assay based on human uterine leiomyoma tissue. Here we tested whether human, porcine, mouse or rat hearts as well as porcine tongue tissues could be similarly used to study carcinoma cell invasion in vitro. Three invasive human oral tongue squamous cell carcinoma (HSC-3, SCC-25 and SCC-15), melanoma (G-361) and ductal breast adenocarcinoma (MDA-MB-231) cell lines, and co-cultures of HSC-3 and carcinoma-associated or normal oral fibroblasts were assayed. Myoma tissue, both native and lyophilized, promoted invasion and growth of the cancer cells. However, the healthy heart or tongue matrices were unable to induce the invasion of any type of cancer cells tested. Moreover, when studied in more detail, small molecular weight fragments derived from heart tissue rinsing media inhibited HSC-3 horizontal migration. Proteome analysis of myoma rinsing media, on the other hand, revealed migration enhancing factors. These results highlight the important role of matrix composition for cancer invasion studies in vitro and further demonstrate the unique properties of human myoma organotypic model. PMID:27090016

  6. The Extracellular Matrix in Bronchopulmonary Dysplasia: Target and Source.

    PubMed

    Mižíková, Ivana; Morty, Rory E

    2015-01-01

    Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth that contributes significantly to morbidity and mortality in neonatal intensive care units. BPD results from life-saving interventions, such as mechanical ventilation and oxygen supplementation used to manage preterm infants with acute respiratory failure, which may be complicated by pulmonary infection. The pathogenic pathways driving BPD are not well-delineated but include disturbances to the coordinated action of gene expression, cell-cell communication, physical forces, and cell interactions with the extracellular matrix (ECM), which together guide normal lung development. Efforts to further delineate these pathways have been assisted by the use of animal models of BPD, which rely on infection, injurious mechanical ventilation, or oxygen supplementation, where histopathological features of BPD can be mimicked. Notable among these are perturbations to ECM structures, namely, the organization of the elastin and collagen networks in the developing lung. Dysregulated collagen deposition and disturbed elastin fiber organization are pathological hallmarks of clinical and experimental BPD. Strides have been made in understanding the disturbances to ECM production in the developing lung, but much still remains to be discovered about how ECM maturation and turnover are dysregulated in aberrantly developing lungs. This review aims to inform the reader about the state-of-the-art concerning the ECM in BPD, to highlight the gaps in our knowledge and current controversies, and to suggest directions for future work in this exciting and complex area of lung development (patho)biology. PMID:26779482

  7. Extracellular Matrix and the Mechanics of Large Artery Development

    PubMed Central

    Cheng, Jeffrey K.; Wagenseil, Jessica E.

    2012-01-01

    The large, elastic arteries, as their name suggests, provide elastic distention and recoil during the cardiac cycle in vertebrate animals. The arteries are distended from the pressure of ejecting blood during active contraction of the left ventricle (LV) during systole, and recoil to their original dimensions during relaxation of the LV during diastole. The cyclic distension occurs with minimal energy loss, due to the elastic properties of one of the major structural extracellular matrix (ECM) components, elastin. The maximum distension is limited to prevent damage to the artery by another major ECM component, collagen. The mix of ECM components in the wall largely determines the passive mechanical behavior of the arteries and the subsequent load on the heart during systole. While much research has focused on initial artery formation, there has been less attention on the continuing development of the artery to produce the mature composite wall complete with endothelial cells (ECs), smooth muscle cells (SMCs), and the necessary mix of ECM components for proper cardiovascular function. This review focuses on the physiology of large artery development, including SMC differentiation and ECM production. The effects of hemodynamic forces and ECM deposition on the evolving arterial structure and function are discussed. Human diseases and mouse models with genetic mutations in ECM proteins that affect large artery development are summarized. A review of constitutive models and growth and remodeling theories is presented, along with future directions to improve understanding of ECM and the mechanics of large artery development. PMID:22584609

  8. Targeting the neural extracellular matrix in neurological disorders.

    PubMed

    Soleman, S; Filippov, M A; Dityatev, A; Fawcett, J W

    2013-12-01

    The extracellular matrix (ECM) is known to regulate important processes in neuronal cell development, activity and growth. It is associated with the structural stabilization of neuronal processes and synaptic contacts during the maturation of the central nervous system. The remodeling of the ECM during both development and after central nervous system injury has been shown to affect neuronal guidance, synaptic plasticity and their regenerative responses. Particular interest has focused on the inhibitory role of chondroitin sulfate proteoglycans (CSPGs) and their formation into dense lattice-like structures, termed perineuronal nets (PNNs), which enwrap sub-populations of neurons and restrict plasticity. Recent studies in mammalian systems have implicated CSPGs and PNNs in regulating and restricting structural plasticity. The enzymatic degradation of CSPGs or destabilization of PNNs has been shown to enhance neuronal activity and plasticity after central nervous system injury. This review focuses on the role of the ECM, CSPGs and PNNs; and how developmental and pharmacological manipulation of these structures have enhanced neuronal plasticity and aided functional recovery in regeneration, stroke, and amblyopia. In addition to CSPGs, this review also points to the functions and potential therapeutic value of these and several other key ECM molecules in epileptogenesis and dementia.

  9. The Extracellular Matrix Contributes to Mechanotransduction in Uterine Fibroids

    PubMed Central

    Leppert, Phyllis C.; Jayes, Friederike L.; Segars, James H.

    2014-01-01

    The role of the extracellular matrix (ECM) and mechanotransduction as an important signaling factor in the human uterus is just beginning to be appreciated. The ECM is not only the substance that surrounds cells, but ECM stiffness will either compress cells or stretch them resulting in signals converted into chemical changes within the cell, depending on the amount of collagen, cross-linking, and hydration, as well as other ECM components. In this review we present evidence that the stiffness of fibroid tissue has a direct effect on the growth of the tumor through the induction of fibrosis. Fibrosis has two characteristics: (1) resistance to apoptosis leading to the persistence of cells and (2) secretion of collagen and other components of the ECM such a proteoglycans by those cells leading to abundant disposition of highly cross-linked, disoriented, and often widely dispersed collagen fibrils. Fibrosis affects cell growth by mechanotransduction, the dynamic signaling system whereby mechanical forces initiate chemical signaling in cells. Data indicate that the structurally disordered and abnormally formed ECM of uterine fibroids contributes to fibroid formation and growth. An appreciation of the critical role of ECM stiffness to fibroid growth may lead to new strategies for treatment of this common disease. PMID:25110476

  10. Extracellular matrix content of ruptured anterior cruciate ligament tissue.

    PubMed

    Young, Kate; Samiric, Tom; Feller, Julian; Cook, Jill

    2011-08-01

    Anterior cruciate ligaments (ACLs) can rupture with simple movements, suggesting that structural changes in the ligament may reduce the loading capacity of the ligament. We aimed to investigate if proteoglycan and collagen levels were different between ruptured and non-ruptured ACLs. We also compared changes in ruptured tissue over time. During arthroscopic knee reconstruction surgery 24 ruptured ACLs were collected from participants (10 females; 14 males; mean age 24 years). Four non-ruptured ACLs were obtained from participants undergoing total knee replacement surgery (one female, three males; mean age 66 years). Western blot analysis was used to characterise core proteins of aggrecan, versican, decorin and biglycan and glycosaminoglycan assays were also conducted. Collagen levels were measured by hydroxyproline (OHPr) assays. Significantly lower levels of collagen, were found in ruptured ACL compared to non-ruptured ACL (p=0.004). Lower levels of both small and large proteoglycans were found in ruptured than non-ruptured ACLs. No correlation was found between time since rupture and proteoglycan or collagen levels. Ruptured ACLs had less collagen and proteoglycans than non-ruptured ACLs. These changes indicate either extracellular matrix protein levels were reduced prior to rupture or levels decreased immediately after rupture. It is possible that the composition and structure of ACLs that rupture are different to normal ACLs, potentially reducing the tissue's ability to withstand loading. An enhanced understanding of the aetiology of ACL injury could help identify individuals who may be predisposed to rupture.

  11. Extracellular matrix of connective tissues in the heads of teleosts.

    PubMed Central

    Benjamin, M; Ralphs, J R

    1991-01-01

    The distribution of extracellular matrix molecules (chondroitin and keratan sulphates, type II collagen) is described in cranial connective tissues of teleosts. Hyaline cartilage was similar to that in mammals and usually contained all 3 molecules. The more cellular cartilages that are not normally present in mammals were more variable in composition. Scleral cartilage closely resembled hyaline cartilage, Zellknorpel in the gill filaments resembled it in some species but not in others, and elastic/cell-rich and hyaline-cell cartilages were unlike hyaline cartilage. These variations may be related to functional or developmental differences between the tissues. Bone and chondroid bone also varied in composition between species. Whilst both tissues contained chondroitin sulphate, bone contained type II collagen in 5 of the 12 species examined. This suggests that cartilage components are more widespread in teleost bone than has previously been shown. Type II collagen also occurred in dense connective tissues of some species. Notably, where this molecule was present in one of these tissues, it was present in all. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:1817131

  12. Extracellular matrix stiffness dictates Wnt expression through integrin pathway.

    PubMed

    Du, Jing; Zu, Yan; Li, Jing; Du, Shuyuan; Xu, Yipu; Zhang, Lang; Jiang, Li; Wang, Zhao; Chien, Shu; Yang, Chun

    2016-02-08

    It is well established that extracellular matrix (ECM) stiffness plays a significant role in regulating the phenotypes and behaviors of many cell types. However, the mechanism underlying the sensing of mechanical cues and subsequent elasticity-triggered pathways remains largely unknown. We observed that stiff ECM significantly enhanced the expression level of several members of the Wnt/β-catenin pathway in both bone marrow mesenchymal stem cells and primary chondrocytes. The activation of β-catenin by stiff ECM is not dependent on Wnt signals but is elevated by the activation of integrin/ focal adhesion kinase (FAK) pathway. The accumulated β-catenin then bound to the wnt1 promoter region to up-regulate the gene transcription, thus constituting a positive feedback of the Wnt/β-catenin pathway. With the amplifying effect of positive feedback, this integrin-activated β-catenin/Wnt pathway plays significant roles in mediating the enhancement of Wnt signal on stiff ECM and contributes to the regulation of mesenchymal stem cell differentiation and primary chondrocyte phenotype maintenance. The present integrin-regulated Wnt1 expression and signaling contributes to the understanding of the molecular mechanisms underlying the regulation of cell behaviors by ECM elasticity.

  13. Extracellular matrix stiffness dictates Wnt expression through integrin pathway

    PubMed Central

    Du, Jing; Zu, Yan; Li, Jing; Du, Shuyuan; Xu, Yipu; Zhang, Lang; Jiang, Li; Wang, Zhao; Chien, Shu; Yang, Chun

    2016-01-01

    It is well established that extracellular matrix (ECM) stiffness plays a significant role in regulating the phenotypes and behaviors of many cell types. However, the mechanism underlying the sensing of mechanical cues and subsequent elasticity-triggered pathways remains largely unknown. We observed that stiff ECM significantly enhanced the expression level of several members of the Wnt/β-catenin pathway in both bone marrow mesenchymal stem cells and primary chondrocytes. The activation of β-catenin by stiff ECM is not dependent on Wnt signals but is elevated by the activation of integrin/ focal adhesion kinase (FAK) pathway. The accumulated β-catenin then bound to the wnt1 promoter region to up-regulate the gene transcription, thus constituting a positive feedback of the Wnt/β-catenin pathway. With the amplifying effect of positive feedback, this integrin-activated β-catenin/Wnt pathway plays significant roles in mediating the enhancement of Wnt signal on stiff ECM and contributes to the regulation of mesenchymal stem cell differentiation and primary chondrocyte phenotype maintenance. The present integrin-regulated Wnt1 expression and signaling contributes to the understanding of the molecular mechanisms underlying the regulation of cell behaviors by ECM elasticity. PMID:26854061

  14. Substrate stiffness regulates extracellular matrix deposition by alveolar epithelial cells

    PubMed Central

    Eisenberg, Jessica L; Safi, Asmahan; Wei, Xiaoding; Espinosa, Horacio D; Budinger, GR Scott; Takawira, Desire; Hopkinson, Susan B; Jones, Jonathan CR

    2012-01-01

    Aim The aim of the study was to address whether a stiff substrate, a model for pulmonary fibrosis, is responsible for inducing changes in the phenotype of alveolar epithelial cells (AEC) in the lung, including their deposition and organization of extracellular matrix (ECM) proteins. Methods Freshly isolated lung AEC from male Sprague Dawley rats were seeded onto polyacrylamide gel substrates of varying stiffness and analyzed for expression and organization of adhesion, cytoskeletal, differentiation, and ECM components by Western immunoblotting and confocal immunofluorescence microscopy. Results We observed that substrate stiffness influences cell morphology and the organization of focal adhesions and the actin cytoskeleton. Surprisingly, however, we found that substrate stiffness has no influence on the differentiation of type II into type I AEC, nor does increased substrate stiffness lead to an epithelial–mesenchymal transition. In contrast, our data indicate that substrate stiffness regulates the expression of the α3 laminin subunit by AEC and the organization of both fibronectin and laminin in their ECM. Conclusions An increase in substrate stiffness leads to enhanced laminin and fibronectin assembly into fibrils, which likely contributes to the disease phenotype in the fibrotic lung. PMID:23204878

  15. The Extracellular Matrix in Bronchopulmonary Dysplasia: Target and Source

    PubMed Central

    Mižíková, Ivana; Morty, Rory E.

    2015-01-01

    Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth that contributes significantly to morbidity and mortality in neonatal intensive care units. BPD results from life-saving interventions, such as mechanical ventilation and oxygen supplementation used to manage preterm infants with acute respiratory failure, which may be complicated by pulmonary infection. The pathogenic pathways driving BPD are not well-delineated but include disturbances to the coordinated action of gene expression, cell–cell communication, physical forces, and cell interactions with the extracellular matrix (ECM), which together guide normal lung development. Efforts to further delineate these pathways have been assisted by the use of animal models of BPD, which rely on infection, injurious mechanical ventilation, or oxygen supplementation, where histopathological features of BPD can be mimicked. Notable among these are perturbations to ECM structures, namely, the organization of the elastin and collagen networks in the developing lung. Dysregulated collagen deposition and disturbed elastin fiber organization are pathological hallmarks of clinical and experimental BPD. Strides have been made in understanding the disturbances to ECM production in the developing lung, but much still remains to be discovered about how ECM maturation and turnover are dysregulated in aberrantly developing lungs. This review aims to inform the reader about the state-of-the-art concerning the ECM in BPD, to highlight the gaps in our knowledge and current controversies, and to suggest directions for future work in this exciting and complex area of lung development (patho)biology. PMID:26779482

  16. Modulation of leukocyte behavior by an inflamed extracellular matrix.

    PubMed

    Schor, H; Vaday, G G; Lider, O

    2000-01-01

    Inflammation is a response of the immune system to foreign insult or physical damage. Various cellular and humoral components of the immune system are recruited from the vascular system and are translocated through endothelium, and into extracellular matrix (ECM) compartments of inflamed tissues. This translocation is orchestrated by various types of accessory signals, in the form of soluble or complexed molecules, which evoke remarkable transitions in leukocyte activities. Recruited inflammatory cells give rise to mechanisms of migration, including the secretion of enzymes and other pro-inflammatory mediators and the alteration of their adhesive contacts with the ECM. Hence, migrating cells secrete enzymes, chemokines, and cytokines which interact with the ECM, and thereby, provide the cells with intrinsic signals for coordinating their responses. Resultant products of enzymatic modifications to the ECM microenvironment, such as cytokine- and ECM-derived molecules, may be also part of a cell-signaling mechanism that provides leukocytes with information about the nature of their inflammatory activity; such a mechanism may give the immune system data that can be cognitively interpreted for consequential activities. This article reviews the findings that support this notion and describe the dynamic interactions between participants of the inflammatory processes. PMID:11097214

  17. EVALUATION OF THE EXTRACELLULAR MATRIX OF INJURED SUPRASPINATUS IN RATS

    PubMed Central

    Almeida, Luiz Henrique Oliveira; Ikemoto, Roberto; Mader, Ana Maria; Pinhal, Maria Aparecida Silva; Munhoz, Bruna; Murachovsky, Joel

    2016-01-01

    ABSTRACT Objective: To evaluate the evolution of injuries of the supraspinatus muscle by immunohistochemistry (IHC) and anatomopathological analysis in animal model (Wistar rats). Methods: Twenty-five Wistar rats were submitted to complete injury of the supraspinatus tendon, then subsequently sacrificed in groups of five animals at the following periods: immediately after the injury, 24h after the injury, 48h after, 30 days after and three months after the injury. All groups underwent histological and IHC analysis. Results: Regarding vascular proliferation and inflammatory infiltrate, we found a statistically significant difference between groups 1(control group) and 2 (24h after injury). IHC analysis showed that expression of vascular endothelial growth factor (VEGF) showed a statistically significant difference between groups 1 and 2, and collagen type 1 (Col-1) evaluation presented a statistically significant difference between groups 1 and 4. Conclusion: We observed changes in the extracellular matrix components compatible with remodeling and healing. Remodeling is more intense 24h after injury. However, VEGF and Col-1 are substantially increased at 24h and 30 days after the injury, respectively. Level of Evidence I, Experimental Study. PMID:26997907

  18. Quercetin Attenuates Lactate Production and Extracellular Matrix Secretion in Keratoconus

    PubMed Central

    McKay, T. B.; Lyon, D.; Sarker-Nag, A.; Priyadarsini, S.; Asara, J. M.; Karamichos, D.

    2015-01-01

    Keratoconus(KC) is an ecstatic corneal disease leading to corneal-thinning and the formation of a cone-like cornea. Elevated lactate levels, increased oxidative stress, and myofibroblast formation have all been previously reported. In the current study, we assess the role of Quercetin on collagen secretion and myofibroblast formation in KC in vitro. Human corneal fibroblasts(HCFs) and human keratoconus cells(HKCs) were treated with a stable Vitamin C derivative and cultured for 4 weeks, stimulating formation of a self-assembled extracellular matrix. All samples were analyzed using Western blots and targeted tandem mass spectrometry. Our data showed that Quercetin significantly down regulates myofibroblast differentiation and fibrotic markers, such as α-smooth muscle actin (α-SMA) and Collagen III (Col III), in both HCFs and HKCs. Collagen III secretion was reduced 80% in both HCFs and HKCs following Quercetin treatment. Furthermore, Quercetin reduced lactate production by HKCs to normal HCF levels. Quercetin down regulated TGF-βR2 and TGF-β2 expression in HKCs suggesting a significant link to the TGF-β pathway. These results assert that Quercetin is a key regulator of fibrotic markers and ECM assembly by modulating cellular metabolism and TGF-β signaling. Our study suggests that Quercetin is a potential therapeutic for treatment of corneal dystrophies, such as KC. PMID:25758533

  19. The Biology of the Escherichia coli Extracellular Matrix.

    PubMed

    Hufnagel, David A; Depas, William H; Chapman, Matthew R

    2015-06-01

    Escherichia coli is one of the world's best-characterized organisms, because it has been extensively studied for over a century. However, most of this work has focused on E. coli grown under laboratory conditions that do not faithfully simulate its natural environments. Therefore, the historical perspectives on E. coli physiology and life cycle are somewhat skewed toward experimental systems that feature E. coli growing logarithmically in a test tube. Typically a commensal bacterium, E. coli resides in the lower intestines of a slew of animals. Outside of the lower intestine, E. coli can adapt and survive in a very different set of environmental conditions. Biofilm formation allows E. coli to survive, and even thrive, in environments that do not support the growth of planktonic populations. E. coli can form biofilms virtually everywhere: in the bladder during a urinary tract infection, on in-dwelling medical devices, and outside of the host on plants and in the soil. The E. coli extracellular matrix (ECM), primarily composed of the protein polymer named curli and the polysaccharide cellulose, promotes adherence to organic and inorganic surfaces and resistance to desiccation, the host immune system, and other antimicrobials. The pathways that govern E. coli biofilm formation, cellulose production, and curli biogenesis will be discussed in this article, which concludes with insights into the future of E. coli biofilm research and potential therapies. PMID:26185090

  20. [Glycation of extracellular matrix proteins and its role in atherosclerosis].

    PubMed

    Kuzan, Aleksandra; Chwiłkowska, Agnieszka; Kobielarz, Magdalena; Pezowicz, Celina; Gamian, Andrzej

    2012-10-29

    Glycation consists in formation of advanced glycation end-products (AGE) during non-enzymatic reaction between reducing sugars and proteins, lipids or nucleic acids. This review is focused mainly on glycation of collagen and its role in acceleration of vascular disease. Collagen is an extracellular matrix protein characterized by unique structure forming fibrils with great anti-tensile and anti-breaking strength. The protein builds the connective tissue and is responsible for biomechanical properties of blood vessels. It is reported that higher content of glycated collagen correlates with lower elasticity and greater toughness of the vessel walls and, as a consequence, a faster rate of atherosclerosis development. Numerous mechanisms connected with AGE formation are involved in atherogenesis, among others: receptor-mediated production of free radicals, triggering an inflammatory process, activation of leukocytes and thrombocytes, facilitation of LDL binding, change in level of growth factors, adhesion molecules, MMP and some other proteins' expression. The coverages allow the development of therapeutic strategies to prevent or slow down the pathological processes connected with glycation of collagen and other proteins in the artery wall. The main strategies are based on limitation of exogenous AGE, consumption of products which contain rutin, treatment with drugs which inhibit AGE formation, such as pyridoxamine, and chemicals which are able to cleave already formed AGE protein-protein crosslinks, such as ALT-711.

  1. Expression of genes encoding extracellular matrix proteins: a macroarray study.

    PubMed

    Futyma, Konrad; Miotła, Paweł; Różyńska, Krystyna; Zdunek, Małgorzata; Semczuk, Andrzej; Rechberger, Tomasz; Wojcierowski, Jacek

    2014-12-01

    Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs.

  2. Biofilm-specific extracellular matrix proteins of nontypeable Haemophilus influenzae.

    PubMed

    Wu, Siva; Baum, Marc M; Kerwin, James; Guerrero, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

    2014-12-01

    Nontypeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen, can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24- and 96-h NTHi biofilms contained polysaccharides and proteinaceous components as detected by nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24-h biofilms, two were found only in 96-h biofilms, and fifteen were present in the ECM of both 24- and 96-h NTHi biofilms. All proteins identified were either associated with bacterial membranes or cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation.

  3. Extracellular matrix, biotensegrity and tumor microenvironment. An update and overview.

    PubMed

    Noguera, R; Nieto, O A; Tadeo, I; Fariñas, F; Alvaro, T

    2012-06-01

    The extracellular matrix (ECM) constitutes a three-dimensional network that surrounds all cells, organs and tissues in the body. It forms a biophysical filter for protection, nutrition and cell innervation, as well as the medium for facilitating immune response, angiogenesis, fibrosis and tissue regeneration. It is the mechanism by which mechanical forces are transmitted to the basement membrane which, through the integrins, supports the tensegrity system and activates the epigenetic mechanisms of the cell. A review and update on current knowledge on this topic reveals how disturbance of the ECM leads to a loss of efficient filtering, nutrition, elimination, and cell denervation functions, in addition to loss of regeneration capacity and disorders in mechanotransduction. Furthermore, such disturbance results in a loss of substrate, and with it the ability to provide a proper immune response against tumor, toxic and infectious agents. Reciprocal communication between ECM stromal and parenchymatous cells directs gene expression. The oncogenic capacity of the stroma derives from the associated cells as well as from the tumor cells, the angiogenic microenvironment and from an alteration in tensegrity; all of which are dependent on the ECM. It has been shown that the malignant phenotype is reversible by correction of the altered cues of the ECM. PMID:22473691

  4. Translational control of the fibroblast-extracellular matrix association

    PubMed Central

    Nho, Richard Seonghun; Polunovsky, Vitaly

    2013-01-01

    Pulmonary fibrosis is a severe lung disease characterized by sustained propagation of lung fibroblasts and relentless accumulation of extracellular matrix (ECM). Idiopathic pulmonary fibrosis (IPF) is the most severe chronic form of pulmonary fibrosis and results both in the gradual exchange of normal lung parenchyma with fibrotic tissue and in the irreversible impairment of gas exchange in the lung. Despite the urgency for novel therapies in IPF treatment, there is no effective and proven medical therapy available. Molecular mechanisms underlying IPF pathogenesis include aberrant ECM signaling through the canonical integrin/PI3K/Akt/mTORC1 signal transduction pathway. One important and well-characterized downstream effector of this pathway is the cellular protein synthesis machinery. Here we will review the recent advances in our understanding of the function of ECM and integrin receptor signaling in development of IPF and will present evidence indicating that the dysregulation of the eIF4F-mediated translational apparatus is an important factor in the development and progression of IPF and other fibrotic disorders. We further discuss the perspectives and challenges to curbing this deadly disease by targeting aberrant translation. PMID:26824013

  5. Extracellular matrix, mechanotransduction and structural hierarchies in heart tissue engineering.

    PubMed

    Parker, Kevin K; Ingber, Donald E

    2007-08-29

    The spatial and temporal scales of cardiac organogenesis and pathogenesis make engineering of artificial heart tissue a daunting challenge. The temporal scales range from nanosecond conformational changes responsible for ion channel opening to fibrillation which occurs over seconds and can lead to death. Spatial scales range from nanometre pore sizes in membrane channels and gap junctions to the metre length scale of the whole cardiovascular system in a living patient. Synchrony over these scales requires a hierarchy of control mechanisms that are governed by a single common principle: integration of structure and function. To ensure that the function of ion channels and contraction of muscle cells lead to changes in heart chamber volume, an elegant choreography of metabolic, electrical and mechanical events are executed by protein networks composed of extracellular matrix, transmembrane integrin receptors and cytoskeleton which are functionally connected across all size scales. These structural control networks are mechanoresponsive, and they process mechanical and chemical signals in a massively parallel fashion, while also serving as a bidirectional circuit for information flow. This review explores how these hierarchical structural networks regulate the form and function of living cells and tissues, as well as how microfabrication techniques can be used to probe this structural control mechanism that maintains metabolic supply, electrical activation and mechanical pumping of heart muscle. Through this process, we delineate various design principles that may be useful for engineering artificial heart tissue in the future.

  6. Extracellular matrix remodelling after coxsackievirus B3-induced murine myocarditis.

    PubMed Central

    Gómez, R. M.; Castagnino, C. G.; Berría, M. I.

    1992-01-01

    Weanling inbred Balb/c mice were intraperitoneally inoculated with a myocarditic variant of coxsackievirus B3. At days 1, 2, 4, 6, 8, 10, 14, 24 and 30 post-infection (p.i.), myocardial tissue was harvested for viral infectivity titrations and histological studies, including routine techniques (haematoxylin-eosin, Masson trichrome and von Kossa) and specialized procedures (silver impregnation for reticulin, picrosirius red stain for collagen and immunoperoxidase labelling for laminin). Virus was isolated as from day 2, reached maximal infectivity at days 6-8 and decreased gradually to become undetectable by day 14. Early histological findings during the 1st week consisted mainly of scattered foci of necrotic myocytes showing calcium deposits; slight mononuclear cell infiltration and fragmentation of both reticulin fibres and pericellular laminin were also present. From the 2nd up to 4th week p.i., inflammatory reaction abated concomitantly with the gradual development of fibrosis, as evidenced by reticulin fibre thickening, irregular laminin distribution and collagen fibre increase. Our results suggest that viral-induced necrosis is able to trigger marked extracellular matrix remodelling even in the case of minimal inflammation. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:1329915

  7. Targeting the neural extracellular matrix in neurological disorders.

    PubMed

    Soleman, S; Filippov, M A; Dityatev, A; Fawcett, J W

    2013-12-01

    The extracellular matrix (ECM) is known to regulate important processes in neuronal cell development, activity and growth. It is associated with the structural stabilization of neuronal processes and synaptic contacts during the maturation of the central nervous system. The remodeling of the ECM during both development and after central nervous system injury has been shown to affect neuronal guidance, synaptic plasticity and their regenerative responses. Particular interest has focused on the inhibitory role of chondroitin sulfate proteoglycans (CSPGs) and their formation into dense lattice-like structures, termed perineuronal nets (PNNs), which enwrap sub-populations of neurons and restrict plasticity. Recent studies in mammalian systems have implicated CSPGs and PNNs in regulating and restricting structural plasticity. The enzymatic degradation of CSPGs or destabilization of PNNs has been shown to enhance neuronal activity and plasticity after central nervous system injury. This review focuses on the role of the ECM, CSPGs and PNNs; and how developmental and pharmacological manipulation of these structures have enhanced neuronal plasticity and aided functional recovery in regeneration, stroke, and amblyopia. In addition to CSPGs, this review also points to the functions and potential therapeutic value of these and several other key ECM molecules in epileptogenesis and dementia. PMID:24012743

  8. Brain extracellular matrix retains connectivity in neuronal networks

    PubMed Central

    Bikbaev, Arthur; Frischknecht, Renato; Heine, Martin

    2015-01-01

    The formation and maintenance of connectivity are critically important for the processing and storage of information in neuronal networks. The brain extracellular matrix (ECM) appears during postnatal development and surrounds most neurons in the adult mammalian brain. Importantly, the removal of the ECM was shown to improve plasticity and post-traumatic recovery in the CNS, but little is known about the mechanisms. Here, we investigated the role of the ECM in the regulation of the network activity in dissociated hippocampal cultures grown on microelectrode arrays (MEAs). We found that enzymatic removal of the ECM in mature cultures led to transient enhancement of neuronal activity, but prevented disinhibition-induced hyperexcitability that was evident in age-matched control cultures with intact ECM. Furthermore, the ECM degradation followed by disinhibition strongly affected the network interaction so that it strongly resembled the juvenile pattern seen in naïve developing cultures. Taken together, our results demonstrate that the ECM plays an important role in retention of existing connectivity in mature neuronal networks that can be exerted through synaptic confinement of glutamate. On the other hand, removal of the ECM can play a permissive role in modification of connectivity and adaptive exploration of novel network architecture. PMID:26417723

  9. Renal extracellular matrix alterations in lead-treated rats.

    PubMed

    Sánchez, S; Pérez Aguilar, R; Genta, S; Aybar, M; Villecco, E; Sánchez Riera, A

    2001-01-01

    Although the nephrotoxic effects of lead are well documented, the subcellular mechanisms of its action on the kidney remain unclear. The aim of the present work was to investigate the effects of chronic lead exposure on the expression of laminin-1 and fibronectin in the kidney of lead-treated rats. Western immunoblotting of the kidney extracts revealed that experimental exposure to lead resulted in a marked decrease in the intensity of the bands corresponding to laminin-1 and an increase in the intensity of the band corresponding to fibronectin. Immunohistochemical studies demonstrated a weak labelling to laminin-1 and a strong labelling to fibronectin in all renal basement membranes together with a decrease in their thickness. Other ultrastructural alterations found were a diminution in the amount of endothelial fenestrae, an increased fusion of foot processes in epithelial cells of the glomerulus and the presence of intranuclear inclusion bodies in the proximal tubule cells. Lead intoxication might be responsible for the above alterations in the renal extracellular matrix that could play an important role in the pathogenesis of lead nephropathy. PMID:11746185

  10. Tissue Extracellular Matrix Nanoparticle Presentation in Electrospun Nanofibers

    PubMed Central

    Gibson, Matt; Mao, Hai-Quan; Elisseeff, Jennifer

    2014-01-01

    Biomaterials derived from the decellularization of mature tissues retain biological and architectural features that profoundly influence cellular activity. However, the clinical utility of such materials remains limited as the shape and physical properties are difficult to control. In contrast, scaffolds based on synthetic polymers can be engineered to exhibit specific physical properties, yet often suffer from limited biological functionality. This study characterizes composite materials that present decellularized extracellular matrix (DECM) particles in combination with synthetic nanofibers and examines the ability of these materials to influence stem cell differentiation. Mechanical processing of decellularized tissues yielded particles with diameters ranging from 71 to 334 nm. Nanofiber scaffolds containing up to 10% DECM particles (wt/wt) derived from six different tissues were engineered and evaluated to confirm DECM particle incorporation and to measure bioactivity. Scaffolds containing bone, cartilage, and fat promoted osteogenesis at 1 and 3 weeks compared to controls. In contrast, spleen and lung DECM significantly reduced osteogenic outcomes compared to controls. These findings highlight the potential to incorporate appropriate source DECM nanoparticles within nanofiber composites to design a scaffold with bioactivity targeted to specific applications. PMID:24971329

  11. Brain extracellular matrix retains connectivity in neuronal networks.

    PubMed

    Bikbaev, Arthur; Frischknecht, Renato; Heine, Martin

    2015-09-29

    The formation and maintenance of connectivity are critically important for the processing and storage of information in neuronal networks. The brain extracellular matrix (ECM) appears during postnatal development and surrounds most neurons in the adult mammalian brain. Importantly, the removal of the ECM was shown to improve plasticity and post-traumatic recovery in the CNS, but little is known about the mechanisms. Here, we investigated the role of the ECM in the regulation of the network activity in dissociated hippocampal cultures grown on microelectrode arrays (MEAs). We found that enzymatic removal of the ECM in mature cultures led to transient enhancement of neuronal activity, but prevented disinhibition-induced hyperexcitability that was evident in age-matched control cultures with intact ECM. Furthermore, the ECM degradation followed by disinhibition strongly affected the network interaction so that it strongly resembled the juvenile pattern seen in naïve developing cultures. Taken together, our results demonstrate that the ECM plays an important role in retention of existing connectivity in mature neuronal networks that can be exerted through synaptic confinement of glutamate. On the other hand, removal of the ECM can play a permissive role in modification of connectivity and adaptive exploration of novel network architecture.

  12. Mechanocompatible Polymer-Extracellular-Matrix Composites for Vascular Tissue Engineering.

    PubMed

    Jiang, Bin; Suen, Rachel; Wang, Jiao-Jing; Zhang, Zheng J; Wertheim, Jason A; Ameer, Guillermo A

    2016-07-01

    Small-diameter vascular grafts developed from vascular extracellular matrix (ECM) can potentially be used for bypass surgeries and other vascular reconstruction and repair procedures. The addition of heparin to the ECM improves graft hemocompatibility but often involves chemical cross-linking, which increases ECM mechanical stiffness compared to native arteries. Herein, the importance of maintaining ECM mechanocompatibility is demonstrated, and a mechanocompatible strategy to immobilize heparin onto the ECM via a biodegradable elastomer is described. Specifically, poly(1,8-octamethylene citrate)-co-cysteine is hybridized to the ECM, forming a polymer-ECM composite that allows for heparin immobilization via maleimide-thiol "click" chemistry. Heparinized composites reduce platelet adhesion by >60% in vitro, without altering the elastic modulus of the ECM. In a rat abdominal aortic interposition model, intimal hyperplasia in heparinized mechanocompatible grafts is 65% lower when compared to ECM-only control grafts at four weeks. In contrast, grafts that are heparinized with carbodiimide chemistry exhibit increased intimal hyperplasia (4.2-fold) and increased macrophage infiltration (3.5-fold) compared to ECM-only control grafts. All grafts show similar, partial endothelial cell coverage and little to no ECM remodeling. Overall, a mechanocompatible strategy to improve ECM thromboresistance is described and the importance of ECM mechanical properties for proper in vivo graft performance is highlighted. PMID:27109033

  13. The Biology of the Escherichia coli Extracellular Matrix

    PubMed Central

    Hufnagel, David A.; DePas, William H.; Chapman, Matthew R.

    2015-01-01

    Chapter Summary Escherichia coli (E. coli) is one of the world’s best-characterized organisms, as it has been extensively studied for over a century. However, most of this work has focused on E. coli grown under laboratory conditions that do not faithfully simulate its natural environments. Therefore, the historical perspectives on E. coli physiology and life cycle are somewhat skewed toward experimental systems that feature E. coli growing logarithmically in a test tube. Typically a commensal bacterium, E. coli resides in the lower intestines of a slew of animals. Outside of the lower intestine, E. coli can adapt and survive in a very different set of environmental conditions. Biofilm formation allows E. coli to survive, and even thrive, in environments that do not support the growth of planktonic populations. E. coli can form biofilms virtually everywhere; in the bladder during a urinary tract infection, on in-dwelling medical devices, and outside of the host on plants and in the soil. The E. coli extracellular matrix, primarily composed of the protein polymer named curli and the polysaccharide cellulose, promotes adherence to organic and inorganic surfaces, and resistance to desiccation, the host immune system and other antimicrobials. The pathways that govern E. coli biofilm formation, cellulose production, and curli biogenesis will be discussed in this book chapter, which concludes with insights into the future of E. coli biofilm research and potential therapies. PMID:26185090

  14. Enhancement of Tenogenic Differentiation of Human Adipose Stem Cells by Tendon-Derived Extracellular Matrix

    PubMed Central

    Yang, Guang; Rothrauff, Benjamin B.; Lin, Hang; Gottardi, Riccardo; Alexander, Peter G.; Tuan, Rocky S.

    2014-01-01

    Mesenchymal stem cells (MSCs) have gained increasing research interest for their potential in improving healing and regeneration of injured tendon tissues. Developing functional three-dimensional (3D) scaffolds to promote MSC proliferation and differentiation is a critical requirement in tendon tissue engineering. Tendon extracellular matrix has been shown to maintain the tenogenic potential of tendon stem cells and stimulate tenogenesis of human adipose stem cells (hASCs) in 2D culture. This study aims at characterizing the biological composition of urea-extracted fraction of tendon ECM (tECM) and its tenogenic effect on hASCs cultured in a 3D collagen scaffold under uniaxial tension. The tECM obtained was cell-free and rich in ECM proteins. hASCs seeded in tECM supplemented scaffold exhibited significantly increased proliferation and tenogenic differentiation. The presence of tECM also greatly suppressed the osteogenic differentiation of hASCs triggered by uniaxial tension. In addition, tECM-supplemented constructs displayed enhanced mechanical strength, accompanied by reduced expression and activity of MMPs in the seeded hASCs, indicating a regulatory activity of tECM in cell-mediated scaffold remodeling. These findings support the utility of tECM in creating bio-functional scaffolds for tendon tissue engineering. PMID:24044998

  15. Linearized texture of three-dimensional extracellular matrix is mandatory for bladder cancer cell invasion

    PubMed Central

    Alfano, Massimo; Nebuloni, Manuela; Allevi, Raffaele; Zerbi, Pietro; Longhi, Erika; Lucianò, Roberta; Locatelli, Irene; Pecoraro, Angela; Indrieri, Marco; Speziali, Chantal; Doglioni, Claudio; Milani, Paolo; Montorsi, Francesco; Salonia, Andrea

    2016-01-01

    In the fields of biomaterials and tissue engineering simulating the native microenvironment is of utmost importance. As a major component of the microenvironment, the extracellular matrix (ECM) contributes to tissue homeostasis, whereas modifications of native features are associated with pathological conditions. Furthermore, three-dimensional (3D) geometry is an important feature of synthetic scaffolds favoring cell stemness, maintenance and differentiation. We analyzed the 3D structure, geometrical measurements and anisotropy of the ECM isolated from (i) human bladder mucosa (basal lamina and lamina propria) and muscularis propria; and, (ii) bladder carcinoma (BC). Next, binding and invasion of bladder metastatic cell line was observed on synthetic scaffold recapitulating anisotropy of tumoral ECM, but not on scaffold with disorganized texture typical of non-neoplastic lamina propria. This study provided information regarding the ultrastructure and geometry of healthy human bladder and BC ECMs. Likewise, using synthetic scaffolds we identified linearization of the texture as a mandatory feature for BC cell invasion. Integrating microstructure and geometry with biochemical and mechanical factors could support the development of an innovative synthetic bladder substitute or a tumoral scaffold predictive of chemotherapy outcomes. PMID:27779205

  16. In vivo screening of extracellular matrix components produced under multiple experimental conditions implanted in one animal.

    PubMed

    Higuera, Gustavo A; Hendriks, Jeanine A A; van Dalum, Joost; Wu, Ling; Schotel, Roka; Moreira-Teixeira, Liliana; van den Doel, Mirella; Leijten, Jeroen C H; Riesle, Jens; Karperien, Marcel; van Blitterswijk, Clemens A; Moroni, Lorenzo

    2013-06-01

    Animal experiments help to progress and ensure safety of an increasing number of novel therapies, drug development and chemicals. Unfortunately, these also lead to major ethical concerns, costs and limited experimental capacity. We foresee a coercion of all these issues by implantation of well systems directly into vertebrate animals. Here, we used rapid prototyping to create wells with biomaterials to create a three-dimensional (3D) well-system that can be used in vitro and in vivo. First, the well sizes and numbers were adjusted for 3D cell culture and in vitro screening of molecules. Then, the functionality of the wells was evaluated in vivo under 36 conditions for tissue regeneration involving human mesenchymal stem cells (hMSCs) and bovine primary chondrocytes (bPCs) screened in one animal. Each biocompatible well was controlled to contain μl-size volumes of tissue, which led to tissue penetration from the host and tissue formation under implanted conditions. We quantified both physically and biologically the amounts of extracellular matrix (ECM) components found in each well. Using this new concept the co-culture of hMSCs and bPCs was identified as a positive hit for cartilage tissue repair, which was a comparable result using conventional methods. The in vivo screening of candidate conditions opens an entirely new range of experimental possibilities, which significantly abates experimental animal use and increases the pace of discovery of medical treatments.

  17. Excitation propagation in three-dimensional engineered hearts using decellularized extracellular matrix.

    PubMed

    Yasui, Haruyo; Lee, Jong-Kook; Yoshida, Akira; Yokoyama, Teruki; Nakanishi, Hiroyuki; Miwa, Keiko; Naito, Atsuhiko T; Oka, Toru; Akazawa, Hiroshi; Nakai, Junichi; Miyagawa, Shigeru; Sawa, Yoshiki; Sakata, Yasushi; Komuro, Issei

    2014-09-01

    Engineering of three-dimensional (3D) cardiac tissues using decellularized extracellular matrix could be a new technique to create an "organ-like" structure of the heart. To engineer artificial hearts functionally comparable to native hearts, however, much remain to be solved including stable excitation-propagation. To elucidate the points, we examined conduction properties of engineered tissues. We repopulated the decellularized hearts with neonatal rat cardiac cells and then, we observed excitation-propagation of spontaneous beatings using high resolution cameras. We also conducted immunofluorescence staining to examine morphological aspects. Live tissue imaging revealed that GFP-labeled-isolated cardiac cells were migrated into interstitial spaces through extravasation from coronary arteries. Engineered hearts repopulated with Ca(2+)-indicating protein (GCaMP2)-expressing cardiac cells were subjected to optical imaging experiments. Although the engineered hearts generally showed well-organized stable excitation-propagation, the hearts also demonstrated arrhythmogenic propensity such as disorganized propagation. Immunofluorescence study revealed randomly-mixed alignment of cardiomyocytes, endothelial cells and smooth muscle cells. The recellularized hearts also showed disarray of cardiomyocytes and markedly decreased expression of connexin43. In conclusion, we successfully demonstrated that the recellularized hearts showed dynamic excitation-propagation as a "whole organ". Our strategy could provide prerequisite information to construct a 3D-engineered heart, functionally comparable to the native heart.

  18. Enhancement of tenogenic differentiation of human adipose stem cells by tendon-derived extracellular matrix.

    PubMed

    Yang, Guang; Rothrauff, Benjamin B; Lin, Hang; Gottardi, Riccardo; Alexander, Peter G; Tuan, Rocky S

    2013-12-01

    Mesenchymal stem cells (MSCs) have gained increasing research interest for their potential in improving healing and regeneration of injured tendon tissues. Developing functional three-dimensional (3D) scaffolds to promote MSC proliferation and differentiation is a critical requirement in tendon tissue engineering. Tendon extracellular matrix has been shown to maintain the tenogenic potential of tendon stem cells and stimulate tenogenesis of human adipose stem cells (hASCs) in 2D culture. This study aims at characterizing the biological composition of urea-extracted fraction of tendon ECM (tECM) and its tenogenic effect on hASCs cultured in a 3D collagen scaffold under uniaxial tension. The tECM obtained was cell-free and rich in ECM proteins. hASCs seeded in tECM-supplemented scaffold exhibited significantly increased proliferation and tenogenic differentiation. The presence of tECM also greatly suppressed the osteogenic differentiation of hASCs triggered by uniaxial tension. In addition, tECM-supplemented constructs displayed enhanced mechanical strength, accompanied by reduced expression and activity of MMPs in the seeded hASCs, indicating a regulatory activity of tECM in cell-mediated scaffold remodeling. These findings support the utility of tECM in creating bio-functional scaffolds for tendon tissue engineering.

  19. Odontogenic Induction of Dental Stem Cells by Extracellular Matrix-Inspired Three-Dimensional Scaffold

    PubMed Central

    Ravindran, Sriram; Zhang, Youbin; Huang, Chun-Chieh

    2014-01-01

    Currently, root canal therapy is the only clinical treatment available to treat damaged or necrotic dental pulp tissue arising from caries. This treatment results in the loss of tooth vitality. Somatic dental stem cell-based tissue engineering approaches can alleviate this problem by preserving tooth vitality. Dental stem cells are multipotent and under appropriate conditions could be used for dental pulp tissue engineering. Successful use of these cells in pulp repair requires a combination of growth factors and appropriate scaffolds to induce cell differentiation. In this study, we demonstrate the odontogenic differentiation of human dental pulp stem cells (DPSCs) and the human periodontal ligament stem cells when cultured on a decellularized 3D extracellular matrix (ECM) scaffold without the need for exogenous addition of growth factors. Subcutaneous implantation of the ECM scaffolds containing DPSCs showed the formation of dental pulp-like tissue with cells expressing dentin sialoprotein (DSP) and dentin phosphophoryn (DPP). Additionally, we also show that the ECM scaffold can be exploited as a tool to study the extracellular function of multifunctional proteins. These promising results demonstrate the feasibility of developing these biomimetic scaffolds for treatment of dental caries. PMID:23859633

  20. Extracellular matrix production and calcium carbonate precipitation by coral cells in vitro.

    PubMed

    Helman, Yael; Natale, Frank; Sherrell, Robert M; Lavigne, Michèle; Starovoytov, Valentin; Gorbunov, Maxim Y; Falkowski, Paul G

    2008-01-01

    The evolution of multicellularity in animals required the production of extracellular matrices that serve to spatially organize cells according to function. In corals, three matrices are involved in spatial organization: (i) an organic ECM, which facilitates cell-cell and cell-substrate adhesion; (ii) a skeletal organic matrix (SOM), which facilitates controlled deposition of a calcium carbonate skeleton; and (iii) the calcium carbonate skeleton itself, which provides the structural support for the 3D organization of coral colonies. In this report, we examine the production of these three matrices by using an in vitro culturing system for coral cells. In this system, which significantly facilitates studies of coral cell physiology, we demonstrate in vitro excretion of ECM by primary (nondividing) tissue cultures of both soft (Xenia elongata) and hard (Montipora digitata) corals. There are structural differences between the ECM produced by X. elongata cell cultures and that of M. digitata, and ascorbic acid, a critical cofactor for proline hydroxylation, significantly increased the production of collagen in the ECM of the latter species. We further demonstrate in vitro production of SOM and extracellular mineralized particles in cell cultures of M. digitata. Inductively coupled plasma mass spectrometry analysis of Sr/Ca ratios revealed the particles to be aragonite. De novo calcification was confirmed by following the incorporation of (45)Ca into acid labile macromolecules. Our results demonstrate the ability of isolated, differentiated coral cells to undergo fundamental processes required for multicellular organization. PMID:18162537

  1. Differential expression of extracellular matrix and growth factors by embryoid bodies in hydrodynamic and static cultures.

    PubMed

    Fridley, Krista M; Nair, Rekha; McDevitt, Todd C

    2014-12-01

    During development, cell fate specification and tissue development are orchestrated by the sequential presentation of soluble growth factors (GF) and extracellular matrix (ECM) molecules. Similarly, differentiation of stem cells in vitro relies upon the temporal presence of extracellular cues within the microenvironment. Hydrodynamic culture systems are not limited by volume restrictions and therefore offer several practical advantages for scalability over static cultures; however, hydrodynamic cultures expose cells to physical parameters not present in static culture, such as fluid shear stress and mass transfer through convective forces. In this study, the differences between static and hydrodynamic culture conditions on the expression of ECM and GF molecules during the differentiation of mouse embryonic stem cells were examined at both the gene and protein level. The expression of ECM and GF genes exhibited an early decrease in static cultures based on heat map and hierarchical clustering analysis and a relative delayed increase in hydrodynamic cultures. Although the temporal patterns of specific ECM and GF protein expression were comparable between static and hydrodynamic cultures, several notable differences in the magnitudes of expression were observed at similar time points. These results describe the establishment of an analytical framework that can be used to examine the expression patterns of ECM and GF molecules expressed by pluripotent stem cells undergoing differentiation as 3D multicellular aggregates under different culture conditions, and suggest that physical parameters of stem cell microenvironments can alter endogenous ECM and GF expression profiles that may, in turn, influence cell fate decisions. PMID:25423310

  2. Brain extracellular matrix meets COST--matrix for European research networks.

    PubMed

    Gajović, Srećko; Pochet, Roland

    2014-01-01

    Today's researchers are faced with a change from curiosity-driven to mandate-driven research. These two approaches are well combined within scientific networks (Actions) supported by the European Cooperation in Science and Technology (COST) program. The functioning of COST Actions, although directed only to networking, has a substantial impact on European science and can be compared to the functioning of the extracellular matrix in the brain, which although scarce plays a key role in initiation, maintenance, and plasticity of intercellular interactions in the nervous system. COST networks enable interdisciplinary approach and support early-stage researchers, which is a vital asset for the advancement of science. PMID:25410370

  3. Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix.

    PubMed

    Stout, David A; Toyjanova, Jennet; Franck, Christian

    2015-01-01

    The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis. PMID:26131645

  4. Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix

    PubMed Central

    1978-01-01

    Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro. PMID:363726

  5. The Role of the Extracellular Matrix Components in Cutaneous Wound Healing

    PubMed Central

    Mencner, Łukasz; Komosinska-Vassev, Katarzyna

    2014-01-01

    Wound healing is the physiologic response to tissue trauma proceeding as a complex pathway of biochemical reactions and cellular events, secreted growth factors, and cytokines. Extracellular matrix constituents are essential components of the wound repair phenomenon. Firstly, they create a provisional matrix, providing a structural integrity of matrix during each stage of healing process. Secondly, matrix molecules regulate cellular functions, mediate the cell-cell and cell-matrix interactions, and serve as a reservoir and modulator of cytokines and growth factors' action. Currently known mechanisms, by which extracellular matrix components modulate each stage of the process of soft tissue remodeling after injury, have been discussed. PMID:24772435

  6. 3D self-consistent modeling of a matrix source of negative hydrogen ions.

    PubMed

    Tarnev, Kh; Demerdjiev, A; Shivarova, A; Lishev, St

    2016-02-01

    The paper is in the scope of studies on the rf driving of a matrix source of negative hydrogen ions: a matrix of small radius discharges with planar-coil inductive driving and single aperture extraction from each discharge. The results from a three-dimensional model, in which plasma description is coupled to electrodynamics, confirm former conclusion that a single coil driving of the whole matrix by a zigzag coil with an omega-shaped conductor on the bottom of each discharge tube ensures efficient rf power deposition to the plasma. The latter is due to similarities with the rf driving of a single discharge by a single planar coil, shown by the obtained induced current and spatial distribution of the plasma parameters. Distinctions associated with the coil configuration as a single coil for the whole matrix are also discussed. PMID:26932005

  7. Process-induced extracellular matrix alterations affect the mechanisms of soft tissue repair and regeneration

    PubMed Central

    Xu, Hui; Sandor, Maryellen; Lombardi, Jared

    2013-01-01

    Extracellular matrices derived from animal tissues for human tissue repairs are processed by various methods of physical, chemical, or enzymatic decellularization, viral inactivation, and terminal sterilization. The mechanisms of action in tissue repair vary among bioscaffolds and are suggested to be associated with process-induced extracellular matrix modifications. We compared three non-cross-linked, commercially available extracellular matrix scaffolds (Strattice, Veritas, and XenMatrix), and correlated extracellular matrix alterations to in vivo biological responses upon implantation in non-human primates. Structural evaluation showed significant differences in retaining native tissue extracellular matrix histology and ultrastructural features among bioscaffolds. Tissue processing may cause both the condensation of collagen fibers and fragmentation or separation of collagen bundles. Calorimetric analysis showed significant differences in the stability of bioscaffolds. The intrinsic denaturation temperature was measured to be 51°C, 38°C, and 44°C for Strattice, Veritas, and XenMatrix, respectively, demonstrating more extracellular matrix modifications in the Veritas and XenMatrix scaffolds. Consequently, the susceptibility to collagenase degradation was increased in Veritas and XenMatrix when compared to their respective source tissues. Using a non-human primate model, three bioscaffolds were found to elicit different biological responses, have distinct mechanisms of action, and yield various outcomes of tissue repair. Strattice permitted cell repopulation and was remodeled over 6 months. Veritas was unstable at body temperature, resulting in rapid absorption with moderate inflammation. XenMatrix caused severe inflammation and sustained immune reactions. This study demonstrates that extracellular matrix alterations significantly affect biological responses in soft tissue repair and regeneration. The data offer useful insights into the rational design of

  8. Magnetic properties of 3D nanocomposites consisting of an opal matrix with embedded spinel ferrite particles

    NASA Astrophysics Data System (ADS)

    Rinkevich, A. B.; Korolev, A. V.; Samoylovich, M. I.; Kleshcheva, S. M.; Perov, D. V.

    2016-02-01

    The magnetic properties of 3D nanocomposites representing Mn-Zn, Ni-Zn, Co-Zn, La-Co-Zn, and Nd-Co-Zn spinel ferrite particles embedded in the interspherical spaces of opal matrices are studied. Experimental data are obtained in the temperature interval 2-300 K by measuring the magnetization at a static magnetic field strength of up to 50 kOe and the ac magnetic susceptibility at an alternating magnetic field amplitude of 4 kOe and a frequency of 80 Hz.

  9. Regulation of human mesenchymal stem cells differentiation into chondrocytes in extracellular matrix-based hydrogel scaffolds.

    PubMed

    Du, Mingchun; Liang, Hui; Mou, Chenchen; Li, Xiaoran; Sun, Jie; Zhuang, Yan; Xiao, Zhifeng; Chen, Bing; Dai, Jianwu

    2014-02-01

    To induce human mesenchymal stem cells (hMSCs) to differentiate into chondrocytes in three-dimensional (3D) microenvironments, we developed porous hydrogel scaffolds using the cartilage extracellular matrix (ECM) components of chondroitin sulfate (CS) and collagen (COL). The turbidity and viscosity experiments indicated hydrogel could form through pH-triggered co-precipitation when pH=2-3. Enzyme-linked immunosorbent assay (ELISA) confirmed the hydrogel scaffolds could controllably release growth factors as envisaged. Transforming growth factor-β (TGF-β) was released to stimulate hMSCs differentiation into chondrocytes; and then collagen binding domain-basic fibroblast growth factor (CBD-bFGF) was released to improve the differentiation and preserve the chondrocyte phenotype. In in vitro cell culture experiments, the differentiation processes were compared in different microenvironments: 2D culture in culture plate as control, 3D culture in the fabricated scaffolds without growth factors (CC), the samples with CBD-bFGF (CC-C), the samples with TGF-β (CC-T), the samples with CBD-bFGF/TGF-β (CC-CT). Real-time polymerase chain reaction (RT-PCR) revealed the hMSC marker genes of CD44 and CD105 decreased; at the same time the chondrocyte marker genes of collagen type II and aggrecan increased, especially in the CC-CT sample. Immunostaining results further confirmed the hMSC marker protein of CD 44 disappeared and the chondrocyte marker protein of collagen type II emerged over time in the CC-CT sample. These results imply the ECM-based hydrogel scaffolds with growth factors can supply suitable 3D cell niches for hMSCs differentiation into chondrocytes and the differentiation process can be regulated by the controllably released growth factors. PMID:24231133

  10. Modulation of 3D Fibrin Matrix Stiffness by Intrinsic Fibrinogen–Thrombin Compositions and by Extrinsic Cellular Activity

    PubMed Central

    Duong, Haison; Wu, Benjamin

    2009-01-01

    Fibrin is a substance formed through catalytic conversion of coagulation constituents: fibrinogen and thrombin. The kinetics of the two constituents determines the structural properties of the fibrin architecture. We have shown previously that changing the fibrinogen and thrombin concentrations in the final three-dimensional (3D) fibrin matrix influenced cell proliferation and differentiation. In this study, we further examined the effect of changing fibrinogen and thrombin concentrations in the absence or presence of fibroblasts on the structural modulus or stiffness of 3D fibrin matrices. We have prepared fibroblast-free and fibroblast-embedded 3D fibrin matrices of different fibrinogen and thrombin formulations, and tested the stiffness of these constructs using standard mechanical testing assays. Results showed that there was a corresponding increase in stiffness with increasing thrombin and fibrinogen concentrations; the increase was more notable with fibrinogen and to a lesser degree with thrombin. The effect of fibroblasts on the stiffness of the fibrin construct was also examined. We have observed a small increase in the stiffness of the fibroblast-incorporated fibrin construct as they proliferated and exhibited spreading morphology. To our knowledge, this is the first comprehensive report detailing the relationship between fibrinogen and thrombin concentrations, cell proliferation, and stiffness in 3D fibrin matrices. The data obtained may lead to optimally design suitable bioscaffolds where we can control both cell proliferation and structural integrity for a variety of tissue engineering applications. PMID:19309239

  11. Milk extracellular vesicles accelerate osteoblastogenesis but impair bone matrix formation.

    PubMed

    Oliveira, Marina C; Arntz, Onno J; Blaney Davidson, Esmeralda N; van Lent, Peter L E M; Koenders, Marije I; van der Kraan, Peter M; van den Berg, Wim B; Ferreira, Adaliene V M; van de Loo, Fons A J

    2016-04-01

    The claimed beneficial effect of milk on bone is still a matter for debate. Recently extracellular vesicles (EVs) that contain proteins and RNA were discovered in milk, but their effect on bone formation has not yet been determined. We demonstrated previously that bovine milk-derived EVs (BMEVs) have immunoregulatory properties. Our aim was to evaluate the effect of BMEVs on osteogenesis by mice and human mesenchymal stem cells (hMSCs). Oral delivery of two concentrations of BMEVs to female DBA/1J mice during 7weeks did not alter the tibia trabecular bone area; however, the osteocytes number increased. In addition, the highest dose of BMEVs markedly increased the woven bone tissue, which is more brittle. The exposure of hMSCs to BMEVs during 21days resulted in less mineralization but higher cell proliferation. Interestingly BMEVs reduced the collagen production, but enhanced the expression of genes characteristic for immature osteoblasts. A kinetic study showed that BMEVs up-regulated many osteogenic genes within the first 4days. However, the production of type I collagen and expression of its genes (COL1A1 and COL1A2) were markedly reduced at days 21 and 28. At day 28, BMEVs again lead to higher proliferation, but mineralization was significantly increased. This was associated with increased expression of sclerostin, a marker for osteocytes, and reduced osteonectin, which is associated to bone matrix formation. Our study adds BMEVs to the list of milk components that can affect bone formation and may shed new light on the contradictory claims of milk on bone formation.

  12. Extracellular Matrix and Dermal Fibroblast Function in the Healing Wound

    PubMed Central

    Tracy, Lauren E.; Minasian, Raquel A.; Caterson, E.J.

    2016-01-01

    Significance: Fibroblasts play a critical role in normal wound healing. Various extracellular matrix (ECM) components, including collagens, fibrin, fibronectin, proteoglycans, glycosaminoglycans, and matricellular proteins, can be considered potent protagonists of fibroblast survival, migration, and metabolism. Recent Advances: Advances in tissue culture, tissue engineering, and ex vivo models have made the examination and precise measurements of ECM components in wound healing possible. Likewise, the development of specific transgenic animal models has created the opportunity to characterize the role of various ECM molecules in healing wounds. In addition, the recent characterization of new ECM molecules, including matricellular proteins, dermatopontin, and FACIT collagens (Fibril-Associated Collagens with Interrupted Triple helices), further demonstrates our cursory knowledge of the ECM in coordinated wound healing. Critical Issues: The manipulation and augmentation of ECM components in the healing wound is emerging in patient care, as demonstrated by the use of acellular dermal matrices, tissue scaffolds, and wound dressings or topical products bearing ECM proteins such as collagen, hyaluronan (HA), or elastin. Once thought of as neutral structural proteins, these molecules are now known to directly influence many aspects of cellular wound healing. Future Directions: The role that ECM molecules, such as CCN2, osteopontin, and secreted protein, acidic and rich in cysteine, play in signaling homing of fibroblast progenitor cells to sites of injury invites future research as we continue investigating the heterotopic origin of certain populations of fibroblasts in a healing wound. Likewise, research into differently sized fragments of the same polymeric ECM molecule is warranted as we learn that fragments of molecules such as HA and tenascin-C can have opposing effects on dermal fibroblasts. PMID:26989578

  13. 3D ToF-SIMS Analysis of Peptide Incorporation into MALDI Matrix Crystals with Sub-micrometer Resolution.

    PubMed

    Körsgen, Martin; Pelster, Andreas; Dreisewerd, Klaus; Arlinghaus, Heinrich F

    2016-02-01

    The analytical sensitivity in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is largely affected by the specific analyte-matrix interaction, in particular by the possible incorporation of the analytes into crystalline MALDI matrices. Here we used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to visualize the incorporation of three peptides with different hydrophobicities, bradykinin, Substance P, and vasopressin, into two classic MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA). For depth profiling, an Ar cluster ion beam was used to gradually sputter through the matrix crystals without causing significant degradation of matrix or biomolecules. A pulsed Bi3 ion cluster beam was used to image the lateral analyte distribution in the center of the sputter crater. Using this dual beam technique, the 3D distribution of the analytes and spatial segregation effects within the matrix crystals were imaged with sub-μm resolution. The technique could in the future enable matrix-enhanced (ME)-ToF-SIMS imaging of peptides in tissue slices at ultra-high resolution. Graphical Abstract ᅟ. PMID:26419771

  14. 3D ToF-SIMS Analysis of Peptide Incorporation into MALDI Matrix Crystals with Sub-micrometer Resolution

    NASA Astrophysics Data System (ADS)

    Körsgen, Martin; Pelster, Andreas; Dreisewerd, Klaus; Arlinghaus, Heinrich F.

    2016-02-01

    The analytical sensitivity in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is largely affected by the specific analyte-matrix interaction, in particular by the possible incorporation of the analytes into crystalline MALDI matrices. Here we used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to visualize the incorporation of three peptides with different hydrophobicities, bradykinin, Substance P, and vasopressin, into two classic MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA). For depth profiling, an Ar cluster ion beam was used to gradually sputter through the matrix crystals without causing significant degradation of matrix or biomolecules. A pulsed Bi3 ion cluster beam was used to image the lateral analyte distribution in the center of the sputter crater. Using this dual beam technique, the 3D distribution of the analytes and spatial segregation effects within the matrix crystals were imaged with sub-μm resolution. The technique could in the future enable matrix-enhanced (ME)-ToF-SIMS imaging of peptides in tissue slices at ultra-high resolution.

  15. Microfabrication of extracellular matrix structures using multipohoton-excited photochemistry: Application to modeling ovarian tissue in vitro

    NASA Astrophysics Data System (ADS)

    Ajeti, Visar

    The extracellular matrix plays a crucial role in tissue development, differentiation and homeostasis by providing the necessary biophysical and biochemical cues for the cells. In tumors, the composition and the structure of the microenvironment is thought to be manipulated by the cancers cells to support proliferative growth and enhanced migration as means of facilitated metastasis. Current in vitro tools to address these mechanistic events in tumor progression are lacking in part due to the difficulty in recapitulating the complexity of the composition and nanoarchitecture of the tumor microenvironment. In this thesis, we explore the feasibility of multiphoton-excited photochemistry as a fabrication tool for generating in vitro scaffolds that are highly repeatable, biologically relevant and relatively affordable in a research setting. The power of this technique lays in the capabilities of crosslinking whole extracellular matrix proteins in three dimensions (3D) to recreate key topographical features of the tissue with sub-micron resolution and high fidelity. The technological developments we present here enable direct translation of matrix topographies by using the high resolution image data of the tissue samples as a fabrication template. To this effect, we have applied the fabrication technique to generate gradients of crosslinked proteins as means of studying the role of haptotaxis in ovarian and breast cancers. Our findings show that cancer cells modulate their migration velocity and persistence in response to the changes in the composition of the extracellular matrix. In addition, we have examined structural features of the stroma in relation to cancer migration dynamics. We find that by recreating highly aligned nanoarchitectural features prevalent in cancer stroma, we see permissive and enhanced cell migration with cell morphologies similar to in vivo. We believe multiphoton fabrication to be an enabling tool in the next generation of tissue scaffolding

  16. Experimental studies of cobalt ferrite nanoparticles doped silica matrix 3D magneto-photonic crystals

    NASA Astrophysics Data System (ADS)

    Abou Diwan, E.; Royer, F.; Kekesi, R.; Jamon, D.; Blanc-Mignon, M. F.; Neveu, S.; Rousseau, J. J.

    2013-05-01

    In this paper, we present the synthesis and the optical properties of 3D magneto-photonic structures. The elaboration process consists in firstly preparing then infiltrating polystyrene direct opals with a homogeneous solution of sol-gel silica precursors doped by cobalt ferrite nanoparticles, and finally dissolving the polystyrene spheres. Scanning Electron Microscopy (SEM) images of the prepared samples clearly evidence a periodic arrangement. Using a home-made polarimetric optical bench, the transmittance as a function of the wavelength, the Faraday rotation as a function of the applied magnetic field, and the Faraday ellipticity as a function of the wavelength and as a function of the applied magnetic field were measured. The existence of deep photonic band gaps (PBG), the unambiguous magnetic character of the samples and the qualitative modification of the Faraday ellipticity in the area of the PBG are evidenced.

  17. Multi-ray-based system matrix generation for 3D PET reconstruction.

    PubMed

    Moehrs, Sascha; Defrise, Michel; Belcari, Nicola; Guerra, Alberto Del; Bartoli, Antonietta; Fabbri, Serena; Zanetti, Gianluigi

    2008-12-01

    Iterative image reconstruction algorithms for positron emission tomography (PET) require a sophisticated system matrix (model) of the scanner. Our aim is to set up such a model offline for the YAP-(S)PET II small animal imaging tomograph in order to use it subsequently with standard ML-EM (maximum-likelihood expectation maximization) and OSEM (ordered subset expectation maximization) for fully three-dimensional image reconstruction. In general, the system model can be obtained analytically, via measurements or via Monte Carlo simulations. In this paper, we present the multi-ray method, which can be considered as a hybrid method to set up the system model offline. It incorporates accurate analytical (geometric) considerations as well as crystal depth and crystal scatter effects. At the same time, it has the potential to model seamlessly other physical aspects such as the positron range. The proposed method is based on multiple rays which are traced from/to the detector crystals through the image volume. Such a ray-tracing approach itself is not new; however, we derive a novel mathematical formulation of the approach and investigate the positioning of the integration (ray-end) points. First, we study single system matrix entries and show that the positioning and weighting of the ray-end points according to Gaussian integration give better results compared to equally spaced integration points (trapezoidal integration), especially if only a small number of integration points (rays) are used. Additionally, we show that, for a given variance of the single matrix entries, the number of rays (events) required to calculate the whole matrix is a factor of 20 larger when using a pure Monte-Carlo-based method. Finally, we analyse the quality of the model by reconstructing phantom data from the YAP-(S)PET II scanner. PMID:19001696

  18. Multi-ray-based system matrix generation for 3D PET reconstruction

    NASA Astrophysics Data System (ADS)

    Moehrs, Sascha; Defrise, Michel; Belcari, Nicola; DelGuerra, Alberto; Bartoli, Antonietta; Fabbri, Serena; Zanetti, Gianluigi

    2008-12-01

    Iterative image reconstruction algorithms for positron emission tomography (PET) require a sophisticated system matrix (model) of the scanner. Our aim is to set up such a model offline for the YAP-(S)PET II small animal imaging tomograph in order to use it subsequently with standard ML-EM (maximum-likelihood expectation maximization) and OSEM (ordered subset expectation maximization) for fully three-dimensional image reconstruction. In general, the system model can be obtained analytically, via measurements or via Monte Carlo simulations. In this paper, we present the multi-ray method, which can be considered as a hybrid method to set up the system model offline. It incorporates accurate analytical (geometric) considerations as well as crystal depth and crystal scatter effects. At the same time, it has the potential to model seamlessly other physical aspects such as the positron range. The proposed method is based on multiple rays which are traced from/to the detector crystals through the image volume. Such a ray-tracing approach itself is not new; however, we derive a novel mathematical formulation of the approach and investigate the positioning of the integration (ray-end) points. First, we study single system matrix entries and show that the positioning and weighting of the ray-end points according to Gaussian integration give better results compared to equally spaced integration points (trapezoidal integration), especially if only a small number of integration points (rays) are used. Additionally, we show that, for a given variance of the single matrix entries, the number of rays (events) required to calculate the whole matrix is a factor of 20 larger when using a pure Monte-Carlo-based method. Finally, we analyse the quality of the model by reconstructing phantom data from the YAP-(S)PET II scanner.

  19. Extracellular vesicles of calcifying turkey leg tendon characterized by immunocytochemistry and high voltage electron microscopic tomography and 3-D graphic image reconstruction

    NASA Technical Reports Server (NTRS)

    Landis, W. J.; Hodgens, K. J.; McKee, M. D.; Nanci, A.; Song, M. J.; Kiyonaga, S.; Arena, J.; McEwen, B.

    1992-01-01

    To gain insight into the structure and possible function of extracellular vesicles in certain calcifying vertebrate tissues, normally mineralizing leg tendons from the domestic turkey, Meleagris gallopavo, have been studied in two separate investigations, one concerning the electron microscopic immunolocalization of the 66 kDa phosphoprotein, osteopontin, and the other detailing the organization and distribution of mineral crystals associated with the vesicles as determined by high voltage microscopic tomography and 3-D graphic image reconstruction. Immunolabeling shows that osteopontin is related to extracellular vesicles of the tendon in the sense that its initial presence appears coincident with the development of mineral associated with the vesicle loci. By high voltage electron microscopy and 3-D imaging techniques, mineral crystals are found to consist of small irregularly shaped particles somewhat randomly oriented throughout individual vesicles sites. Their appearance is different from that found for the mineral observed within calcifying tendon collagen, and their 3-D disposition is not regularly ordered. Possible spatial and temporal relationships of vesicles, osteopontin, mineral, and collagen are being examined further by these approaches.

  20. Targeting the extracellular matrix: matricellular proteins regulate cell-extracellular matrix communication within distinct niches of the intervertebral disc.

    PubMed

    Bedore, Jake; Leask, Andrew; Séguin, Cheryle A

    2014-07-01

    The so-called "matricellular" proteins have recently emerged as important regulators of cell-extracellular matrix (ECM) interactions. These proteins modulate a variety of cell functions through a range of interactions with cell-surface receptors, hormones, proteases and structural components of the ECM. As such, matricellular proteins are crucial regulators of cell phenotype, and consequently tissue function. The distinct cell types and microenvironments that together form the IVD provide an excellent paradigm to study how matricellular proteins mediate communication within and between adjacent tissue types. In recent years, the role of several matricellular proteins in the intervertebral disc has been explored in vivo using mutant mouse models in which the expression of target matricellular proteins was deleted from either one or all compartments of the intervertebral disc. The current review outlines what is presently known about the roles of the matricellular proteins belonging to the CCN family, SPARC (Secreted Protein, Acidic, and Rich in Cysteine), and thrombospondin (TSP) 2 in regulating intervertebral disc cell-ECM interactions, ECM synthesis and disc tissue homeostasis using genetically modified mouse models. Furthermore, we provide a brief overview of recent preliminary studies of other matricellular proteins including, periostin (POSTN) and tenascin (TN). Each specific tissue type of the IVD contains a different matricellular protein signature, which varies based on the specific stage of development, maturity or disease. A growing body of direct genetic evidence links IVD development, maintenance and repair to the coordinate interaction of matricellular proteins within their respective niches and suggests that several of these signaling modulators hold promise in the development of diagnostics and/or therapeutics targeting intervertebral disc aging and/or degeneration.

  1. FERM3D: A finite element R-matrix electron molecule scattering code

    NASA Astrophysics Data System (ADS)

    Tonzani, Stefano

    2007-01-01

    FERM3D is a three-dimensional finite element program, for the elastic scattering of a low energy electron from a general polyatomic molecule, which is converted to a potential scattering problem. The code is based on tricubic polynomials in spherical coordinates. The electron-molecule interaction is treated as a sum of three terms: electrostatic, exchange, and polarization. The electrostatic term can be extracted directly from ab initio codes ( GAUSSIAN 98 in the work described here), while the exchange term is approximated using a local density functional. A local polarization potential based on density functional theory [C. Lee, W. Yang, R.G. Parr, Phys. Rev. B 37 (1988) 785] describes the long range attraction to the molecular target induced by the scattering electron. Photoionization calculations are also possible and illustrated in the present work. The generality and simplicity of the approach is important in extending electron-scattering calculations to more complex targets than it is possible with other methods. Program summaryTitle of program:FERM3D Catalogue identifier:ADYL_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/ADYL_v1_0 Program obtainable from: CPC Program Library, Queen's University of Belfast, N. Ireland Computer for which the program is designed and others on which it has been tested:Intel Xeon, AMD Opteron 64 bit, Compaq Alpha Operating systems or monitors under which the program has been tested:HP Tru64 Unix v5.1, Red Hat Linux Enterprise 3 Programming language used:Fortran 90 Memory required to execute with typical data:900 MB (neutral CO 2), 2.3 GB (ionic CO 2), 1.4 GB (benzene) No. of bits in a word:32 No. of processors used:1 Has the code been vectorized?:No No. of lines in distributed program, including test data, etc.:58 383 No. of bytes in distributed program, including test data, etc.:561 653 Distribution format:tar.gzip file CPC Program library subprograms used:ADDA, ACDP Nature of physical problem:Scattering of an

  2. Avoiding detrimental human immune response against Mammalian extracellular matrix implants.

    PubMed

    Galili, Uri

    2015-04-01

    This review describes the antibodies formed against mammalian extracellular matrix (ECM) implants in humans and proposes methods for avoiding the detrimental effects of these antibodies. There are two types of antibodies against ECM implants: (i) The natural anti-Gal antibody constituting ∼1% of immunoglobulins in humans. This antibody binds to a carbohydrate antigen called the α-gal epitope with the structure Galα1-3Galβ1-4GlcNAc-R. The α-gal epitope is abundant in nonprimate mammals, including on ECM proteins and proteoglycans. Moreover, anti-Gal antibody titers increase within 2-4 weeks by 10- to 100-folds in human recipients of mammalian implants or xenografts expressing α-gal epitopes. (ii) Anti-non gal antibodies formed against ECM peptide sequences differing from those in homologous proteins in humans. Most homologous proteins in mammals contain immunogenic peptides that elicit anti-non gal antibody production when introduced into humans. Formation of anti-non gal antibodies is much slower than that of elicited anti-Gal antibodies. Both anti-Gal and anti-non gal antibodies are detrimental to ECM implant regeneration in humans by binding to the ECM and directing extensive macrophage-mediated degradation of the implant. In addition, antibodies binding to ECM proteins/proteoglycans may hinder stem cells interaction with the ECM, which is required for directing stem cell differentiation. The anti-Gal immunological barrier can be avoided by using mammalian ECM implants lacking α-gal epitopes. Such implants can be engineered by enzymatic destruction of α-gal epitopes with recombinant α-galactosidase. Alternatively, implants may be obtained from α1,3galactosyltransferase knockout donor species that lack α-gal epitopes. Since postimplantation production of anti-non gal antibodies is a slow process, the detrimental effects of these antibodies may be avoided by accelerating stem cells recruitment into implants, thus accelerating the regeneration process

  3. Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate

    PubMed Central

    Leight, Jennifer L.; Alge, Daniel L.; Maier, Andrew J.; Anseth, Kristi S.

    2014-01-01

    Incorporation of degradable moieties into synthetic hydrogels has greatly increased the utility of these three-dimensional matrices for in vitro cell culture as well as tissue engineering applications. A common method for introducing degradability is the inclusion of oligopeptides sensitive to cleavage by matrix metalloproteinases (MMPs), enabling cell-mediated remodeling and migration within the material. While this strategy has been effective, characterization and measurement of cell-mediated degradation in these materials has remained challenging. There are 20+ MMP family members whose activity is regulated in space and time by a number of biochemical and biophysical cues. Thus, the typical approach of characterizing cleavage of degradable moieties in solution with recombinant enzymes does not easily translate to three dimensional cell-mediated matrix remodeling. To address this challenge, we report here the synthesis of a cell-laden hydrogel matrix functionalized with a fluorogenic peptide substrate to provide real-time, quantitative monitoring of global MMP activity. Using this system, stimulation of MMP activity was observed with growth factor treatment in mammary epithelial cells and compared to classical zymography results. Further, the effect of biophysical cues on MMP activity of human mesenchymal stem cells was also investigated where more rigid hydrogels were observed to increase MMP activity. The regulation of MMP activity by these biochemical and biophysical cues highlights the need for in situ, real time measurement of hydrogel degradation, and use of these functionalized hydrogels will aid in future rational design of degradable synthetic hydrogels for in vitro cell studies and tissue engineering applications. PMID:23830581

  4. Shear resonance mode decoupling to determine the characteristic matrix of piezoceramics for 3-D modeling.

    PubMed

    Pardo, Lorena; García, Alvaro; de Espinosa, Francisco Montero; Brebøl, Klaus

    2011-03-01

    The determination of the characteristic frequencies of an electromechanical resonance does not provide enough data to obtain the material properties of piezoceramics, including all losses, from complex impedance measurements. Values of impedance around resonance and antiresonance frequencies are also required to calculate the material losses. Uncoupled resonances are needed for this purpose. The shear plates used for the material characterization present unavoidable mode coupling of the shear mode and other modes of the plate. A study of the evolution of the complex material coefficients as the coupling of modes evolves with the change in the aspect ratio (lateral dimension/thickness) of the plate is presented here. These are obtained using software. A soft commercial PZT ceramic was used in this study and several shear plates amenable to material characterization were obtained in the range of aspect ratios below 15. The validity of the material properties for 3-D modeling of piezoceramics is assessed by means of finite element analysis, which shows that uncoupled resonances are virtually pure thickness-driven shear modes.

  5. Extracellular matrix remodelling in response to venous hypertension: proteomics of human varicose veins

    PubMed Central

    Barallobre-Barreiro, Javier; Oklu, Rahmi; Lynch, Marc; Fava, Marika; Baig, Ferheen; Yin, Xiaoke; Barwari, Temo; Potier, David N.; Albadawi, Hassan; Jahangiri, Marjan; Porter, Karen E.; Watkins, Michael T.; Misra, Sanjay; Stoughton, Julianne; Mayr, Manuel

    2016-01-01

    Aims Extracellular matrix remodelling has been implicated in a number of vascular conditions, including venous hypertension and varicose veins. However, to date, no systematic analysis of matrix remodelling in human veins has been performed. Methods and results To understand the consequences of venous hypertension, normal and varicose veins were evaluated using proteomics approaches targeting the extracellular matrix. Varicose saphenous veins removed during phlebectomy and normal saphenous veins obtained during coronary artery bypass surgery were collected for proteomics analysis. Extracellular matrix proteins were enriched from venous tissues. The proteomics analysis revealed the presence of >150 extracellular matrix proteins, of which 48 had not been previously detected in venous tissue. Extracellular matrix remodelling in varicose veins was characterized by a loss of aggrecan and several small leucine-rich proteoglycans and a compensatory increase in collagen I and laminins. Gene expression analysis of the same tissues suggested that the remodelling process associated with venous hypertension predominantly occurs at the protein rather than the transcript level. The loss of aggrecan in varicose veins was paralleled by a reduced expression of aggrecanases. Chymase and tryptase β1 were among the up-regulated proteases. The effect of these serine proteases on the venous extracellular matrix was further explored by incubating normal saphenous veins with recombinant enzymes. Proteomics analysis revealed extensive extracellular matrix degradation after digestion with tryptase β1. In comparison, chymase was less potent and degraded predominantly basement membrane-associated proteins. Conclusion The present proteomics study provides unprecedented insights into the expression and degradation of structural and regulatory components of the vascular extracellular matrix in varicosis. PMID:27068509

  6. Multi-scale Characterisation of the 3D Microstructure of a Thermally-Shocked Bulk Metallic Glass Matrix Composite.

    PubMed

    Zhang, Wei; Bodey, Andrew J; Sui, Tan; Kockelmann, Winfried; Rau, Christoph; Korsunsky, Alexander M; Mi, Jiawei

    2016-01-01

    Bulk metallic glass matrix composites (BMGMCs) are a new class of metal alloys which have significantly increased ductility and impact toughness, resulting from the ductile crystalline phases distributed uniformly within the amorphous matrix. However, the 3D structures and their morphologies of such composite at nano and micrometre scale have never been reported before. We have used high density electric currents to thermally shock a Zr-Ti based BMGMC to different temperatures, and used X-ray microtomography, FIB-SEM nanotomography and neutron diffraction to reveal the morphologies, compositions, volume fractions and thermal stabilities of the nano and microstructures. Understanding of these is essential for optimizing the design of BMGMCs and developing viable manufacturing methods. PMID:26725519

  7. Multi-scale Characterisation of the 3D Microstructure of a Thermally-Shocked Bulk Metallic Glass Matrix Composite

    NASA Astrophysics Data System (ADS)

    Zhang, Wei; Bodey, Andrew J.; Sui, Tan; Kockelmann, Winfried; Rau, Christoph; Korsunsky, Alexander M.; Mi, Jiawei

    2016-01-01

    Bulk metallic glass matrix composites (BMGMCs) are a new class of metal alloys which have significantly increased ductility and impact toughness, resulting from the ductile crystalline phases distributed uniformly within the amorphous matrix. However, the 3D structures and their morphologies of such composite at nano and micrometre scale have never been reported before. We have used high density electric currents to thermally shock a Zr-Ti based BMGMC to different temperatures, and used X-ray microtomography, FIB-SEM nanotomography and neutron diffraction to reveal the morphologies, compositions, volume fractions and thermal stabilities of the nano and microstructures. Understanding of these is essential for optimizing the design of BMGMCs and developing viable manufacturing methods.

  8. Multi-scale Characterisation of the 3D Microstructure of a Thermally-Shocked Bulk Metallic Glass Matrix Composite

    PubMed Central

    Zhang, Wei; Bodey, Andrew J.; Sui, Tan; Kockelmann, Winfried; Rau, Christoph; Korsunsky, Alexander M.; Mi, Jiawei

    2016-01-01

    Bulk metallic glass matrix composites (BMGMCs) are a new class of metal alloys which have significantly increased ductility and impact toughness, resulting from the ductile crystalline phases distributed uniformly within the amorphous matrix. However, the 3D structures and their morphologies of such composite at nano and micrometre scale have never been reported before. We have used high density electric currents to thermally shock a Zr-Ti based BMGMC to different temperatures, and used X-ray microtomography, FIB-SEM nanotomography and neutron diffraction to reveal the morphologies, compositions, volume fractions and thermal stabilities of the nano and microstructures. Understanding of these is essential for optimizing the design of BMGMCs and developing viable manufacturing methods. PMID:26725519

  9. Enzyme-Immobilized 3D-Printed Reactors for Online Monitoring of Rat Brain Extracellular Glucose and Lactate.

    PubMed

    Su, Cheng-Kuan; Yen, Shuo-Chih; Li, Tzu-Wen; Sun, Yuh-Chang

    2016-06-21

    In this study we constructed a highly sensitive system for in vivo monitoring of the concentrations of rat brain extracellular glucose and lactate. This system involved microdialysis (MD) sampling and fluorescence determination in conjunction with a novel sample derivatization scheme in which glucose oxidase and lactate oxidase were immobilized in ABS flow bioreactors (manufactured through low-cost three-dimensional printing (3DP)), via fused deposition modeling, for online oxidization of sampled glucose and lactate, respectively, in rat brain microdialysate. After optimizing the experimental conditions for MD sampling, the manufacture of the designed flow reactors, the enzyme immobilization procedure, and the online derivatization scheme, the available sampling frequency was 15 h(-1) and the system's detection limits reached as low as 0.060 mM for glucose and 0.059 mM for lactate, based on a 20-μL conditioned microdialysate; these characteristics were sufficient to reliably determine the concentrations of extracellular glucose and lactate in the brains of living rats. To demonstrate the system's applicability, we performed (i) spike analyses of offline-collected rat brain microdialysate and (ii) in vivo dynamic monitoring of the extracellular glucose and lactate in living rat brains, in addition to triggering neuronal depolarization by perfusing a high-K(+) medium from the implanted MD probe. Our analytical results and demonstrations confirm that postprinting functionalization of analytical devices manufactured using 3DP technology can be a powerful strategy for extending the diversity and adaptability of currently existing analytical configurations. PMID:27232384

  10. Enzyme-Immobilized 3D-Printed Reactors for Online Monitoring of Rat Brain Extracellular Glucose and Lactate.

    PubMed

    Su, Cheng-Kuan; Yen, Shuo-Chih; Li, Tzu-Wen; Sun, Yuh-Chang

    2016-06-21

    In this study we constructed a highly sensitive system for in vivo monitoring of the concentrations of rat brain extracellular glucose and lactate. This system involved microdialysis (MD) sampling and fluorescence determination in conjunction with a novel sample derivatization scheme in which glucose oxidase and lactate oxidase were immobilized in ABS flow bioreactors (manufactured through low-cost three-dimensional printing (3DP)), via fused deposition modeling, for online oxidization of sampled glucose and lactate, respectively, in rat brain microdialysate. After optimizing the experimental conditions for MD sampling, the manufacture of the designed flow reactors, the enzyme immobilization procedure, and the online derivatization scheme, the available sampling frequency was 15 h(-1) and the system's detection limits reached as low as 0.060 mM for glucose and 0.059 mM for lactate, based on a 20-μL conditioned microdialysate; these characteristics were sufficient to reliably determine the concentrations of extracellular glucose and lactate in the brains of living rats. To demonstrate the system's applicability, we performed (i) spike analyses of offline-collected rat brain microdialysate and (ii) in vivo dynamic monitoring of the extracellular glucose and lactate in living rat brains, in addition to triggering neuronal depolarization by perfusing a high-K(+) medium from the implanted MD probe. Our analytical results and demonstrations confirm that postprinting functionalization of analytical devices manufactured using 3DP technology can be a powerful strategy for extending the diversity and adaptability of currently existing analytical configurations.

  11. Targeted rehabilitation after extracellular matrix scaffold transplantation for the treatment of volumetric muscle loss.

    PubMed

    Gentile, Natalie E; Stearns, Kristen M; Brown, Elke H P; Rubin, J Peter; Boninger, Michael L; Dearth, Christopher L; Ambrosio, Fabrisia; Badylak, Stephen F

    2014-11-01

    Rehabilitation therapy is an important aspect of recovery after volumetric muscle loss. However, the traditional rehabilitation approach involves a period of rest and passive loading followed by gradual active loading. Extracellular matrix is a naturally occurring material consisting of structural proteins that provide mechanical strength, structural support, and functional molecules with diverse bioactive properties. There is evidence to suggest that the addition of aggressive regenerative rehabilitation protocols immediately after surgical implantation of an extracellular matrix scaffold to an area of volumetric muscle loss has significant benefits for extracellular matrix remodeling. Rehabilitation exercises likely provide the needed mechanical signals to encourage cell migration and site-specific differentiation in the temporal framework required for constructive remodeling. Herein, the authors review the literature and present an example of an aggressive rehabilitation program implemented immediately after extracellular matrix transplantation into a severely injured quadriceps muscle.

  12. Fibulin's organization into the extracellular matrix of fetal lung fibroblasts is dependent on fibronectin matrix assembly.

    PubMed

    Roman, J; McDonald, J A

    1993-05-01

    Fibulin is a newly described extracellular matrix (ECM) glycoprotein whose function has not been elucidated. We have observed that cultured fetal lung fibroblasts produce fibulin and have postulated that its expression may be important during lung development. To begin to understand the potential function of fibulin in lung development, we examined its expression and distribution in cultured fetal lung fibroblasts. Immunofluorescence staining of cultured fibroblasts revealed that fibulin was distributed upon their surface in a fibrillar array resembling fibronectin (FN), another ECM glycoprotein expressed by fetal lung fibroblasts and implicated in lung and heart development. Detection of fibulin by immunofluorescence staining of nonpermeabilized cells, its immunoprecipitation from 125I-cell surface-labeled fibroblasts, pulse-chase analysis, and temperature-induced phase separation studies revealed that fibulin is an ECM peripheral membrane protein that is synthesized and secreted by cultured fetal lung fibroblasts shortly after plating and incorporated into their matrix in a divalent cation-dependent manner. Because fibulin co-distributes with both FN and the FN receptor, the integrin alpha 5 beta 1, we examined the possibility that fibulin was interacting with both components. Dissociation of FN receptors from FN fibers with anti-FN receptor antibodies did not affect fibulin's distribution, suggesting that fibulin binds FN and that this interaction is not affected by the state of FN receptor binding. Finally, inhibition of FN matrix assembly prevented the deposition of fibulin, providing further support for FN-fibulin interactions and suggesting that fibulin deposition is dependent on FN matrix assembly. PMID:8481235

  13. Probing cell-matrix interactions in RGD-decorated macroporous poly (ethylene glycol) hydrogels for 3D chondrocyte culture.

    PubMed

    Zhang, Jingjing; Mujeeb, Ayeesha; Du, Yanan; Lin, Jianhao; Ge, Zigang

    2015-06-01

    Macroporous hydrogels have shown great promise as scaffolds for cartilage engineering by facilitating nutrition transport and tissue in growth. Cell-matrix adhesion-a fundamental process in tissue engineering-has shown a profound effect on subsequent cell phenotype, extracellular matrix (ECM) accumulation, and tissue reorganization. In this study, arginine-glycine-aspartic acid (RGD) was introduced to macroporous hydrogels of poly (ethylene glycol) (PEG) to fabricate PEG-G400 (with 0.4mM RGD) and PEG-G2000 (2mM RGD) to probe the cell-matrix interactions within hydrogels. Primary chondrocytes demonstrated a slightly stretched morphology with increasing RGD concentration and PEG-G2000 hydrogels boosted cell viability, proliferation, and deposition of collagen II and GAG, in comparison to the PEG-G400 and PEG-RED groups. Results also revealed chondrocytes within the cell aggregates underwent dedifferentiation and hypertrophy within RGD incorporated hydrogels, as evidenced by the high level of gene expression of collagen I on day 14 and strong immunohistological staining of collagen X and collagen I on day 35. Evidently, a high concentration of RGD (2mM RGD) enhanced cell-matrix interactions through elevating the expression of integrin β1 and vinculin. Thus, the integration of RGD in macroporous hydrogels with a concentration of 2 mM may be sufficient for improving cell functionality, with a slight probability of dedifferentiation and hypertrophy of chondrocytes. PMID:26107534

  14. Modifications in stromal extracellular matrix of aged corneas can be induced by ultraviolet A irradiation

    PubMed Central

    Gendron, Sébastien P; Rochette, Patrick J

    2015-01-01

    With age, structural and functional changes can be observed in human cornea. Some studies have shown a loss of corneal transparency and an increase in turbidity associated with aging. These changes are caused by modifications in the composition and arrangement of extracellular matrix in the corneal stroma. In human skin, it is well documented that exposure to solar radiation, and mainly to the UVA wavelengths, leads to phenotypes of photoaging characterized by alteration in extracellular matrix of the dermis. Although the cornea is also exposed to solar radiation, the extracellular matrix modifications observed in aging corneas have been mainly attributed to chronological aging and not to solar exposure. To ascertain the real implication of UVA exposure in extracellular matrix changes observed with age in human cornea, we have developed a model of photoaging by chronically exposing corneal stroma keratocytes with a precise UVA irradiation protocol. Using this model, we have analyzed UVA-induced transcriptomic and proteomic changes in corneal stroma. Our results show that cumulative UVA exposure causes changes in extracellular matrix that are found in corneal stromas of aged individuals, suggesting that solar exposure catalyzes corneal aging. Indeed, we observe a downregulation of collagen and proteoglycan gene expression and a reduction in proteoglycan production and secretion in response to cumulative UVA exposure. This study provides the first evidence that chronic ocular exposure to sunlight affects extracellular matrix composition and thus plays a role in corneal changes observed with age. PMID:25728164

  15. Interaction between Cartilage Oligomeric Matrix Protein and Extracellular Matrix Protein 1 Mediates Endochondral Bone Growth

    PubMed Central

    Kong, Li; Tian, Qingyun; Guo, Fengjin; Mucignat, Maria T.; Perris, Roberto; Sercu, Sandy; Merregaert, Joseph; Di Cesare, Paul E.; Liu, Chuan-ju

    2010-01-01

    In an effort to define the biological functions of COMP, a functional genetic screen was performed. This led to the identification of extracellular matrix protein 1 (ECM1) as a novel COMP-associated partner. COMP directly binds to ECM1 both in vitro and in vivo. The EGF domain of COMP and the C-terminus of ECM1 mediate the interaction between them. COMP and ECM1 Colocalize in the Growth Plates in Vivo. ECM1 inhibits chondrocyte hypertrophy, matrix mineralization, and endochondral bone formation, and COMP overcomes the inhibition by ECM1. In addition, COMP-mediated neutralization of ECM1 inhibition depends on their interaction, since COMP largely fails to overcome the ECM1 inhibition in the presence of the EGF domain of COMP, which disturbs the association of COMP and ECM1. These findings provide the first evidence linking the association of COMP and ECM1 and the biological significance underlying the interaction between them in regulating endochondral bone growth. PMID:20138147

  16. Cellular contractility and extracellular matrix stiffness regulate matrix metalloproteinase activity in pancreatic cancer cells.

    PubMed

    Haage, Amanda; Schneider, Ian C

    2014-08-01

    The pathogenesis of cancer is often driven by local invasion and metastasis. Recently, mechanical properties of the tumor microenvironment have been identified as potent regulators of invasion and metastasis, while matrix metalloproteinases (MMPs) are classically known as significant enhancers of cancer cell migration and invasion. Here we have been able to sensitively measure MMP activity changes in response to specific extracellular matrix (ECM) environments and cell contractility states. Cells of a pancreatic cancer cell line, Panc-1, up-regulate MMP activities between 3- and 10-fold with increased cell contractility. Conversely, they down-regulate MMP activities when contractility is blocked to levels seen with pan-MMP activity inhibitors. Similar, albeit attenuated, responses are seen in other pancreatic cancer cell lines, BxPC-3 and AsPC-1. In addition, MMP activity was modulated by substrate stiffness, collagen gel concentration, and the degree of collagen cross-linking, when cells were plated on collagen gels ranging from 0.5 to 5 mg/ml that span the physiological range of substrate stiffness (50-2000 Pa). Panc-1 cells showed enhanced MMP activity on stiffer substrates, whereas BxPC-3 and AsPC-1 cells showed diminished MMP activity. In addition, eliminating heparan sulfate proteoglycans using heparinase completely abrogated the mechanical induction of MMP activity. These results demonstrate the first functional link between MMP activity, contractility, and ECM stiffness and provide an explanation as to why stiffer environments result in enhanced cell migration and invasion.

  17. Extracellular Matrix-Based Biohybrid Materials for Engineering Compliant, Matrix-Dense Tissues.

    PubMed

    Bracaglia, Laura G; Fisher, John P

    2015-11-18

    An ideal tissue engineering scaffold should not only promote, but take an active role in, constructive remodeling and formation of site appropriate tissue. Extracellular matrix (ECM)-derived proteins provide unmatched cellular recognition, and therefore influence cellular response towards predicted remodeling behaviors. Materials built with only these proteins, however, can degrade rapidly or begin too weak to substitute for compliant, matrix-dense tissues. The focus of this Progress Report is on biohybrid materials that incorporate polymer components with ECM-derived proteins, to produce a substrate with desired mechanical and degradation properties, as well as actively guide tissue remodeling. Materials are described through four fabrication methods: 1) polymer and ECM-protein fibers woven together, 2) polymer and ECM proteins combined in a bilayer, 3) cell-built ECM on polymer scaffold, and 4) ECM proteins and polymers combined in a single hydrogel. Scaffolds from each fabrication method can achieve characteristics suitable for different types of tissue. In vivo testing has shown progressive remodeling in injury models, and suggests ECM-based biohybrid materials promote a prohealing immune response over single component alternatives. The prohealing immune response is associated with lasting success and long term host maintenance of the implant.

  18. Monocytes increase human cardiac myofibroblast-mediated extracellular matrix remodeling through TGF-β1.

    PubMed

    Mewhort, Holly E M; Lipon, Brodie D; Svystonyuk, Daniyil A; Teng, Guoqi; Guzzardi, David G; Silva, Claudia; Yong, V Wee; Fedak, Paul W M

    2016-03-15

    Following myocardial infarction (MI), cardiac myofibroblasts remodel the extracellular matrix (ECM), preventing mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis, and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity; however, the mechanisms are unclear. We assessed the influence of peripheral blood monocytes on human cardiac myofibroblast activity in a three-dimensional (3D) ECM microenvironment. Human cardiac myofibroblasts isolated from surgical biopsies of the right atrium and left ventricle were seeded into 3D collagen matrices. Peripheral blood monocytes were isolated from healthy human donors and cocultured with myofibroblasts. Monocytes increased myofibroblast activity measured by collagen gel contraction (baseline: 57.6 ± 5.9% vs. coculture: 65.2 ± 7.1% contraction; P < 0.01) and increased local ECM remodeling quantified by confocal microscopy. Under coculture conditions that allow indirect cellular interaction via paracrine factors but prevent direct cell-cell contact, monocytes had minimal effects on myofibroblast activity (17.9 ± 11.1% vs. 6.4 ± 7.0% increase, respectively; P < 0.01). When cells were cultured under direct contact conditions, multiplex analysis of the coculture media revealed an increase in the paracrine factors TGF-β1 and matrix metalloproteinase 9 compared with baseline (122.9 ± 10.1 pg/ml and 3,496.0 ± 190.4 pg/ml, respectively, vs. 21.5 ± 16.3 pg/ml and 183.3 ± 43.9 pg/ml; P < 0.001). TGF-β blockade abolished the monocyte-induced increase in cardiac myofibroblast activity. These data suggest that direct cell-cell interaction between monocytes and cardiac myofibroblasts stimulates TGF-β-mediated myofibroblast activity and increases remodeling of local matrix. Peripheral blood monocyte interaction with human cardiac myofibroblasts stimulates myofibroblast activity through release of TGF-β1

  19. Tumor cell-driven extracellular matrix remodeling drives haptotaxis during metastatic progression

    PubMed Central

    Oudin, Madeleine J.; Jonas, Oliver; Kosciuk, Tatsiana; Broye, Liliane C.; Guido, Bruna C.; Wyckoff, Jeff; Riquelme, Daisy; Lamar, John M.; Asokan, Sreeja B.; Whittaker, Charlie; Ma, Duanduan; Langer, Robert; Cima, Michael J.; Wisinski, Kari B.; Hynes, Richard O.; Lauffenburger, Douglas A.; Keely, Patricia J.; Bear, James E.; Gertler, Frank B.

    2016-01-01

    Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo. Haptotaxis on FN gradients requires direct interaction between α5β1 integrin and Mena, an actin regulator, and involves increases in focal complex signaling and tumor-cell-mediated extracellular matrix (ECM) remodeling. Compared to Mena, higher levels of the pro-metastatic MenaINV isoform associate with α5, which enables 3D haptotaxis of tumor cells towards the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MenaINV and FN levels were correlated in two breast cancer cohorts, and high levels of MenaINV were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor-cell-intrinsic mechanism that promotes metastasis through ECM remodeling and ECM guided directional migration. PMID:26811325

  20. Extracellular Matrix Components Regulate Cellular Polarity and Tissue Structure in the Developing and Mature Retina

    PubMed Central

    Varshney, Shweta; Hunter, Dale D.; Brunken, William J.

    2015-01-01

    While genetic networks and other intrinsic mechanisms regulate much of retinal development, interactions with the extracellular environment shape these networks and modify their output. The present review has focused on the role of one family of extracellular matrix molecules and their signaling pathways in retinal development. In addition to their effects on the developing retina, laminins play a role in maintaining Müller cell polarity and compartmentalization, thereby contributing to retinal homeostasis. This article which is intended for the clinical audience, reviews the fundamentals of retinal development, extracellular matrix organization and the role of laminins in retinal development. The role of laminin in cortical development is also briefly discussed. PMID:26730321

  1. Ultrastructural localization of extracellular matrix proteins in liver biopsies using ultracryomicrotomy and immuno-gold labelling.

    PubMed

    Burt, A D; Griffiths, M R; Schuppan, D; Voss, B; MacSween, R N

    1990-01-01

    We describe a technique for the localization of extracellular matrix proteins in wedge and needle biopsy specimens of human liver. Using ultra-thin (50-70 nm) sections of puncture perfusion fixed tissue, extracellular matrix proteins were localized using a protein A-gold labelling procedure. We obtained good preservation of ultrastructural detail and specific labelling for collagen types I, III and IV and fibronectin. The method represents a sensitive means of identifying the extracellular distribution and the cellular origin of these proteins in normal and diseased human liver.

  2. Comparison of basis functions for 3D PET reconstruction using a Monte Carlo system matrix

    NASA Astrophysics Data System (ADS)

    Cabello, Jorge; Rafecas, Magdalena

    2012-04-01

    In emission tomography, iterative statistical methods are accepted as the reconstruction algorithms that achieve the best image quality. The accuracy of these methods relies partly on the quality of the system response matrix (SRM) that characterizes the scanner. The more physical phenomena included in the SRM, the higher the SRM quality, and therefore higher image quality is obtained from the reconstruction process. High-resolution small animal scanners contain as many as 103-104 small crystal pairs, while the field of view (FOV) is divided into hundreds of thousands of small voxels. These two characteristics have a significant impact on the number of elements to be calculated in the SRM. Monte Carlo (MC) methods have gained popularity as a way of calculating the SRM, due to the increased accuracy achievable, at the cost of introducing some statistical noise and long simulation times. In the work presented here the SRM is calculated using MC methods exploiting the cylindrical symmetries of the scanner, significantly reducing the simulation time necessary to calculate a high statistical quality SRM and the storage space necessary. The use of cylindrical symmetries makes polar voxels a convenient basis function. Alternatively, spherically symmetric basis functions result in improved noise properties compared to cubic and polar basis functions. The quality of reconstructed images using polar voxels, spherically symmetric basis functions on a polar grid, cubic voxels and post-reconstruction filtered polar and cubic voxels is compared from a noise and spatial resolution perspective. This study demonstrates that polar voxels perform as well as cubic voxels, reducing the simulation time necessary to calculate the SRM and the disk space necessary to store it. Results showed that spherically symmetric functions outperform polar and cubic basis functions in terms of noise properties, at the cost of slightly degraded spatial resolution, larger SRM file size and longer

  3. The Synergistic Effects of Matrix Stiffness and Composition on the Response of Chondroprogenitor Cells in a 3D Precondensation Microenvironment.

    PubMed

    Carrion, Bita; Souzanchi, Mohammad F; Wang, Victor T; Tiruchinapally, Gopinath; Shikanov, Ariella; Putnam, Andrew J; Coleman, Rhima M

    2016-05-01

    Improve functional quality of cartilage tissue engineered from stem cells requires a better understanding of the functional evolution of native cartilage tissue. Therefore, a biosynthetic hydrogel was developed containing RGD, hyaluronic acid and/or type-I collagen conjugated to poly(ethylene glycol) acrylate to recapitulate the precondensation microenvironment of the developing limb. Conjugation of any combination of the three ligands did not alter the shear moduli or diffusion properties of the PEG hydrogels; thus, the influence of ligand composition on chondrogenesis could be investigated in the context of varying matrix stiffness. Gene expression of ligand receptors (CD44 and the b1-integrin) as well as markers of condensation (cell clustering and N-cadherin gene expression) and chondrogenesis (Col2a1 gene expression and sGAG production) by chondroprogenitor cells in this system were modulated by both matrix stiffness and ligand composition, with the highest gene expression occurring in softer hydrogels containing all three ligands. Cell proliferation in these 3D matrices for 7 d prior to chondrogenic induction increased the rate of sGAG production in a stiffness-dependent manner. This biosynthetic hydrogel supports the features of early limb-bud condensation and chondrogenesis and is a novel platform in which the influence of the matrix physicochemical properties on these processes can be elucidated.

  4. Three-Dimensional Adult Cardiac Extracellular Matrix Promotes Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    PubMed

    Fong, Ashley H; Romero-López, Mónica; Heylman, Christopher M; Keating, Mark; Tran, David; Sobrino, Agua; Tran, Anh Q; Pham, Hiep H; Fimbres, Cristhian; Gershon, Paul D; Botvinick, Elliot L; George, Steven C; Hughes, Christopher C W

    2016-08-01

    Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular, human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency, their self-renewal potential, and their ability to create patient-specific cell lines. Unfortunately, pluripotent stem cell-derived CMs are immature, with characteristics more closely resembling fetal CMs than adult CMs, and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation, as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold, compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes, Junctin, CaV1.2, NCX1, HCN4, SERCA2a, Triadin, and CASQ2. Consistent with this, we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine, likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together, these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease. PMID:27392582

  5. Influence of Cartilage Extracellular Matrix Molecules on Cell Phenotype and Neocartilage Formation

    PubMed Central

    Grogan, Shawn P.; Chen, Xian; Sovani, Sujata; Taniguchi, Noboru; Colwell, Clifford W.; Lotz, Martin K.

    2014-01-01

    Interaction between chondrocytes and the cartilage extracellular matrix (ECM) is essential for maintaining the cartilage's role as a low-friction and load-bearing tissue. In this study, we examined the influence of cartilage zone-specific ECM on human articular chondrocytes (HAC) in two-dimensional and three-dimensional (3D) environments. Two culture systems were used. SYSTEM 1: HAC were cultured on cell-culture plates that had been precoated with the following ECM molecules for 7 days: decorin, biglycan, tenascin C (superficial zone), collagen type II, hyaluronan (HA) (middle and deep zones), and osteopontin (deep zone). Uncoated standard culture plates were used as controls. Expanded cells were examined for phenotypic changes using real-time polymerase chain reaction. In addition, expanded cells were placed into high-density pellet cultures for 14 days. Neocartilage formation was assessed via gene expression and histology evaluations. SYSTEM 2: HAC that were cultured on untreated plates and encapsulated in a 3D alginate scaffold were mixed with one of the zone-specific ECM molecules. Cell viability, gene expression, and histology assessments were conducted on 14-day-old tissues. In HAC monolayer culture, exposure to decorin, HA, and osteopontin increased COL2A1 and aggrecan messenger RNA (mRNA) levels compared with controls. Biglycan up-regulated aggrecan without a significant impact on COL2A1 expression; Tenascin C reduced COL2A1 expression. Neocartilage formed after preculture on tenascin C and collagen type II expressed higher COL2A1 mRNA compared with control pellets. Preculture of HAC on HA decreased both COL2A1 and aggrecan expression levels compared with controls, which was consistent with histology. Reduced proteoglycan 4 (PRG4) mRNA levels were observed in HAC pellets that had been precultured with biglycan and collagen type II. Exposing HAC to HA directly in 3D-alginate culture most effectively induced neocartilage formation, showing increased COL2A1

  6. The Matrix Reloaded: How Sensing the Extracellular Matrix Synchronizes Bacterial Communities

    PubMed Central

    Steinberg, Nitai

    2015-01-01

    In response to chemical communication, bacterial cells often organize themselves into complex multicellular communities that carry out specialized tasks. These communities are frequently referred to as biofilms, which involve the collective behavior of different cell types. Like cells of multicellular eukaryotes, the biofilm cells are surrounded by self-produced polymers that constitute the extracellular matrix (ECM), which binds them to each other and to the surface. In multicellular eukaryotes, it has been evident for decades that cell-ECM interactions control multiple cellular processes during development. While cells both in biofilms and in multicellular eukaryotes are surrounded by ECM and activate various genetic programs, until recently it has been unclear whether cell-ECM interactions are recruited in bacterial communicative behaviors. In this review, we describe the examples reported thus far for ECM involvement in control of cell behavior throughout the different stages of biofilm formation. The studies presented in this review have provided a newly emerging perspective of the bacterial ECM as an active player in regulation of biofilm development. PMID:25825428

  7. Angiogenic Type I Collagen Extracellular Matrix Integrated with Recombinant Bacteriophages Displaying Vascular Endothelial Growth Factors.

    PubMed

    Yoon, Junghyo; Korkmaz Zirpel, Nuriye; Park, Hyun-Ji; Han, Sewoon; Hwang, Kyung Hoon; Shin, Jisoo; Cho, Seung-Woo; Nam, Chang-Hoon; Chung, Seok

    2016-01-21

    Here, a growth-factor-integrated natural extracellular matrix of type I collagen is presented that induces angiogenesis. The developed matrix adapts type I collagen nanofibers integrated with synthetic colloidal particles of recombinant bacteriophages that display vascular endothelial growth factor (VEGF). The integration is achieved during or after gelation of the type I collagen and the matrix enables spatial delivery of VEGF into a desired region. Endothelial cells that contact the VEGF are found to invade into the matrix to form tube-like structures both in vitro and in vivo, proving the angiogenic potential of the matrix.

  8. Extracellular Matrix and Regenerative Therapies from the Cardiac Perspective.

    PubMed

    Dogan, Arin; Parmaksız, Mahmut; Elçin, A Eser; Elçin, Y Murat

    2016-04-01

    Cardiovascular diseases are the leading cause of death and a major cause of financial burden. Regenerative therapies for heart diseases bring the promise of alternative treatment modalities for myocardial infarction, ischemic heart disease, and congestive heart failure. Although, clinical trials attest to the safety of stem cell injection therapies, researchers need to overcome the underlying mechanisms that are limiting the success of future regenerative options. This article aims to review the basic scientific concepts in the field of mechanobiology and the effects of extracellular functions on stem cell fate. PMID:26668014

  9. Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment.

    PubMed

    Bush, Katie A; Downing, Brett R; Walsh, Sarah E; Pins, George D

    2007-02-01

    The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.

  10. Formation of atypical podosomes in extravillous trophoblasts regulates extracellular matrix degradation

    PubMed Central

    Patel, Anand; Dash, Philip R.

    2012-01-01

    Throughout pregnancy the cytotrophoblast, the stem cell of the placenta, gives rise to the differentiated forms of trophoblasts. The two main cell lineages are the syncytiotrophoblast and the invading extravillous trophoblast. A successful pregnancy requires extravillous trophoblasts to migrate and invade through the decidua and then remodel the maternal spiral arteries. Many invasive cells use specialised cellular structures called invadopodia or podosomes in order to degrade extracellular matrix. Despite being highly invasive cells, the presence of invadapodia or podosomes has not previously been investigated in trophoblasts. In this study these structures have been identified and characterised in extravillous trophoblasts. The role of specialised invasive structures in trophoblasts in the degradation of the extracellular matrix was compared with well characterised podosomes and invadopodia in other invasive cells and the trophoblast specific structures were characterised by using a sensitive matrix degradation assay which enabled visualisation of the structures and their dynamics. We show trophoblasts form actin rich protrusive structures which have the ability to degrade the extracellular matrix during invasion. The degradation ability and dynamics of the structures closely resemble podosomes, but have unique characteristics that have not previously been described in other cell types. The composition of these structures does not conform to the classic podosome structure, with no distinct ring of plaque proteins such as paxillin or vinculin. In addition, trophoblast podosomes protrude more deeply into the extracellular matrix than established podosomes, resembling invadopodia in this regard. We also show several significant pathways such as Src kinase, MAPK kinase and PKC along with MMP-2 and 9 as key regulators of extracellular matrix degradation activity in trophoblasts, while podosome activity was regulated by the rigidity of the extracellular matrix. PMID

  11. Role of cell-matrix interactions on VIC phenotype and tissue deposition in 3D PEG hydrogels.

    PubMed

    Gould, Sarah T; Anseth, Kristi S

    2013-10-16

    Valvular interstitial cells (VICs) respond to 3D matrix interactions in a complex manner, but understanding these effects on VIC function better is important for applications ranging from valve tissue engineering to studying valve disease. Here, we encapsulated VICs in poly(ethylene glycol) (PEG) hydrogels modified with three different adhesive ligands, derived from fibronectin (RGDS), elastin (VGVAPG) and collagen-1 (P15). By day 14, VICs became significantly more elongated in RGDS-containing gels compared to VGVAPG or P15. This difference in cell morphology appeared to correlate with global matrix metalloproteinase (MMP) activity, as VICs encapsulated in RGDS-functionalized hydrogels secreted higher levels of active MMP at day 2. VIC activation to a myofibroblast phenotype was also characterized by staining for α-smooth muscle actin (αSMA) at day 14. The percentage of αSMA(+) VICs in the VGVAPG gels was the highest (56%) compared to RGDS (33%) or P15 (38%) gels. Matrix deposition and composition were also characterized at days 14 and 42 and found to depend on the initial hydrogel composition. All gel formulations had similar levels of collagen, elastin and chondroitin sulphate deposited as the porcine aortic valve. However, the composition of collagen deposited by VICs in VGVAPG-functionalized gels had a significantly higher collagen-X:collagen-1 ratio, which is associated with stenotic valves. Taken together, these data suggest that peptide-functionalized PEG hydrogels are a useful system for culturing VICs three-dimensionally and, with the ability to systematically alter biochemical and biophysical properties, this platform may prove useful in manipulating VIC function for valve regeneration. Copyright © 2013 John Wiley & Sons, Ltd. PMID:24130082

  12. Anisotropic properties of the enamel organic extracellular matrix.

    PubMed

    do Espírito Santo, Alexandre R; Novaes, Pedro D; Line, Sérgio R P

    2006-05-01

    Enamel biosynthesis is initiated by the secretion, processing, and self-assembly of a complex mixture of proteins. This supramolecular ensemble controls the nucleation of the crystalline mineral phase. The detection of anisotropic properties by polarizing microscopy has been extensively used to detect macromolecular organizations in ordinary histological sections. The aim of this work was to study the birefringence of enamel organic matrix during the development of rat molar and incisor teeth. Incisor and molar teeth of rats were fixed in 2% paraformaldehyde/0.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS), pH 7.2, and decalcified in 5% nitric acid/4% formaldehyde. After paraffin embedding, 5-microm-thick sections were obtained, treated with xylene, and hydrated. Form birefringence curves were obtained after measuring optical retardations in imbibing media, with different refractive indices. Our observations showed that enamel organic matrix of rat incisor and molar teeth is strongly birefringent, presenting an ordered supramolecular structure. The birefringence starts during the early secretion phase and disappears at the maturation phase. The analysis of enamel organic matrix birefringence may be used to detect the effects of genetic and environmental factors on the supramolecular orientation of enamel matrix and their effects on the structure of mature enamel.

  13. Modeling and Analysis of Granite Matrix Pore Structure and Hydraulic Characteristics in 2D and 3D Networks

    NASA Astrophysics Data System (ADS)

    Gvozdik, L.; Polak, M.; Zaruba, J.; Vanecek, M.

    2010-12-01

    A geological environment labeled as a Granite massif represents in terms of groundwater flow and transport a distinct hydrogeological environment from that of sedimentary basins, the characterisation of which is generally more complex and uncertain. Massifs are composed of hard crystalline rocks with the very low effective porosity. Due to their rheological properties such rocks are predisposed to brittle deformation resulting from changes in stress conditions. Our specific research project (Research on the influence of intergrangular porosity on deep geological disposal: geological formations, methodology and the development of measurement apparatus) is focussed on the problem of permeable zones within apparently undisturbed granitic rock matrix. The project including the both laboratory and in-situ tracer tests study migration along and through mineral grains in fresh and altered granite. The objective of the project is to assess whether intergranular porosity is a general characteristic of the granitic rock matrix or subject to significant evolution resulting from geochemical and/or hydrogeochemical processes, geotechnical and/or mechanical processes. Moreover, the research is focussed on evaluating methods quantifying intergranular porosity by both physical testing and mathematical modelling using verified standard hydrological software tools. Groundwater flow in microfractures and intergranular pores in granite rock matrix were simulated in three standard hydrogeological modeling programs with completely different conceptual approaches: MODFLOW (Equivalent Continuum concept), FEFLOW (Discrete Fracture and Equivalent Continuum concepts) and NAPSAC (Discrete Fracture Network concept). Specialized random fracture generators were used for creation of several 2D and 3D models in each of the chosen program. Percolation characteristics of these models were tested and analyzed. Several scenarios of laboratory tests of the rock samples permeability made in triaxial

  14. Detection of extracellular matrix modification in cancer models with inverse spectroscopic optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Spicer, Graham L. C.; Azarin, Samira M.; Yi, Ji; Young, Scott T.; Ellis, Ronald; Bauer, Greta M.; Shea, Lonnie D.; Backman, Vadim

    2016-10-01

    In cancer biology, there has been a recent effort to understand tumor formation in the context of the tissue microenvironment. In particular, recent progress has explored the mechanisms behind how changes in the cell-extracellular matrix ensemble influence progression of the disease. The extensive use of in vitro tissue culture models in simulant matrix has proven effective at studying such interactions, but modalities for non-invasively quantifying aspects of these systems are scant. We present the novel application of an imaging technique, Inverse Spectroscopic Optical Coherence Tomography, for the non-destructive measurement of in vitro biological samples during matrix remodeling. Our findings indicate that the nanoscale-sensitive mass density correlation shape factor D of cancer cells increases in response to a more crosslinked matrix. We present a facile technique for the non-invasive, quantitative study of the micro- and nano-scale structure of the extracellular matrix and its host cells.

  15. Engineering strategies to recapitulate epithelial morphogenesis within synthetic three-dimensional extracellular matrix with tunable mechanical properties

    NASA Astrophysics Data System (ADS)

    Miroshnikova, Y. A.; Jorgens, D. M.; Spirio, L.; Auer, M.; Sarang-Sieminski, A. L.; Weaver, V. M.

    2011-04-01

    The mechanical properties (e.g. stiffness) of the extracellular matrix (ECM) influence cell fate and tissue morphogenesis and contribute to disease progression. Nevertheless, our understanding of the mechanisms by which ECM rigidity modulates cell behavior and fate remains rudimentary. To address this issue, a number of two and three-dimensional (3D) hydrogel systems have been used to explore the effects of the mechanical properties of the ECM on cell behavior. Unfortunately, many of these systems have limited application because fiber architecture, adhesiveness and/or pore size often change in parallel when gel elasticity is varied. Here we describe the use of ECM-adsorbed, synthetic, self-assembling peptide (SAP) gels that are able to recapitulate normal epithelial acini morphogenesis and gene expression in a 3D context. By exploiting the range of viscoelasticity attainable with these SAP gels, and their ability to recreate native-like ECM fibril topology with minimal variability in ligand density and pore size, we were able to reconstitute normal and tumor-like phenotypes and gene expression patterns in nonmalignant mammary epithelial cells. Accordingly, this SAP hydrogel system presents the first tunable system capable of independently assessing the interplay between ECM stiffness and multi-cellular epithelial phenotype in a 3D context. Originally submitted for the special focus issue on physical oncology.

  16. Engineering strategies to recapitulate epithelial morphogenesis within synthetic three-dimensional extracellular matrix with tunable mechanical properties.

    PubMed

    Miroshnikova, Y A; Jorgens, D M; Spirio, L; Auer, M; Sarang-Sieminski, A L; Weaver, V M

    2011-04-01

    The mechanical properties (e.g. stiffness) of the extracellular matrix (ECM) influence cell fate and tissue morphogenesis and contribute to disease progression. Nevertheless, our understanding of the mechanisms by which ECM rigidity modulates cell behavior and fate remains rudimentary. To address this issue, a number of two and three-dimensional (3D) hydrogel systems have been used to explore the effects of the mechanical properties of the ECM on cell behavior. Unfortunately, many of these systems have limited application because fiber architecture, adhesiveness and/or pore size often change in parallel when gel elasticity is varied. Here we describe the use of ECM-adsorbed, synthetic, self-assembling peptide (SAP) gels that are able to recapitulate normal epithelial acini morphogenesis and gene expression in a 3D context. By exploiting the range of viscoelasticity attainable with these SAP gels, and their ability to recreate native-like ECM fibril topology with minimal variability in ligand density and pore size, we were able to reconstitute normal and tumor-like phenotypes and gene expression patterns in nonmalignant mammary epithelial cells. Accordingly, this SAP hydrogel system presents the first tunable system capable of independently assessing the interplay between ECM stiffness and multi-cellular epithelial phenotype in a 3D context.

  17. Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones

    SciTech Connect

    Hansen, R K; Bissell, M J

    2000-06-01

    The changes in tissue architecture that accompany the development of breast cancer have been the focus of investigations aimed at developing new cancer therapeutics. As we learn more about the normal mammary gland, we have begun to understand the complex signaling pathways underlying the dramatic shifts in the structure and function of breast tissue. Integrin-, growth factor-, and steroid hormone-signaling pathways all play an important part in maintaining tissue architecture; disruption of the delicate balance of signaling results in dramatic changes in the way cells interact with each other and with the extracellular matrix, leading to breast cancer. The extracellular matrix itself plays a central role in coordinating these signaling processes. In this review, we consider the interrelationships between the extracellular matrix, integrins, growth factors, and steroid hormones in mammary gland development and function.

  18. Adherence of extracellular matrix components to modified surfaces of titanium alloys

    NASA Astrophysics Data System (ADS)

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

    2009-04-01

    The adherence of biological materials on metal surfaces is of special importance in biology and medicine. The underlying interactions between surface and biological materials (e.g. extracellular matrix components or cells) are responsible for the application as a medical device. Numerous products are made of pure titanium and titanium alloys. This paper shows the influence of a laser production technology on machined surfaces of TiAl6V4 and the resulting adherence of biological material on the basis of the surface characterisation. In this study, different machined TiAl6V4 surfaces were used for coatings with extracellular matrix components. For this process, different coating with collagen I monomers and a complex mixture of extracellular matrix proteins derived from the dermal-epidermal basement membrane zone were analysed. The efficiency of the coating was analysed by different methods and the results are presented in this paper.

  19. Endothelial Cell-Derived Basic Fibroblast Growth Factor: Synthesis and Deposition into Subendothelial Extracellular Matrix

    NASA Astrophysics Data System (ADS)

    Vlodavsky, Israel; Folkman, Judah; Sullivan, Robert; Fridman, Rafael; Ishai-Michaeli, Rivka; Sasse, Joachim; Klagsbrun, Michael

    1987-04-01

    Bovine aortic and corneal endothelial cells synthesize a growth factor that remains mostly cell-associated but can also be extracted from the subendothelial extracellular matrix (ECM) deposited by these cells. The endothelial cell-derived growth factors extracted from cell lysates and from the extracellular matrix appear to be structurally related to basic fibroblast growth factor by the criteria that they (i) bind to heparin-Sepharose and are eluted at 1.4-1.6 M NaCl, (ii) have a molecular weight of about 18,400, (iii) cross-react with anti-basic fibroblast growth factor antibodies when analyzed by electrophoretic blotting and immunoprecipitation, and (iv) are potent mitogens for bovine aortic and capillary endothelial cells. It is suggested that endothelium can store growth factors capable of autocrine growth promotion in two ways: by sequestering growth factor within the cell and by incorporating it into the underlying extracellular matrix.

  20. A volume of intersection approach for on-the-fly system matrix calculation in 3D PET image reconstruction

    NASA Astrophysics Data System (ADS)

    Lougovski, A.; Hofheinz, F.; Maus, J.; Schramm, G.; Will, E.; van den Hoff, J.

    2014-02-01

    The aim of this study is the evaluation of on-the-fly volume of intersection computation for system’s geometry modelling in 3D PET image reconstruction. For this purpose we propose a simple geometrical model in which the cubic image voxels on the given Cartesian grid are approximated with spheres and the rectangular tubes of response (ToRs) are approximated with cylinders. The model was integrated into a fully 3D list-mode PET reconstruction for performance evaluation. In our model the volume of intersection between a voxel and the ToR is only a function of the impact parameter (the distance between voxel centre to ToR axis) but is independent of the relative orientation of voxel and ToR. This substantially reduces the computational complexity of the system matrix calculation. Based on phantom measurements it was determined that adjusting the diameters of the spherical voxel size and the ToR in such a way that the actual voxel and ToR volumes are conserved leads to the best compromise between high spatial resolution, low noise, and suppression of Gibbs artefacts in the reconstructed images. Phantom as well as clinical datasets from two different PET systems (Siemens ECAT HR+ and Philips Ingenuity-TF PET/MR) were processed using the developed and the respective vendor-provided (line of intersection related) reconstruction algorithms. A comparison of the reconstructed images demonstrated very good performance of the new approach. The evaluation showed the respective vendor-provided reconstruction algorithms to possess 34-41% lower resolution compared to the developed one while exhibiting comparable noise levels. Contrary to explicit point spread function modelling our model has a simple straight-forward implementation and it should be easy to integrate into existing reconstruction software, making it competitive to other existing resolution recovery techniques.

  1. Cutting edge: members of the Staphylococcus aureus extracellular fibrinogen-binding protein family inhibit the interaction of C3d with complement receptor 2.

    PubMed

    Ricklin, Daniel; Ricklin-Lichtsteiner, Salome K; Markiewski, Maciej M; Geisbrecht, Brian V; Lambris, John D

    2008-12-01

    Staphylococcus aureus expresses a highly diversified arsenal of immune evasion proteins, many of which target the complement system. The extracellular fibrinogen-binding protein (Efb) and the Efb homologous protein (Ehp) have previously been demonstrated to bind to C3 and inhibit complement activation and amplification. In this study we present the first evidence that Efb and Ehp are also capable of inhibiting the interaction of C3d with complement receptor 2 (CR2), which plays an important role in B cell activation and maturation. The C-terminal domain of Efb efficiently blocked this interaction both in surface plasmon resonance-based competition studies and cellular assays and prevented the CR2-mediated stimulation of B cells. Furthermore, analyses of the available structural data were consistent with a molecular mechanism that reflects both steric and electrostatic effects on the C3d-CR2 interaction. Our study therefore suggests that S. aureus may disrupt both the innate and adaptive immune responses with a single protein module. PMID:19017934

  2. Biglycan fragmentation in pathologies associated with extracellular matrix remodeling by matrix metalloproteinases

    PubMed Central

    2013-01-01

    Background The proteoglycan biglycan (BGN) is involved in collagen fibril assembly and its fragmentation is likely to be associated with collagen turnover during the pathogenesis of diseases which involve dysregulated extracellular matrix remodeling (ECMR), such as rheumatoid arthritis (RA) and liver fibrosis. The scope of the present study was to develop a novel enzyme-linked immunosorbent assay (ELISA) for the measurement of a MMP-9 and MMP-12-generated biglycan neo-epitope and to test its biological validity in a rat model of RA and in two rat models of liver fibrosis, chosen as models of ECMR. Results Biglycan was cleaved in vitro by MMP-9 and -12 and the 344′YWEVQPATFR′353 peptide (BGM) was chosen as a potential neo-epitope. A technically sound competitive ELISA for the measurement of BGM was generated and the assay was validated in a bovine cartilage explant culture (BEX), in a collagen induced model of rheumatoid arthritis (CIA) and in two different rat models of liver fibrosis: the carbon tetrachloride (CCL4)-induced fibrosis model, and the bile duct ligation (BDL) model. Significant elevation in serum BGM was found in CIA rats compared to controls, in rats treated with CCL4 for 16 weeks and 20 weeks compared to the control groups as well as in all groups of rats subject to BDL compared with sham operated groups. Furthermore, there was a significant correlation of serum BGM levels with the extent of liver fibrosis determined by the Sirius red staining of liver sections in the CCL4 model. Conclusion We demonstrated that the specific tissue remodeling product of MMPs-degraded biglycan, namely the neo-epitope BGM, is correlated with pathological ECMR. This assay represents both a novel marker of ECM turnover and a potential new tool to elucidate biglycan role during the pathological processes associated with ECMR. PMID:23635022

  3. Symposium: Role of the extracellular matrix in mammary development. Regulation of milk protein and basement membrane gene expression: The influence of the extracellular matrix

    SciTech Connect

    Aggeler, J.; Park, C.S.; Bissell, M.J.

    1988-10-01

    Synthesis and secretion of milk proteins ({alpha}-casein, {beta}-casein, {gamma}-casein, and transferrin) by cultured primary mouse mammary epithelial cells is modulated by the extracellular matrix. In cells grown on released or floating type I collagen gels, mRNA for {beta}-casein and transferrin is increased as much as 30-fold over cells grown on plastic. Induction of {beta}-casein expression depends strongly on the presence of lactogenic hormones, especially prolactin, in the culture. When cells are plated onto partially purified reconstituted basement membrane, dramatic changes in morphology and milk protein gene expression are observed. Cells cultured on the matrix for 6 to 8 d in the presence of prolactin, insulin, and hydrocortisone form hollow spheres and duct-like structures that are completely surrounded by matrix. The cells lining these spheres appear actively secretory and are oriented with their apices facing the lumen. Hybridization experiments indicate that mRNA for {beta}-casein can be increased as much as 70-fold in these cultures. Because > 90% of the cultured cells synthesize immunoreactive {beta}-casein, as compared with only 40% of cells in the late pregnant gland, the matrix appears to be able to induce protein expression in previously silent cells. Synthesis of laminin and assembly of a mammary-specific basal lamina by cells cultured on different extracellular matrices also appears to depend on the presence of lactogenic hormones. These studies provide support for the concept of dynamic reciprocity in which complex interactions between extracellular matrix and the cellular cytoskeleton contribute to the induction and maintenance of tissue-specific gene expression in the mammary gland.

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

    PubMed

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

    1994-07-01

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

  5. An extracellular matrix-based mechanism of rapid neutrophil extracellular trap formation in response to Candida albicans.

    PubMed

    Byrd, Angel S; O'Brien, Xian M; Johnson, Courtney M; Lavigne, Liz M; Reichner, Jonathan S

    2013-04-15

    The armament of neutrophil-mediated host defense against pathogens includes the extrusion of a lattice of DNA and microbicidal enzymes known as neutrophil extracellular traps (NETs). The receptor/ligand interactions and intracellular signaling mechanisms responsible for elaborating NETs were determined for the response to Candida albicans. Because the host response of extravasated neutrophils to mycotic infections within tissues necessitates contact with extracellular matrix, this study also identified a novel and significant regulatory role for the ubiquitous matrix component fibronectin (Fn) in NET release. We report that recognition of purified fungal pathogen-associated molecular pattern β-glucan by human neutrophils causes rapid (≤ 30 min) homotypic aggregation and NET release by a mechanism that requires Fn. Alone, immobilized β-glucan induces reactive oxygen species (ROS) production but not NET release, whereas in the context of Fn, ROS production is suppressed and NETs are extruded. NET release to Fn with β-glucan is robust, accounting for 17.2 ± 3.4% of total DNA in the cell population. Release is dependent on β-glucan recognition by complement receptor 3 (CD11b/CD18), but not Dectin-1, or ROS. The process of NET release included filling of intracellular vesicles with nuclear material that was eventually extruded. We identify a role for ERK in homotypic aggregation and NET release. NET formation to C. albicans hyphae was also found to depend on β-glucan recognition by complement receptor 3, require Fn and ERK but not ROS, and result in hyphal destruction. We report a new regulatory mechanism of NETosis in which the extracellular matrix is a key component of the rapid antifungal response. PMID:23509360

  6. Comparison of 3D Reconstructive Technologies Used for Morphometric Research and the Translation of Knowledge Using a Decision Matrix

    ERIC Educational Resources Information Center

    Martin, Charys M.; Roach, Victoria A.; Nguyen, Ngan; Rice, Charles L.; Wilson, Timothy D.

    2013-01-01

    The use of three-dimensional (3D) models for education, pre-operative assessment, presurgical planning, and measurement have become more prevalent. With the increase in prevalence of 3D models there has also been an increase in 3D reconstructive software programs that are used to create these models. These software programs differ in…

  7. Extracellular Matrix Ligand and Stiffness Modulate Immature Nucleus Pulposus Cell-Cell Interactions

    PubMed Central

    Gilchrist, Christopher L.; Darling, Eric M.; Chen, Jun; Setton, Lori A.

    2011-01-01

    The nucleus pulposus (NP) of the intervertebral disc functions to provide compressive load support in the spine, and contains cells that play a critical role in the generation and maintenance of this tissue. The NP cell population undergoes significant morphological and phenotypic changes during maturation and aging, transitioning from large, vacuolated immature cells arranged in cell clusters to a sparse population of smaller, isolated chondrocyte-like cells. These morphological and organizational changes appear to correlate with the first signs of degenerative changes within the intervertebral disc. The extracellular matrix of the immature NP is a soft, gelatinous material containing multiple laminin isoforms, features that are unique to the NP relative to other regions of the disc and that change with aging and degeneration. Based on this knowledge, we hypothesized that a soft, laminin-rich extracellular matrix environment would promote NP cell-cell interactions and phenotypes similar to those found in immature NP tissues. NP cells were isolated from porcine intervertebral discs and cultured in matrix environments of varying mechanical stiffness that were functionalized with various matrix ligands; cellular responses to periods of culture were assessed using quantitative measures of cell organization and phenotype. Results show that soft (<720 Pa), laminin-containing extracellular matrix substrates promote NP cell morphologies, cell-cell interactions, and proteoglycan production in vitro, and that this behavior is dependent upon both extracellular matrix ligand and substrate mechanical properties. These findings indicate that NP cell organization and phenotype may be highly sensitive to their surrounding extracellular matrix environment. PMID:22087260

  8. Degenerated human intervertebral discs contain autoantibodies against extracellular matrix proteins.

    PubMed

    Capossela, S; Schläfli, P; Bertolo, A; Janner, T; Stadler, B M; Pötzel, T; Baur, M; Stoyanov, J V

    2014-04-04

    Degeneration of intervertebral discs (IVDs) is associated with back pain and elevated levels of inflammatory cells. It has been hypothesised that discogenic pain is a direct result of vascular and neural ingrowth along annulus fissures, which may expose the avascular nucleus pulposus (NP) to the systemic circulation and induce an autoimmune reaction. In this study, we confirmed our previous observation of antibodies in human degenerated and post-traumatic IVDs cultured in vitro. We hypothesised that the presence of antibodies was due to an autoimmune reaction against specific proteins of the disc. Furthermore we identified antigens which possibly trigger an autoimmune response in degenerative disc diseases. We demonstrated that degenerated and post-traumatic IVDs contain IgG antibodies against typical extracellular proteins of the disc, particularly proteins of the NP. We identified IgGs against collagen type II and aggrecan, confirming an autoimmune reaction against the normally immune privileged NP. We also found specific IgGs against collagens types I and V, but not against collagen type III. In conclusion, this study confirmed the association between disc degeneration and autoimmunity, and may open the avenue for future studies on developing prognostic, diagnostic and therapy-monitoring markers for degenerative disc diseases.

  9. Extracellular-matrix tethering regulates stem-cell fate

    NASA Astrophysics Data System (ADS)

    Trappmann, Britta; Gautrot, Julien E.; Connelly, John T.; Strange, Daniel G. T.; Li, Yuan; Oyen, Michelle L.; Cohen Stuart, Martien A.; Boehm, Heike; Li, Bojun; Vogel, Viola; Spatz, Joachim P.; Watt, Fiona M.; Huck, Wilhelm T. S.

    2012-07-01

    To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5 kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions.

  10. Extracellular matrix organization in various regions of rat brain grey matter.

    PubMed

    Brückner, G; Härtig, W; Kacza, J; Seeger, J; Welt, K; Brauer, K

    1996-05-01

    Previous studies revealed the concentration of extracellular matrix proteoglycans in the so-called perineuronal nets on the one hand and in certain zones of the neuropil on the other. This nonhomogeneous distribution suggested a non-random chemical and spatial heterogeneity of the extracellular space. In the present investigation, regions dominated by one of both distribution patterns, i.e. piriform and parietal cortex, reticular thalamic nucleus, medial septum/diagonal band complex and cerebellar nuclei, were selected for correlative light and electron microscopic analysis. The labelling was performed by the use of the N-acetylgalactosamine-binding plant lectin Wisteria floribunda agglutinin visualized by peroxidase staining and additionally by photoconversion of red carbocyanine fluorescence labelling for electron microscopy. The intense labelling of the neuropil of a superficial piriform region, presumably identical with sublayer Ia, was confined to a fine meshwork spreading over the extracellular space between non-myelinated axons, dendrites and glial profiles. In the reticular thalamic nucleus the neuronal cell bodies were embedded in zones of labelled neuropil. In contrast to these patterns, the labelled extracellular matrix in different cortical layers and in the other subcortical regions was concentrated in perineuronal nets as large accumulations at surface areas of the neuronal perikarya and dendrites and the attached presynaptic boutons. Astrocytic processes usually were separated from the neuronal surface by the interposed extracellular material. Despite a great variability, the width of the extracellular space containing the labelled matrix components in all perineuronal nets appeared to be considerably larger than that in the labelled zones of neuropil and the non-labelled microenvironment of other neurons. Our results support the view that differences expressed in topographical and spatial peculiarities of the extracellular matrix constituents are

  11. Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

    NASA Technical Reports Server (NTRS)

    Bancroft, Gregory N.; Sikavitsas, Vassilios I.; van den Dolder, Juliette; Sheffield, Tiffany L.; Ambrose, Catherine G.; Jansen, John A.; Mikos, Antonios G.; McIntire, L. V. (Principal Investigator)

    2002-01-01

    Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

  12. A Prototype PZT Matrix Transducer With Low-Power Integrated Receive ASIC for 3-D Transesophageal Echocardiography.

    PubMed

    Chen, Chao; Raghunathan, Shreyas B; Yu, Zili; Shabanimotlagh, Maysam; Chen, Zhao; Chang, Zu-yao; Blaak, Sandra; Prins, Christian; Ponte, Jacco; Noothout, Emile; Vos, Hendrik J; Bosch, Johan G; Verweij, Martin D; de Jong, Nico; Pertijs, Michiel A P

    2016-01-01

    This paper presents the design, fabrication, and experimental evaluation of a prototype lead zirconium titanate (PZT) matrix transducer with an integrated receive ASIC, as a proof of concept for a miniature three-dimensional (3-D) transesophageal echocardiography (TEE) probe. It consists of an array of 9 ×12 piezoelectric elements mounted on the ASIC via an integration scheme that involves direct electrical connections between a bond-pad array on the ASIC and the transducer elements. The ASIC addresses the critical challenge of reducing cable count, and includes front-end amplifiers with adjustable gains and micro-beamformer circuits that locally process and combine echo signals received by the elements of each 3 ×3 subarray. Thus, an order-of-magnitude reduction in the number of receive channels is achieved. Dedicated circuit techniques are employed to meet the strict space and power constraints of TEE probes. The ASIC has been fabricated in a standard 0.18-μm CMOS process and consumes only 0.44 mW/channel. The prototype has been acoustically characterized in a water tank. The ASIC allows the array to be presteered across ±37° while achieving an overall dynamic range of 77 dB. Both the measured characteristics of the individual transducer elements and the performance of the ASIC are in good agreement with expectations, demonstrating the effectiveness of the proposed techniques.

  13. Intracellular nanomanipulation by a photonic-force microscope with real-time acquisition of a 3D stiffness matrix

    NASA Astrophysics Data System (ADS)

    Bertseva, E.; Singh, A. S. G.; Lekki, J.; Thévenaz, P.; Lekka, M.; Jeney, S.; Gremaud, G.; Puttini, S.; Nowak, W.; Dietler, G.; Forró, L.; Unser, M.; Kulik, A. J.

    2009-07-01

    A traditional photonic-force microscope (PFM) results in huge sets of data, which requires tedious numerical analysis. In this paper, we propose instead an analog signal processor to attain real-time capabilities while retaining the richness of the traditional PFM data. Our system is devoted to intracellular measurements and is fully interactive through the use of a haptic joystick. Using our specialized analog hardware along with a dedicated algorithm, we can extract the full 3D stiffness matrix of the optical trap in real time, including the off-diagonal cross-terms. Our system is also capable of simultaneously recording data for subsequent offline analysis. This allows us to check that a good correlation exists between the classical analysis of stiffness and our real-time measurements. We monitor the PFM beads using an optical microscope. The force-feedback mechanism of the haptic joystick helps us in interactively guiding the bead inside living cells and collecting information from its (possibly anisotropic) environment. The instantaneous stiffness measurements are also displayed in real time on a graphical user interface. The whole system has been built and is operational; here we present early results that confirm the consistency of the real-time measurements with offline computations.

  14. Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

    NASA Astrophysics Data System (ADS)

    Pati, Falguni; Jang, Jinah; Ha, Dong-Heon; Won Kim, Sung; Rhie, Jong-Won; Shim, Jin-Hyung; Kim, Deok-Ho; Cho, Dong-Woo

    2014-06-01

    The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method.

  15. Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

    PubMed Central

    Pati, Falguni; Jang, Jinah; Ha, Dong-Heon; Won Kim, Sung; Rhie, Jong-Won; Shim, Jin-Hyung; Kim, Deok-Ho; Cho, Dong-Woo

    2014-01-01

    The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method. PMID:24887553

  16. Biophysical properties of dermal building-blocks affects extra cellular matrix assembly in 3D endogenous macrotissue.

    PubMed

    Urciuolo, F; Garziano, A; Imparato, G; Panzetta, V; Fusco, S; Casale, C; Netti, P A

    2016-01-29

    The fabrication of functional tissue units is one of the major challenges in tissue engineering due to their in vitro use in tissue-on-chip systems, as well as in modular tissue engineering for the construction of macrotissue analogs. In this work, we aim to engineer dermal tissue micromodules obtained by culturing human dermal fibroblasts into porous gelatine microscaffold. We proved that such stromal cells coupled with gelatine microscaffolds are able to synthesize and to assemble an endogenous extracellular matrix (ECM) resulting in tissue micromodules, which evolve their biophysical features over the time. In particular, we found a time-dependent variation of oxygen consumption kinetic parameters, of newly formed ECM stiffness and of micromodules self-aggregation properties. As consequence when used as building blocks to fabricate larger tissues, the initial tissue micromodules state strongly affects the ECM organization and maturation in the final macrotissue. Such results highlight the role of the micromodules properties in controlling the formation of three-dimensional macrotissue in vitro, defining an innovative design criterion for selecting tissue-building blocks for modular tissue engineering.

  17. Extracellular matrix structure and nano-mechanics determine megakaryocyte function.

    PubMed

    Malara, Alessandro; Gruppi, Cristian; Pallotta, Isabella; Spedden, Elise; Tenni, Ruggero; Raspanti, Mario; Kaplan, David; Tira, Maria Enrica; Staii, Cristian; Balduini, Alessandra

    2011-10-20

    Cell interactions with matrices via specific receptors control many functions, with chemistry, physics, and membrane elasticity as fundamental elements of the processes involved. Little is known about how biochemical and biophysical processes integrate to generate force and, ultimately, to regulate hemopoiesis into the bone marrow-matrix environment. To address this hypothesis, in this work we focus on the regulation of MK development by type I collagen. By atomic force microscopy analysis, we demonstrate that the tensile strength of fibrils in type I collagen structure is a fundamental requirement to regulate cytoskeleton contractility of human MKs through the activation of integrin-α2β1-dependent Rho-ROCK pathway and MLC-2 phosphorylation. Most importantly, this mechanism seemed to mediate MK migration, fibronectin assembly, and platelet formation. On the contrary, a decrease in mechanical tension caused by N-acetylation of lysine side chains in type I collagen completely reverted these processes by preventing fibrillogenesis.

  18. Glycation of extracellular matrix proteins impairs migration of immune cells.

    PubMed

    Haucke, Elisa; Navarrete-Santos, Alexander; Simm, Andreas; Silber, Rolf-Edgar; Hofmann, Britt

    2014-01-01

    The immune response during aging and diabetes is disturbed and may be due to the altered migration of immune cells in an aged tissue. Our study should prove the hypothesis that age and diabetes-related advanced glycation end products (AGEs) have an impact on the migration and adhesion of human T-cells. To achieve our purpose, we used in vitro AGE-modified proteins (soluble albumin and fibronectin [FN]), as well as human collagen obtained from bypass graft. A Boyden chamber was used to study cell migration. Migrated Jurkat T-cells were analyzed by flow cytometry and cell adhesion by crystal violet staining. Actin polymerization was determined by phalloidin-Alexa-fluor 488-labeled antibody and fluorescence microscopy. We found that significantly fewer cells (50%, p = 0.003) migrated through methylglyoxal modified FN. The attachment to FN in the presence of AGE-bovine serum albumin (BSA) was also reduced (p < 0.05). In ex vivo experiments, isolated collagen from human vein graft material negatively affected the migration of the cells depending on the grade of AGE modification of the collagen. Collagen with a low AGE level reduced the cell migration by 30%, and collagen with a high AGE level by 60%. Interaction of the cells with an AGE-modified matrix, but not with soluble AGEs like BSA-AGE per se, was responsible for a disturbed migration. The reduced migration was accompanied by an impaired actin polymerization. We conclude that AGEs-modified matrix protein inhibits cell migration and adhesion of Jurkat T-cells. PMID:24635174

  19. Glycation of extracellular matrix proteins impairs migration of immune cells.

    PubMed

    Haucke, Elisa; Navarrete-Santos, Alexander; Simm, Andreas; Silber, Rolf-Edgar; Hofmann, Britt

    2014-01-01

    The immune response during aging and diabetes is disturbed and may be due to the altered migration of immune cells in an aged tissue. Our study should prove the hypothesis that age and diabetes-related advanced glycation end products (AGEs) have an impact on the migration and adhesion of human T-cells. To achieve our purpose, we used in vitro AGE-modified proteins (soluble albumin and fibronectin [FN]), as well as human collagen obtained from bypass graft. A Boyden chamber was used to study cell migration. Migrated Jurkat T-cells were analyzed by flow cytometry and cell adhesion by crystal violet staining. Actin polymerization was determined by phalloidin-Alexa-fluor 488-labeled antibody and fluorescence microscopy. We found that significantly fewer cells (50%, p = 0.003) migrated through methylglyoxal modified FN. The attachment to FN in the presence of AGE-bovine serum albumin (BSA) was also reduced (p < 0.05). In ex vivo experiments, isolated collagen from human vein graft material negatively affected the migration of the cells depending on the grade of AGE modification of the collagen. Collagen with a low AGE level reduced the cell migration by 30%, and collagen with a high AGE level by 60%. Interaction of the cells with an AGE-modified matrix, but not with soluble AGEs like BSA-AGE per se, was responsible for a disturbed migration. The reduced migration was accompanied by an impaired actin polymerization. We conclude that AGEs-modified matrix protein inhibits cell migration and adhesion of Jurkat T-cells.

  20. Implementing a Matrix-free Analytical Jacobian to Handle Nonlinearities in Models of 3D Lithospheric Deformation

    NASA Astrophysics Data System (ADS)

    Kaus, B.; Popov, A.

    2015-12-01

    The analytical expression for the Jacobian is a key component to achieve fast and robust convergence of the nonlinear Newton-Raphson iterative solver. Accomplishing this task in practice often requires a significant algebraic effort. Therefore it is quite common to use a cheap alternative instead, for example by approximating the Jacobian with a finite difference estimation. Despite its simplicity it is a relatively fragile and unreliable technique that is sensitive to the scaling of the residual and unknowns, as well as to the perturbation parameter selection. Unfortunately no universal rule can be applied to provide both a robust scaling and a perturbation. The approach we use here is to derive the analytical Jacobian for the coupled set of momentum, mass, and energy conservation equations together with the elasto-visco-plastic rheology and a marker in cell/staggered finite difference method. The software project LaMEM (Lithosphere and Mantle Evolution Model) is primarily developed for the thermo-mechanically coupled modeling of the 3D lithospheric deformation. The code is based on a staggered grid finite difference discretization in space, and uses customized scalable solvers form PETSc library to efficiently run on the massively parallel machines (such as IBM Blue Gene/Q). Currently LaMEM relies on the Jacobian-Free Newton-Krylov (JFNK) nonlinear solver, which approximates the Jacobian-vector product using a simple finite difference formula. This approach never requires an assembled Jacobian matrix and uses only the residual computation routine. We use an approximate Jacobian (Picard) matrix to precondition the Krylov solver with the Galerkin geometric multigrid. Because of the inherent problems of the finite difference Jacobian estimation, this approach doesn't always result in stable convergence. In this work we present and discuss a matrix-free technique in which the Jacobian-vector product is replaced by analytically-derived expressions and compare results

  1. Extracellular matrix of smooth muscle cells: interaction of collagen type V with heparan sulfate proteoglycan

    SciTech Connect

    Gay, S.; Hoeoek, M.; Gay, R.E.; Magargal, W.W.; Reynertson, R.H.

    1986-03-05

    Alteration in the extracellular matrix produced by smooth muscle cells may play a role in the development of atherosclerotic lesions. Consequently the authors have initiated studies on the structural organization of the extracellular matrix produced by cultured smooth muscle cells. Immunohisotological examination of this matrix using well-characterized mono- and polyclonal antibodies showed a partial codistribution of heparan sulfate (HS) proteoglycans with a number of different matrix components including collagen types I, III, IV, V and VI, laminin and fibronectin. Subsequent binding studies between isolated matrix proteins and HS showed that the polysaccharide interacts strongly with type V collagen and to a lesser extent with fibronectin as well as collagen types III and VI. The interaction between type V and HS was readily inhibited by heparin and highly sulfated HS but not be dermatan sulfate, chondroitin sulfate or HS with a low sulfate content. Furthermore, (/sup 35/S)-HS proteoglycans isolated from cultured smooth muscle cells could be adsorbed on a column of sepharose conjugated with native type V collagen and eluted in a salt gradient. Hence, the interaction between type V and HS may play a major part in stabilizing the extracellular matrix of the vessel wall.

  2. Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation

    PubMed Central

    Glass, RH; Aggeler, J; Spindle, A; Pederson, RA; Werb, Z

    1983-01-01

    During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17β-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or ε-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0 PMID:6339525

  3. Development-dependent modification of the extracellular matrix by a sulphated glycoprotein in Volvox carteri.

    PubMed

    Wenzl, S; Thym, D; Sumper, M

    1984-04-01

    We report the chemical characterization of the highly sulphated glycoprotein SSG 185 from Volvox carteri. SSG 185 is a hydroxyproline-containing, extracellular glycoprotein. The sulphate residues are clustered within the parent saccharide structure of SSG 185, since on mercaptolysis all the sulphate residues are recovered in a small saccharide fragment containing mannose, arabinose and sulphate (in a molar ratio of 112). SSG 185 is a short-lived molecule, serving as a precursor for a high mol. wt. component of the extracellular matrix. Synthesis of SSG 185 is developmentally controlled. Different SSG 185 variants, with unknown modifications in the sulphated saccharide fragment, are synthesized at different developmental stages or under the influence of the sexual inducer. These modifications remain conserved in the aggregated state of SSG 185, indicating the development-dependent modification of the extracellular matrix. PMID:16453512

  4. Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies

    PubMed Central

    Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Collin, Estelle; Rochev, Yury; Rodriguez, Brian J.; Gorelov, Alexander; Dillon, Simon; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I.

    2015-01-01

    Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro. PMID:25736020

  5. Novel extracellular matrix and microtubule cables associated with pseudopodia of Astrammina rara, a carnivorous Antarctic foraminifer.

    PubMed

    Bowser, S S; DeLaca, T E; Rieder, C L

    1986-02-01

    Astrammina rara is a benthic foraminiferan protozoan which uses an extensive network of fine, branching, and anastomosing pseudopodia to capture and digest metazoans up to 12 mm long. During such predation the pseudopodia appear remarkably elastic and tensile. Electron microscopy has revealed a novel extracellular matrix of thin branching fibers associated with A. rara's pseudopodia, which are otherwise typical in appearance. These fibers are structurally associated with the pseudopodial glycocalyx. Cytoplasmic microtubules are often found in close juxtaposition to the plasma membrane and overlying extracellular fibers. In the main pseudopodial trunks bundles of 30-300 microtubules are surrounded by a lightly staining matrix and linked by occasional bridges. The microtubules follow straight trajectories in distal filopodia, but in pseudopodial trunks they appear to coil around one another to form cables. These microtubular cables and extracellular fibers are novel features of A. rara's pseudopodia and may provide the structural basis for their tensile strength.

  6. Dynamic compressive behavior of human meniscus correlates with its extra-cellular matrix composition.

    PubMed

    Bursac, P; Arnoczky, S; York, A

    2009-01-01

    The menisci of the knee play a significant role in the complex biomechanics of the joint and are critically important in maintaining articular cartilage health. While a general form-function relationship has been identified for the structural orientation of the extra-cellular matrix of the meniscus, the role of individual biochemical components has yet to be fully explored. To determine if correlations exist between the dynamic and static compressive modulus of human menisci and their major extra-cellular matrix constituents (collagen, glycosoaminoglycan and water content), 12 lateral and 11 medial menisci from 13 adult donors were examined. The results showed that in dynamic compression at high loading frequencies (0.1-1 Hz) the menisci behave as a rubber-like elastic material while at lower frequencies (0.01-0.03 Hz) significant viscous dissipation occurs. While regional variations in compressive moduli and extra-cellular matrix composition were observed, the magnitude of both dynamic and static compressive moduli were found to be insensitive to collagen content (p>0.4). However, this magnitude was found to significantly increase with increasing glycosaminoglycan content (p<0.001) and significantly decrease with increasing water content (p<0.001). The results of this study identify significant relationships between the viscoelastic behavior of the meniscus and its extra-cellular matrix composition.

  7. Dynamic compressive behavior of human meniscus correlates with its extra-cellular matrix composition.

    PubMed

    Bursac, P; Arnoczky, S; York, A

    2009-01-01

    The menisci of the knee play a significant role in the complex biomechanics of the joint and are critically important in maintaining articular cartilage health. While a general form-function relationship has been identified for the structural orientation of the extra-cellular matrix of the meniscus, the role of individual biochemical components has yet to be fully explored. To determine if correlations exist between the dynamic and static compressive modulus of human menisci and their major extra-cellular matrix constituents (collagen, glycosoaminoglycan and water content), 12 lateral and 11 medial menisci from 13 adult donors were examined. The results showed that in dynamic compression at high loading frequencies (0.1-1 Hz) the menisci behave as a rubber-like elastic material while at lower frequencies (0.01-0.03 Hz) significant viscous dissipation occurs. While regional variations in compressive moduli and extra-cellular matrix composition were observed, the magnitude of both dynamic and static compressive moduli were found to be insensitive to collagen content (p>0.4). However, this magnitude was found to significantly increase with increasing glycosaminoglycan content (p<0.001) and significantly decrease with increasing water content (p<0.001). The results of this study identify significant relationships between the viscoelastic behavior of the meniscus and its extra-cellular matrix composition. PMID:19581729

  8. Substrates with patterned extracellular matrix and subcellular stiffness gradients reveal local biomechanical responses.

    PubMed

    Tseng, Peter; Di Carlo, Dino

    2014-02-26

    A substrate fabrication process is developed to pattern both the extracellular matrix (ECM) and rigidity at sub-cellular spatial resolution. When growing cells on these substrates, it is found that cells respond locally in their cytoskeleton assembly. The presented method allows unique insight into the biological interpretation of mechanical signals, whereas photolithography-based fabrication is amenable to integration with complex microfabricated substructures.

  9. First demonstration of decorin, an extracellular matrix molecule, in bovine mammary tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the mammary gland, the extracellular matrix (ECM) is secreted by and surrounds cells located in both mammary parenchyma (PAR) and stroma. Decorin is an ECM proteoglycan with cell growth regulatory effects mediated by its ability to interact with growth factors or up-regulation of cyclin-dependent...

  10. Extracellular matrix proteins and the dynamics of dentin formation.

    PubMed

    Butler, William T; Brunn, Jan C; Qin, Chunlin; McKee, Marc D

    2002-01-01

    Dentinogenesis involves controlled reactions that result in conversion of unmineralized predentin to dentin when apatite crystals are formed. This process is dynamic: Maturation events occur within predentin beginning at the proximal layer and progressing to the predentin-dentin (PD) border. One type of controlled reaction is the proteolytic processing of dentin sialophosphoprotein (DSPP) to dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), by cleavage of at least three highly conserved peptide bonds. We postulate that this processing event represents an activation step, resulting in release of DPP, which is active in its effects on formation and growth of apatite crystals. Dentin matrix protein 1 (DPM1), present as a processed fragment (57-kD protein) in bone, is seen in dentin on sodium dodecyl sulfate polyacrylamide gel electrophoresis as one intact protein of 150-200 kD. Anti-57-kD antibodies elicit immunoreactivity in bone, dentin, and cellular cementum. In bone, the reactivity is associated with osteocytes and their cell processes. Similarly, dentin shows reactivity in odontoblasts, predentin, and the odontoblast processes. In summary, the processing of large sialic acid-rich proteins into smaller fragments may be an important part of the controlled conversion of predentin to dentin and osteoid to bone.

  11. Extracellular Matrix Molecular Remodeling in Human Liver Fibrosis Evolution

    PubMed Central

    Baiocchini, Andrea; Montaldo, Claudia; Conigliaro, Alice; Grimaldi, Alessio; Correani, Virginia; Mura, Francesco; Ciccosanti, Fabiola; Rotiroti, Nicolina; Brenna, Alessia; Montalbano, Marzia; D’Offizi, Gianpiero; Capobianchi, Maria Rosaria; Alessandro, Riccardo; Piacentini, Mauro; Schininà, Maria Eugenia; Maras, Bruno; Del Nonno, Franca; Tripodi, Marco; Mancone, Carmine

    2016-01-01

    Chronic liver damage leads to pathological accumulation of ECM proteins (liver fibrosis). Comprehensive characterization of the human ECM molecular composition is essential for gaining insights into the mechanisms of liver disease. To date, studies of ECM remodeling in human liver diseases have been hampered by the unavailability of purified ECM. Here, we developed a decellularization method to purify ECM scaffolds from human liver tissues. Histological and electron microscopy analyses demonstrated that the ECM scaffolds, devoid of plasma and cellular components, preserved the three-dimensional ECM structure and zonal distribution of ECM components. This method has been then applied on 57 liver biopsies of HCV-infected patients at different stages of liver fibrosis according to METAVIR classification. Label-free nLC-MS/MS proteomics and computation biology were performed to analyze the ECM molecular composition in liver fibrosis progression, thus unveiling protein expression signatures specific for the HCV-related liver fibrotic stages. In particular, the ECM molecular composition of liver fibrosis was found to involve dynamic changes in matrix stiffness, flexibility and density related to the dysregulation of predominant collagen, elastic fibers and minor components with both structural and signaling properties. This study contributes to the understanding of the molecular bases underlying ECM remodeling in liver fibrosis and suggests new molecular targets for fibrolytic strategies. PMID:26998606

  12. Physicomechanical properties of the extracellular matrix of a demineralized bone

    NASA Astrophysics Data System (ADS)

    Kirilova, I. A.; Sharkeev, Yu. P.; Nikolaev, S. V.; Podorozhnaya, V. T.; Uvarkin, P. V.; Ratushnyak, A. S.; Chebodaeva, V. V.

    2016-08-01

    The article describes the results of a study of physicomechanical properties of a demineralized bone matrix of human cancellous and compact bones. A demineralized cancellous bone was shown to have the best characteristics of a porous system for colonization of matrices by cells. The ultimate stress and elasticity modulus of samples of demineralized femoral heads isolated in primary hip replacement was demonstrated to vary in wide ranges. The elasticity modulus ranged from 50 to 250 MPa, and the tensile strength varied from 1.1 to 5.5 MPa. Microhardness measurements by the recovered indentation method were not possible because of the viscoelastic properties of a bone material. To study the piezoelectric properties of samples, a measuring system was developed that comprised a measuring chamber with contact electrodes, a system for controlled sample loading, an amplifier-converter unit, and signal recording and processing software. The measurement results were used to determine the dependence of the signal amplitude on the dynamic deformation characteristics. The findings are discussed in terms of the relationship between the mechanical and electrical properties and the structure of the organic bone component.

  13. A Hydrogel Derived From Decellularized Dermal Extracellular Matrix

    PubMed Central

    Wolf, Matthew T.; Daly, Kerry A.; Brennan-Pierce, Ellen P.; Johnson, Scott A.; Carruthers, Christopher; D’Amore, Antonio; Nagarkar, Shailesh P.; Velankar, Sachin S.; Badylak, Stephen F.

    2012-01-01

    The ECM of mammalian tissues has been used as a scaffold to facilitate the repair and reconstruction of numerous tissues. Such scaffolds are prepared in many forms including sheets, powders, and hydrogels. ECM hydrogels provide advantages such as injectability, the ability to fill an irregularly shaped space, and the inherent bioactivity of native matrix. However, material properties of ECM hydrogels and the effect of these properties upon cell behavior are neither well understood nor controlled. The objective of this study was to prepare and determine the structure, mechanics, and the cell response in vitro and in vivo of ECM hydrogels prepared from decellularized porcine dermis and urinary bladder tissues. Dermal ECM hydrogels were characterized by a more dense fiber architecture and greater mechanical integrity than urinary bladder ECM hydrogels, and showed a dose dependent increase in mechanical properties with ECM concentration. In vitro, dermal ECM hydrogels supported greater C2C12 myoblast fusion, and less fibroblast infiltration and less fibroblast mediated hydrogel contraction than urinary bladder ECM hydrogels. Both hydrogels were rapidly infiltrated by host cells, primarily macrophages, when implanted in a rat abdominal wall defect. Both ECM hydrogels degraded by 35 days in vivo, but UBM hydrogels degraded more quickly, and with greater amounts of myogenesis than dermal ECM. These results show that ECM hydrogel properties can be varied and partially controlled by the scaffold tissue source, and that these properties can markedly affect cell behavior. PMID:22789723

  14. Inhaled Steroids Modulate Extracellular Matrix Composition in Bronchial Biopsies of COPD Patients: A Randomized, Controlled Trial

    PubMed Central

    Kunz, Lisette I. Z.; Strebus, Jolanda; Budulac, Simona E.; Lapperre, Therese S.; Sterk, Peter J.; Postma, Dirkje S.; Mauad, Thais; Timens, Wim; Hiemstra, Pieter S.

    2013-01-01

    Rationale Smoking and inflammation contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD), which involves changes in extracellular matrix. This is thought to contribute to airway remodeling and airflow obstruction. We have previously observed that long-term treatment with inhaled corticosteroids can not only reduce bronchial inflammation, but can also attenuate lung function decline in moderate-severe COPD. We hypothesized that inhaled corticosteroids and current smoking modulate bronchial extracellular matrix components in COPD. Objective To compare major extracellular matrix components (elastic fibers; proteoglycans [versican, decorin]; collagens type I and III) in bronchial biopsies 1) after 30-months inhaled steroids treatment or placebo; and 2) between current and ex-smokers with COPD. Methods We included 64 moderate-severe, steroid-naive COPD patients (24/40 (ex)-smokers, 62±7 years, 46 (31–54) packyears, post-bronchodilator forced expiratory volume in one second (FEV1) 62±9% predicted) at baseline in this randomized, controlled trial. 19 and 13 patients received 30-months treatment with fluticasone or placebo, respectively. Bronchial biopsies collected at baseline and after 30 months were studied using (immuno)histochemistry to evaluate extracellular matrix content. Percentage and density of stained area were calculated by digital image analysis. Results 30-Months inhaled steroids increased the percentage stained area of versican (9.6% [CI 0.9 to 18.3%]; p = 0.03) and collagen III (20.6% [CI 3.8 to 37.4%]; p = 0.02) compared to placebo. Increased collagen I staining density correlated with increased post-bronchodilator FEV1 after inhaled steroids treatment (Rs = 0.45, p = 0.04). There were no differences between smokers and ex-smokers with COPD in percentages and densities for all extracellular matrix proteins. Conclusions These data show that long-term inhaled corticosteroids treatment partially changes the

  15. Local fluid transfer regulation in heart extracellular matrix.

    PubMed

    McGee, Maria P; Morykwas, Michael J; Jordan, James E; Wang, Rui; Argenta, Louis C

    2016-06-01

    The interstitial myocardial matrix is a complex and dynamic structure that adapts to local fluctuations in pressure and actively contributes to the heart's fluid exchange and hydration. However, classical physiologic models tend to treat it as a passive conduit for water and solute, perhaps because local interstitial regulatory mechanisms are not easily accessible to experiment in vivo. Here, we examined the interstitial contribution to the fluid-driving pressure ex vivo. Interstitial hydration potentials were determined from influx/efflux rates measured in explants from healthy and ischemia-reperfusion-injured pigs during colloid osmotic pressure titrations. Adaptive responses were further explored by isolating myocardial fibroblasts and measuring their contractile responses to water activity changes in vitro. Results show hydration potentials between 5 and 60 mmHg in healthy myocardia and shifts in excess of 200 mmHg in edematous myocardia after ischemia-reperfusion injury. Further, rates of fluid transfer were temperature-dependent, and in collagen gel contraction assays, myocardial fibroblasts tended to preserve the micro-environment's hydration volume by slowing fluid efflux rates at pressures above 40 mmHg. Our studies quantify components of the fluid-driving forces in the heart interstitium that the classical Starling's equation does not explicitly consider. Measured hydration potentials in healthy myocardia and shifts with edema are larger than predicted from the known values of hydrostatic and colloid osmotic interstitial fluid pressures. Together with fibroblast responses in vitro, they are consistent with regulatory mechanisms that add local biological controls to classic fluid-balance models.

  16. Local fluid transfer regulation in heart extracellular matrix.

    PubMed

    McGee, Maria P; Morykwas, Michael J; Jordan, James E; Wang, Rui; Argenta, Louis C

    2016-06-01

    The interstitial myocardial matrix is a complex and dynamic structure that adapts to local fluctuations in pressure and actively contributes to the heart's fluid exchange and hydration. However, classical physiologic models tend to treat it as a passive conduit for water and solute, perhaps because local interstitial regulatory mechanisms are not easily accessible to experiment in vivo. Here, we examined the interstitial contribution to the fluid-driving pressure ex vivo. Interstitial hydration potentials were determined from influx/efflux rates measured in explants from healthy and ischemia-reperfusion-injured pigs during colloid osmotic pressure titrations. Adaptive responses were further explored by isolating myocardial fibroblasts and measuring their contractile responses to water activity changes in vitro. Results show hydration potentials between 5 and 60 mmHg in healthy myocardia and shifts in excess of 200 mmHg in edematous myocardia after ischemia-reperfusion injury. Further, rates of fluid transfer were temperature-dependent, and in collagen gel contraction assays, myocardial fibroblasts tended to preserve the micro-environment's hydration volume by slowing fluid efflux rates at pressures above 40 mmHg. Our studies quantify components of the fluid-driving forces in the heart interstitium that the classical Starling's equation does not explicitly consider. Measured hydration potentials in healthy myocardia and shifts with edema are larger than predicted from the known values of hydrostatic and colloid osmotic interstitial fluid pressures. Together with fibroblast responses in vitro, they are consistent with regulatory mechanisms that add local biological controls to classic fluid-balance models. PMID:26961911

  17. TGF-beta/extracellular matrix interactions in dentin matrix: a role in regulating sequestration and protection of bioactivity.

    PubMed

    Baker, S M; Sugars, R V; Wendel, M; Smith, A J; Waddington, R J; Cooper, P R; Sloan, A J

    2009-07-01

    TGF-beta isoforms sequestrated in dentin matrix potentially provide a reservoir of bioactive molecules that may influence cell behavior in the dentin-pulp complex following tissue injury. The association of these growth factors with dentin matrix and the influence of such associations on the bioactivity of growth factors are still unclear. We used surface plasmon resonance technology in the BIAcore 3000 system to investigate the binding of TGF-beta isoforms 1 and 3 to purified decorin, biglycan, and EDTA soluble dentin matrix components. TGF-beta isoforms 1 and 3 were immobilized on sensorchips CM4 through amine coupling. For kinetic studies of protein binding, purified decorin and biglycan, isolated EDTA soluble dentin matrix, and dentin matrix immunodepleted of decorin and/or biglycan were injected over TGF-beta isoforms and allowed to interact. Programmed kinetic analysis software provided sensorgrams for each concentration of proteoglycan or dentin matrix extract injected. Purified decorin and biglycan and dentin matrix extract bound to the TGF-beta isoforms. However, the association with TGF-beta3 was much weaker than that with TGF-beta1. After immunoaffinity depletion of the dentin matrix extract, the level of interaction between the dentin matrix extract and TGF-beta was significantly reduced. These results suggest isoform-specific interactions between decorin/biglycan and TGF-beta isoforms 1 and 3, which may explain why TGF-beta3 is not detected in the dentin matrix despite being expressed at higher levels than TGF-beta1 in odontoblasts. These proteoglycans appear to play a significant role in TGF-beta/extracellular matrix interactions and may be important in the sequestration of these growth factors in the dentin matrix.

  18. Brevican: a key proteoglycan in the perisynaptic extracellular matrix of the brain.

    PubMed

    Frischknecht, Renato; Seidenbecher, Constanze I

    2012-07-01

    Brevican is a neural proteoglycan implicated in a multitude of physiological and pathophysiological plasticity processes in the brain. It localizes to neuronal surfaces and contributes to the formation of specific types of extracellular matrix like the perineuronal nets or the perisynaptic or axon initial segment-based matrix in mature neuronal tissue. Via a variable degree of chondroitin sulfate attachment, limited proteolytic cleavage by matrix metalloproteinases, differential splicing and Ca(2+)-dependent binding to interaction partners it acts as a regulator in synaptic plasticity, glioma invasion, post-lesion plasticity or Alzheimer's disease. This review briefly summarizes its gene and protein structure, biochemical interactions and neurobiological functions.

  19. Schur-decomposition for 3D matrix equations and its application in solving radiative discrete ordinates equations discretized by Chebyshev collocation spectral method

    SciTech Connect

    Li Benwen Tian Shuai; Sun Yasong; Hu, Zhang-Mao

    2010-02-20

    The Schur-decomposition for three-dimensional matrix equations is developed and used to directly solve the radiative discrete ordinates equations which are discretized by Chebyshev collocation spectral method. Three methods, say, the spectral methods based on 2D and 3D matrix equation solvers individually, and the standard discrete ordinates method, are presented. The numerical results show the good accuracy of spectral method based on direct solvers. The CPU time cost comparisons against the resolutions between these three methods are made using MATLAB and FORTRAN 95 computer languages separately. The results show that the CPU time cost of Chebyshev collocation spectral method with 3D Schur-decomposition solver is the least, and almost only one thirtieth to one fiftieth CPU time is needed when using the spectral method with 3D Schur-decomposition solver compared with the standard discrete ordinates method.

  20. Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs

    PubMed Central

    Ames, Juliana; Chokshi, Mithil; Aiad, Norman; Sanyal, Sonali; Kawabata, Kimihito C.; Levental, Ilya; Sundararaghavan, Harini G.; Burdick, Jason A.; Janmey, Paul; Miyazono, Kohei; Wells, Rebecca G.; Jones, Peter L.

    2015-01-01

    Abstract Although there are many studies focusing on the molecular pathways underlying lung vascular morphogenesis, the extracellular matrix (ECM)–dependent regulation of mesenchymal cell differentiation in vascular smooth muscle development needs better understanding. In this study, we demonstrate that the paired related homeobox gene transcription factor Prx1 maintains the elastic ECM properties, which are essential for vascular smooth muscle precursor cell differentiation. We have found that Prx1null mouse lungs exhibit defective vascular smooth muscle development, downregulated elastic ECM expression, and compromised transforming growth factor (TGF)–β localization and signaling. Further characterization of ECM properties using decellularized lung ECM scaffolds derived from Prx1 mice demonstrated that Prx1 is required to maintain lung ECM stiffness. The results of cell culture using stiffness-controlled 2-D and 3-D synthetic substrates confirmed that Prx1-dependent ECM stiffness is essential for promotion of smooth muscle precursor differentiation for effective TGF-β stimulation. Supporting these results, both decellularized Prx1null lung ECM and Prx1WT (wild type) ECM scaffolds with blocked TGF-β failed to support mesenchymal cell to 3-D smooth muscle cell differentiation. These results suggest a novel ECM-dependent regulatory pathway of lung vascular development wherein Prx1 regulates lung vascular smooth muscle precursor development by coordinating the ECM biophysical and biochemical properties. PMID:26064466

  1. Effect of spaceflight on the extracellular matrix of skeletal muscle after a crush injury

    NASA Technical Reports Server (NTRS)

    Stauber, W. T.; Fritz, V. K.; Burkovskaia, T. E.; Il'ina-Kakueva, E. I.

    1992-01-01

    The organization and composition of the extracellular matrix were studied in the crush-injured gastrocnemius muscle of rats subjected to 0 G. After 14 days of flight on Cosmos 2044, the gastrocnemius muscle was removed and evaluated by histochemical and immunohistochemical techniques from the five injured flight rodents and various earth-based treatment groups. In general, the repair process was similar in all injured muscle samples with regard to the organization of the extracellular matrix and myofibers. Small and large myofibers were present within an expanded extracellular matrix, indicative of myogenesis and muscle regeneration. In the tail-suspended animals, a more complete repair was observed with nonenlarged area of nonmuscle cells or matrix material visible. In contrast, the muscle samples from the flight animals were less well organized and contained more macrophages and blood vessels in the repair region, indicative of a delayed repair process, but did not demonstrate any chronic inflammation. Myofiber repair did vary in muscles from the different groups, being slowest in the flight animals and most complete in the tail-suspended ones.

  2. Differential expression of extracellular matrix components in the Fallopian tubes throughout the menstrual cycle

    PubMed Central

    2012-01-01

    Background One of the unique characteristics of the female genital tract is the extensive tissue remodeling observed throughout the menstrual cycle. Multiple components of the extracellular matrix take part in this tissue rebuilding; however, the individual components involved have not been identified. Methods In the present study, the expression of extracellular matrix proteins and selected matrix metalloproteinase (MMP) activities in Fallopian tubes (FT) throughout the menstrual cycle were examined by PCR array, immunocytochemistry, zymography and bioinformatics. Results Of the eighty-four genes analyzed, eighty-three were expressed in the FT during at least one stage of the menstrual cycle. We observed a significant increase (>/=2-fold) in ADAMTS1, ADAMTS13, COL7A1, MMP3, MMP9, PECAM1, and THBS3 in the periovulatory phase compared to the follicular phase. Meanwhile, we observed a significant decrease (>/= 2-fold) in COL7A1, ICAM1, ITGA8, MMP16, MMP9, CLEC3B, SELE and TIMP2 in the lutheal phase compared to the periovulatory phase. Immunocytochemistry showed that MMP-3 and MMP-9 were localized in the endosalpinx during all phases of the menstrual cycle. Gelatin zymograms detected non-cycle-dependent protease activity. Conclusions Several extracellular matrix components were regulated throughout the menstrual cycle in a cyclic pattern, suggesting a possible steroid regulation and a role in tissue remodeling and FT functions. PMID:22897899

  3. Extracellular Matrix Protein-Coated Scaffolds Promote the Reversal of Diabetes After Extrahepatic Islet Transplantation

    PubMed Central

    Salvay, David M.; Rives, Christopher B.; Zhang, Xiaomin; Chen, Fei; Kaufman, Dixon B.; Lowe, William L.; Shea, Lonnie D.

    2008-01-01

    Background The survival and function of transplanted pancreatic islets is limited, owing in part to disruption of islet-matrix attachments during the isolation procedure. Using polymer scaffolds as a platform for islet transplantation, we investigated the hypothesis that replacement of key extracellular matrix components known to surround islets in vivo would improve graft function at an extrahepatic implantation site. Methods Microporous polymer scaffolds fabricated from copolymers of lactide and glycolide were adsorbed with collagen IV, fibronectin, laminin-332 or serum proteins before seeding with 125 mouse islets. Islet-seeded scaffolds were then implanted onto the epididymal fat pad of syngeneic mice with streptozotocin-induced diabetes. Nonfasting glucose levels, weight gain, response to glucose challenges, and histology were used to assess graft function for 10 months after transplantation. Results Mice transplanted with islets seeded onto scaffolds adsorbed with collagen IV achieved euglycemia fastest and their response to glucose challenge was similar to normal mice. Fibronectin and laminin similarly promoted euglycemia, yet required more time than collagen IV and less time than serum. Histopathological assessment of retrieved grafts demonstrated that coating scaffolds with specific extracellular matrix proteins increased total islet area in the sections and vessel density within the transplanted islets, relative to controls. Conclusions Extracellular matrix proteins adsorbed to microporous scaffolds can enhance the function of transplanted islets, with collagen IV maximizing graft function relative to the other proteins tested. These scaffolds enable the creation of well-defined microenvironments that promote graft efficacy at extrahepatic sites. PMID:18497687

  4. Extracellular Matrix Stiffness Controls VEGF Signaling and Processing in Endothelial Cells.

    PubMed

    Sack, Kelsey D; Teran, Madelane; Nugent, Matthew A

    2016-09-01

    Vascular endothelial growth factor A (VEGF) drives endothelial cell maintenance and angiogenesis. Endothelial cell behavior is altered by the stiffness of the substrate the cells are attached to suggesting that VEGF activity might be influenced by the mechanical cellular environment. We hypothesized that extracellular matrix (ECM) stiffness modifies VEGF-cell-matrix tethering leading to altered VEGF processing and signaling. We analyzed VEGF binding, internalization, and signaling as a function of substrate stiffness in endothelial cells cultured on fibronectin (Fn) linked polyacrylamide gels. Cell produced extracellular matrices on the softest substrates were least capable of binding VEGF, but the cells exhibited enhanced VEGF internalization and signaling compared to cells on all other substrates. Inhibiting VEGF-matrix binding with sucrose octasulfate decreased cell-internalization of VEGF and, inversely, heparin pre-treatment to enhance Fn-matrix binding of VEGF increased cell-internalization of VEGF regardless of matrix stiffness. β1 integrins, which connect cells to Fn, modulated VEGF uptake in a stiffness dependent fashion. Cells on hard surfaces showed decreased levels of activated β1 and inhibition of β1 integrin resulted in a greater proportional decrease in VEGF internalization than in cells on softer matrices. Extracellular matrix binding is necessary for VEGF internalization. Stiffness modifies the coordinated actions of VEGF-matrix binding and β1 integrin binding/activation, which together are critical for VEGF internalization. This study provides insight into how the microenvironment may influence tissue regeneration and response to injury and disease. J. Cell. Physiol. 231: 2026-2039, 2016. © 2016 Wiley Periodicals, Inc.

  5. An extracellular matrix-based mechanism of rapid neutrophil extracellular trap formation in response to C. albicans1

    PubMed Central

    Byrd, Angel S.; O’Brien, Xian M.; Johnson, Courtney M.; Lavigne, Liz M.; Reichner, Jonathan S.

    2013-01-01

    The armament of neutrophil-mediated host defense against pathogens includes the extrusion of a lattice of DNA and microbicidal enzymes known as Neutrophil Extracellular Traps (NETs). The receptor:ligand interactions and intracellular signaling mechanisms responsible for elaborating NETs were determined for the response to Candida albicans. Since the host response of extravasated neutrophils to mycotic infections within tissues necessitates contact with ECM, this study also identified a novel and significant regulatory role for the ubiquitous matrix component fibronectin (Fn) in NET release. We report that recognition of purified fungal pathogen-associated molecular pattern β-glucan by human neutrophils causes rapid (≤ 30 mins) homotypic aggregation and NET release by a mechanism that requires Fn. Alone, immobilized β-glucan induces reactive oxygen species (ROS) production but not NET release, whereas in the context of Fn, ROS production is suppressed and NETs are extruded. NET release to Fn + β-glucan is robust, accounting for 17.2 ± 3.4% of total DNA in the cell population. Release is dependent on β-glucan recognition by CR3 (CD11b/CD18), but not Dectin-1, or ROS. The process of NET release included filling of intracellular vesicles with nuclear material that was eventually extruded. We identify a role for ERK in homotypic aggregation and NET release. NET formation to C. albicans hyphae was also found to depend on β-glucan recognition by CR3, require Fn and ERK but not ROS, and result in hyphal destruction. We report a new regulatory mechanism of NETosis in which the extracellular matrix is a key component of the rapid anti-fungal response. PMID:23509360

  6. Heterogeneous expression of extracellular matrix molecules in the red nucleus of the rat.

    PubMed

    Rácz, É; Gaál, B; Matesz, C

    2016-05-13

    Previous studies in our laboratory showed that the organization and heterogeneous molecular composition of extracellular matrix is associated with the variable cytoarchitecture, connections and specific functions of the vestibular nuclei and two related areas of the vestibular neural circuits, the inferior olive and prepositus hypoglossi nucleus. The aim of the present study is to reveal the organization and distribution of various molecular components of extracellular matrix in the red nucleus, a midbrain premotor center. Morphologically and functionally the red nucleus is comprised of the magno- and parvocellular parts, with overlapping neuronal population. By using histochemical and immunohistochemical methods, the extracellular matrix appeared as perineuronal net, axonal coat, perisynaptic matrix or diffuse network in the neuropil. In both parts of the red nucleus we have observed positive hyaluronan, tenascin-R, link protein, and lectican (aggrecan, brevican, versican, neurocan) reactions. Perineuronal nets were detected with each of the reactions and the aggrecan showed the most intense staining in the pericellular area. The two parts were clearly distinguished on the basis of neurocan and HAPLN1 expression as they have lower intensity in the perineuronal nets of large cells and in the neuropil of the magnocellular part. Additionally, in contrast to this pattern, the aggrecan was heavily labeled in the magnocellular region sharply delineating from the faintly stained parvocellular area. The most characteristic finding was that the appearance of perineuronal nets was related with the neuronal size independently from its position within the two subdivisions of red nucleus. In line with these statements none of the extracellular matrix molecules were restricted exclusively to the magno- or parvocellular division. The chemical heterogeneity of the perineuronal nets may support the recently accepted view that the red nucleus comprises more different populations of

  7. Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development

    PubMed Central

    Zhang, Weipeng; Sun, Jin; Ding, Wei; Lin, Jinshui; Tian, Renmao; Lu, Liang; Liu, Xiaofen; Shen, Xihui; Qian, Pei-Yuan

    2015-01-01

    Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm. PMID:26029669

  8. Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix.

    PubMed

    Stephenson, Matthew K; Lenihan, Sean; Covarrubias, Roman; Huttinger, Ryan M; Gumina, Richard J; Sawyer, Douglas B; Galindo, Cristi L

    2016-06-14

    Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

  9. Matrix Cracking in 3D Orthogonal Melt-Infiltrated SiC/SiC Composites with Various Z-Fiber Types

    NASA Technical Reports Server (NTRS)

    Morscher, Gregory N.; Yun, Hee Mann; DiCarlo, James A.

    2003-01-01

    The occurrence of matrix cracks in melt-infiltrated SiC/SiC composites with a 3D orthogonal architecture was determined at room temperature for specimens tested in tension oriented in the X-direction (parallel to Z-bundle weave direction) and Y-direction (perpendicular to Z-bundle weave direction) and Y-direction (perpendicular to Z-bundle weave direction). The fiber-types were Sylramic and Sylramic-IBN in the X and Y-directions and lower modulus ZMI, T300, and rayon in the Z-direction. Acoustic emission (AE) was used to monitor the matrix cracking activity. For Y-direction composites, the AE data was used to determine the exact (+/- 0.25 mm) location where matrix cracks occurred in the 3D orthogonal architecture. This enabled the determination of the stress-dependent matrix crack distributions for small but repeatable matrix rich 'unidirectional' and the matrix poor 'cross-ply' regions within the architecture. It was found that matrix cracking initiated at very low stresses (approx. 40 MPa) in the 'unidirectional' regions for the largest z-direction fiber tow composites. Decreasing the size of the z-fiber bundle, increased the stress for matrix cracking in the 'unidirectional' regions. Matrix cracking in the 'cross-ply' regions always occurred at higher stresses than in 'unidirectional' regions, and the stress-dependent matrix crack distribution of the 'cross-ply' regions was always over a wider stress-range than the 'unidirectional' regions. For composites tested in the X-direction, a lower elastic modulus and a narrower and lower stress-range for matrix cracking were observed compared to composites tested in the Y-direction.

  10. [Molecular characteristics of leiomyoma uteri based on selected compounds of the extracellular matrix].

    PubMed

    Auguściak-Duma, Aleksandra; Sieroń, Aleksander L

    2008-01-14

    Leiomyoma is a monoclonal benign tumor. It is often located in the muscle layer of the uterus in women of reproductive age. Its growth is accelerated by pregnancy and hormonal therapy. Its growth also depends on the concentration of sex hormones. Growth factors and cytokines may also participate in the formation of leiomyomas. The modulation of mitotic activity and abnormal extracellular matrix production are key elements of tumor growth. Elements of the TGFbeta superfamily are crucial factors in the proliferation of neoplasmic cells. TGF-beta1 and -beta3 stimulate the synthesis of various components of the extracellular matrix, but they also down-regulate the synthesis of proteinases which degrade the matrix, often leading to excessive overdeposition of connective tissue. Collagen types 1 and 3 are the main structural components of the extracellular matrix. The biosynthesis of collagens requires, among others, the action of procollagen C-endopeptidase, a protein of the BMP-1/mTLD subfamily. BMP-1/mTLD-like proteinases remove the carboxyl propeptides of procollagens 1, 2, and 3. Removal of the C-propeptides decreases the solubility of procollagens about 1000-fold to a concentration critical for their spontaneous self-assembly to collagen fibrils. Different substrates of BMP-1/mTLD are prolysyl oxidase, gamma2 chain of prolaminin, procollagen type VII, miostatin, dentin matrix protein 1, and perlekan. Due to the activation of various substrates by BMP-1/mTLDs, they are important regulators of the production of the extracellular matrix and its quality as well as of antiangiogenic responses by producing a factor from the basal membrane compound called perlekan. The BMP-1/mTLDs influence the formation of dorsal ventral patterning in embryos by releasing BMP-2/4 from the inhibitory protein chordin. Another aspect is induction of the development of muscle and neural tissue by activation of GDF8 and GDF11 as well as the regulation of growth and cell proliferation by

  11. Objective Assessment and Design Improvement of a Staring, Sparse Transducer Array by the Spatial Crosstalk Matrix for 3D Photoacoustic Tomography

    PubMed Central

    Kosik, Ivan; Raess, Avery

    2015-01-01

    Accurate reconstruction of 3D photoacoustic (PA) images requires detection of photoacoustic signals from many angles. Several groups have adopted staring ultrasound arrays, but assessment of array performance has been limited. We previously reported on a method to calibrate a 3D PA tomography (PAT) staring array system and analyze system performance using singular value decomposition (SVD). The developed SVD metric, however, was impractical for large system matrices, which are typical of 3D PAT problems. The present study consisted of two main objectives. The first objective aimed to introduce the crosstalk matrix concept to the field of PAT for system design. Figures-of-merit utilized in this study were root mean square error, peak signal-to-noise ratio, mean absolute error, and a three dimensional structural similarity index, which were derived between the normalized spatial crosstalk matrix and the identity matrix. The applicability of this approach for 3D PAT was validated by observing the response of the figures-of-merit in relation to well-understood PAT sampling characteristics (i.e. spatial and temporal sampling rate). The second objective aimed to utilize the figures-of-merit to characterize and improve the performance of a near-spherical staring array design. Transducer arrangement, array radius, and array angular coverage were the design parameters examined. We observed that the performance of a 129-element staring transducer array for 3D PAT could be improved by selection of optimal values of the design parameters. The results suggested that this formulation could be used to objectively characterize 3D PAT system performance and would enable the development of efficient strategies for system design optimization. PMID:25875177

  12. Helium ion microscopy of enamel crystallites and extracellular tooth enamel matrix

    PubMed Central

    Bidlack, Felicitas B.; Huynh, Chuong; Marshman, Jeffrey; Goetze, Bernhard

    2014-01-01

    An unresolved problem in tooth enamel studies has been to analyze simultaneously and with sufficient spatial resolution both mineral and organic phases in their three dimensional (3D) organization in a given specimen. This study aims to address this need using high-resolution imaging to analyze the 3D structural organization of the enamel matrix, especially amelogenin, in relation to forming enamel crystals. Chemically fixed hemi-mandibles from wild type mice were embedded in LR White acrylic resin, polished and briefly etched to expose the organic matrix in developing tooth enamel. Full-length amelogenin was labeled with specific antibodies and 10 nm immuno-gold. This allowed us to use and compare two different high-resolution imaging techniques for the analysis of uncoated samples. Helium ion microscopy (HIM) was applied to study the spatial organization of organic and mineral structures, while field emission scanning electron microscopy (FE-SEM) in various modes, including backscattered electron detection, allowed us to discern the gold-labeled proteins. Wild type enamel in late secretory to early maturation stage reveals adjacent to ameloblasts a lengthwise parallel alignment of the enamel matrix proteins, including full-length amelogenin proteins, which then transitions into a more heterogeneous appearance with increasing distance from the mineralization front. The matrix adjacent to crystal bundles forms a smooth and lacey sheath, whereas between enamel prisms it is organized into spherical components that are interspersed with rod-shaped protein. These findings highlight first, that the heterogeneous organization of the enamel matrix can be visualized in mineralized en bloc samples. Second, our results illustrate that the combination of these techniques is a powerful approach to elucidate the 3D structural organization of organic matrix molecules in mineralizing tissue in nanometer resolution. PMID:25346697

  13. Soil organic matter and the extracellular microbial matrix show contrasting responses to C and N availability

    PubMed Central

    Redmile-Gordon, M.A.; Evershed, R.P.; Hirsch, P.R.; White, R.P.; Goulding, K.W.T.

    2015-01-01

    An emerging paradigm in soil science suggests microbes can perform ‘N mining’ from recalcitrant soil organic matter (SOM) in conditions of low N availability. However, this requires the production of extracellular structures rich in N (including enzymes and structural components) and thus defies stoichiometric expectation. We set out to extract newly synthesised peptides from the extracellular matrix in soil and compare the amino acid (AA) profiles, N incorporation and AA dynamics in response to labile inputs of contrasting C/N ratio. Glycerol was added both with and without an inorganic source of N (10% 15N labelled NH4NO3) to a soil already containing a large pool of refractory SOM and incubated for 10 days. The resulting total soil peptide (TSP) and extracellular pools were compared using colorimetric methods, gas chromatography, and isotope ratio mass spectrometry. N isotope compositions showed that the extracellular polymeric substance (EPS) contained a greater proportion of products formed de novo than did TSP, with hydrophobic EPS-AAs (leucine, isoleucine, phenylalanine, hydroxyproline and tyrosine) deriving substantially more N from the inorganic source provided. Quantitative comparison between extracts showed that the EPS contained greater relative proportions of alanine, glycine, proline, phenylalanine and tyrosine. The greatest increases in EPS-peptide and EPS-polysaccharide concentrations occurred at the highest C/N ratios. All EPS-AAs responded similarly to treatment whereas the responses of TSP were more complex. The results suggest that extracellular investment of N (as EPS peptides) is a microbial survival mechanism in conditions of low N/high C which, from an evolutionary perspective, must ultimately lead to the tendency for increased N returns to the microbial biomass. A conceptual model is proposed that describes the dynamics of the extracellular matrix in response to the C/N ratio of labile inputs. PMID:26339106

  14. [Extracellular matrix as a microbial virulence factor in the development of human diseases].

    PubMed

    Moryl, Magdalena

    2015-01-01

    Extracellular polymers which build a biofilm matrix possess a complicated structure, where the polysaccharide fraction, composed of homo- or heteropolysaccharides, is the largest. Other important components are proteins, eDNA, glycoproteins and lipids. The matrix has a protective function against the surrounding environment, plays a role in biofilm formation and maturation processes, stabilizes the biofilm structure, and also is a source of nutrients and water for the cells. It is noteworthy that the biofilm matrix is a virulence factor and plays an important role in the pathogenesis of many human diseases. Pseudomonas aeruginosa growing in the lungs of patients with cystic fibrosis produces three major exopolysaccharides (Pel, Psl and alginate) and synthesizes numerous proteins such as lectins and enzymes, e.g. PasP, chitinase, aminopeptidase, and protease IV, which facilitate the tissue colonization. Extracellular polymers play a significant role in the course of caries, which is associated with the development of multi-species biofilm on the teeth surface. The structure of the matrix surrounding that biofilm is complicated--different for each patient. The components of the matrix are constantly changing depending on the environmental conditions, e.g. the presence of sucrose affects the synthesis of mutan and dextran. Infections associated with biofilm formation on implants pose significant medical and economic problems. The main components of the matrices are saccharides (e.g., PIA, EC-TA), as well as surface and extracellular proteins. Studies on the matrix structure and the factors regulating its synthesis are necessary to develop techniques for biofilm eradication and better control of biofilm-related infections.

  15. Moderate cyclic tensile strain alters the assembly of cartilage extracellular matrix proteins in vitro.

    PubMed

    Bleuel, Judith; Zaucke, Frank; Brüggemann, Gert-Peter; Heilig, Juliane; Wolter, Marie-Louise; Hamann, Nina; Firner, Sara; Niehoff, Anja

    2015-06-01

    Mechanical loading influences the structural and mechanical properties of articular cartilage. The cartilage matrix protein collagen II essentially determines the tensile properties of the tissue and is adapted in response to loading. The collagen II network is stabilized by the collagen II-binding cartilage oligomeric matrix protein (COMP), collagen IX, and matrilin-3. However, the effect of mechanical loading on these extracellular matrix proteins is not yet understood. Therefore, the aim of this study was to investigate if and how chondrocytes assemble the extracellular matrix proteins collagen II, COMP, collagen IX, and matrilin-3 in response to mechanical loading. Primary murine chondrocytes were applied to cyclic tensile strain (6%, 0.5 Hz, 30 min per day at three consecutive days). The localization of collagen II, COMP, collagen IX, and matrilin-3 in loaded and unloaded cells was determined by immunofluorescence staining. The messenger ribo nucleic acid (mRNA) expression levels and synthesis of the proteins were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and western blots. Immunofluorescence staining demonstrated that the pattern of collagen II distribution was altered by loading. In loaded chondrocytes, collagen II containing fibrils appeared thicker and strongly co-stained for COMP and collagen IX, whereas the collagen network from unloaded cells was more diffuse and showed minor costaining. Further, the applied load led to a higher amount of COMP in the matrix, determined by western blot analysis. Our results show that moderate cyclic tensile strain altered the assembly of the extracellular collagen network. However, changes in protein amount were only observed for COMP, but not for collagen II, collagen IX, or matrilin-3. The data suggest that the adaptation to mechanical loading is not always the result of changes in RNA and/or protein expression but might also be the result of changes in matrix assembly and structure.

  16. Regulation of Extracellular Matrix Remodeling Proteins by Osteoblasts in Titanium Nanoparticle-Induced Aseptic Loosening Model.

    PubMed

    Xie, Jing; Hou, Yanhua; Fu, Na; Cai, Xiaoxiao; Li, Guo; Peng, Qiang; Lin, Yunfeng

    2015-10-01

    Titanium (Ti)-wear particles, formed at the bone-implant interface, are responsible for aseptic loosening, which is a main cause of total joint replacement failure. There have been many studies on Ti particle-induced function changes in mono-cultured osteoblasts and synovial cells. However, little is known on extracellular matrix remodeling displayed by osteoblasts when in coexistence with Synovial cells. To further mimic the bone-implant interface environment, we firstly established a nanoscaled-Ti particle-induced aseptic loosening system by co-culturing osteoblasts and Synovial cells. We then explored the impact of the Synovial cells on Ti particle-engulfed osteoblasts in the mimicked flamed niche. The matrix metalloproteinases and lysyl oxidases expression levels, two protein families which are critical in osseointegration, were examined under induction by tumor necrosis factor-alpha. It was found that the co-culture between the osteoblasts and Synovial cells markedly increased the migration and proliferation of the osteoblasts, even in the Ti-particle engulfed osteoblasts. Importantly, the Ti-particle engulfed osteoblasts, induced by TNF-alpha after the co-culture, enhanced the release of the matrix metalloproteinases and reduced the expressions of lysyl oxidases. The regulation of extracellular matrix remodeling at the protein level was further assessed by investigations on gene expression of the matrix metalloproteinases and lysyl oxidases, which also suggested that the regulation started at the genetic level. Our research work has therefore revealed the critical role of multi cell-type interactions in the extracellular matrix remodeling within the peri-prosthetic tissues, which provides new insights on aseptic loosening and brings new clues about incomplete osseointegration between the implantation materials and their surrounding bones. PMID:26502645

  17. Designer Extracellular Matrix Based on DNA-Peptide Networks Generated by Polymerase Chain Reaction.

    PubMed

    Finke, Alexander; Bußkamp, Holger; Manea, Marilena; Marx, Andreas

    2016-08-16

    Cell proliferation and differentiation in multicellular organisms are partially regulated by signaling from the extracellular matrix. The ability to mimic an extracellular matrix would allow particular cell types to be specifically recognized, which is central to tissue engineering. We present a new functional DNA-based material with cell-adhesion properties. It is generated by using covalently branched DNA as primers in PCR. These primers were functionalized by click chemistry with the cyclic peptide c(RGDfK), a peptide that is known to predominantly bind to αvβ3 integrins, which are found on endothelial cells and fibroblasts, for example. As a covalent coating of surfaces, this DNA-based material shows cell-repellent properties in its unfunctionalized state and gains adhesiveness towards specific target cells when functionalized with c(RGDfK). These cells remain viable and can be released under mild conditions by DNase I treatment.

  18. How cells sense extracellular matrix stiffness: a material’s perspective

    PubMed Central

    Trappmann, Britta; Chen, Christopher S.

    2014-01-01

    The mechanical properties of the extracellular matrix (ECM) in which cells reside have emerged as an important regulator of cell fate. While materials based on natural ECM have been used to implicate the role of substrate stiffness for cell fate decisions, it is difficult in these matrices to isolate mechanics from other structural parameters. In contrast, fully synthetic hydrogels offer independent control over physical and adhesive properties. New synthetic materials that also recreate the fibrous structural hierarchy of natural matrices are now being designed to study substrate mechanics in more complex ECMs. This perspective examines the ways in which new materials are being used to advance our understanding of how extracellular matrix stiffness impacts cell function. PMID:23611564

  19. Oral lichen sclerosus expressing extracellular matrix proteins and IgG4-positive plasma cells.

    PubMed

    De Aquino Xavier, Flavia Calo; Prates, Alisio Alves; Gurgel, Clarissa Araujo; De Souza, Tulio Geraldo; Andrade, Rodrigo Guimaraes; Goncalves Ramos, Eduardo Antonio; Pedreira Ramalho, Luciana Maria; Dos Santos, Jean Nunes

    2014-09-16

    Lichen sclerosus (LS) is a mucocutaneous disease with uncommon oral involvement. The etiology is not yet well understood, but LS has been associated with autoimmune, genetic, and immunological factors. We report a 47-year-old man with LS that exhibited an asymptomatic white plaque with red patches on the maxillary alveolar mucosa extending to the labial mucosa. He had no other skin disease. Positive immunostaining for tenascin and scarcity of fibronectin suggested extracellular matrix reorganization. Elastin immunostaining indicated a reduction of elastic fibers. Immunoexpression of collagen IV in blood vessels and its absence in the epithelial basement membrane, together with diffuse MMP-9 immunoexpression, suggested altered proteolytic activity. Mast cell staining bordering areas of sclerosis indicated a possible role in the synthesis of collagen. IgG4 positivity in plasma cells suggested a role in the fibrogenesis. This is an unusual presentation of oral LS and we discuss immunohistochemical findings regarding cellular and extracellular matrix components.

  20. How Osteoblasts Sense their Environment: Integrin-Extracellular Matrix Interactions and Mechanical Loading of Bone

    NASA Technical Reports Server (NTRS)

    Globus, Ruth K.; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    Osteoblasts are the cells responsible for forming and replacing bone throughout life. We know that mechanical stimulation through weight-bearing at I gravity on Earth is needed to maintain healthy bone, and that osteoblasts play a critical role in that process. Over the last 9 years in my laboratory at NASA ARC, we have studied the regulation of osteoblast function by interactions between the extracellular matrix and die cell. Using a cell culture approach, we defined the repertoire of adhesion receptors, called integrins, which are expressed on the osteoblast surface, as well as specific extracellular matrix proteins, which are needed for cellular differentiation and survival. We are now extending these observations to determine if integrin signaling is involved in the skeletal responses to disuse and recovery from disuse using the rodent model of hindlimb unloading by tail suspension. Together, our cell culture and animal studies are providing new insight into the regulation of osteoblast function in bone.

  1. Label-free Imaging of Arterial Cells and Extracellular Matrix Using a Multimodal CARS Microscope.

    PubMed

    Wang, Han-Wei; Le, Thuc T; Cheng, Ji-Xin

    2008-04-01

    A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH(2)-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH(2) bonds. The extracellular matrix organization were further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases.

  2. Label-free Imaging of Arterial Cells and Extracellular Matrix Using a Multimodal CARS Microscope

    PubMed Central

    Wang, Han-Wei; Le, Thuc T.; Cheng, Ji-Xin

    2008-01-01

    A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH2-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH2 bonds. The extracellular matrix organization were further confirmed by TPEF signals arising from elastin’s autofluorescence and SFG signals arising from collagen fibrils’ non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases. PMID:19343073

  3. Designer Extracellular Matrix Based on DNA-Peptide Networks Generated by Polymerase Chain Reaction.

    PubMed

    Finke, Alexander; Bußkamp, Holger; Manea, Marilena; Marx, Andreas

    2016-08-16

    Cell proliferation and differentiation in multicellular organisms are partially regulated by signaling from the extracellular matrix. The ability to mimic an extracellular matrix would allow particular cell types to be specifically recognized, which is central to tissue engineering. We present a new functional DNA-based material with cell-adhesion properties. It is generated by using covalently branched DNA as primers in PCR. These primers were functionalized by click chemistry with the cyclic peptide c(RGDfK), a peptide that is known to predominantly bind to αvβ3 integrins, which are found on endothelial cells and fibroblasts, for example. As a covalent coating of surfaces, this DNA-based material shows cell-repellent properties in its unfunctionalized state and gains adhesiveness towards specific target cells when functionalized with c(RGDfK). These cells remain viable and can be released under mild conditions by DNase I treatment. PMID:27410200

  4. Tumor Cell Invasion Can Be Blocked by Modulators of Collagen Fibril Alignment That Control Assembly of the Extracellular Matrix.

    PubMed

    Grossman, Moran; Ben-Chetrit, Nir; Zhuravlev, Alina; Afik, Ran; Bassat, Elad; Solomonov, Inna; Yarden, Yosef; Sagi, Irit

    2016-07-15

    Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECM-associated pathologies. Cancer Res; 76(14); 4249-58. ©2016 AACR.

  5. Biological functionality and mechanistic contribution of extracellular matrix-ornamented three dimensional Ti-6Al-4V mesh scaffolds.

    PubMed

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

    2016-11-01

    The 3D printed metallic implants are considered bioinert in nature because of the absence of bioactive molecules. Thus, surface modification of bioinert materials is expected to favorably promote osteoblast functions and differentiation. In this context, the objective of this study is to fundamentally elucidate the effect of cell-derived decellularized extracellular matrix (dECM) ornamented 3D printed Ti-6Al-4V scaffolds on biological functions, involving cell adhesion, proliferation, and synthesis of vinculin and actin proteins. To mimic the natural ECM environment, the mineralized ECM of osteoblasts was deposited on the Ti-6Al-4V porous scaffolds, fabricated by electron beam melting (EBM) method. The process comprised of osteoblast proliferation, differentiation, and freeze-thaw cycles to obtain decellularized extra cellular matrix (dECM), in vitro. The dECM provided a natural environment to restore the natural cell functionality of osteoblasts that were cultured on dECM ornamented Ti-6Al-4V scaffolds. In comparison to the bare Ti-6Al-4V scaffolds, a higher cell functionality such as cell adhesion, proliferation, and growth including cell-cell and cell-material interaction were observed on dECM ornamented Ti-6Al-4V scaffolds, which were characterized by using markers for focal adhesion and cytoskeleton such as vinculin and actin. Moreover, electron microscopy also indicated higher cell-material interaction and enhanced proliferation of cells on dECM ornamented Ti-6Al-4V scaffolds, supported by MTT assay. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2751-2763, 2016.

  6. Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland

    SciTech Connect

    Ehrhart, E.J.; Gillette, E.L.; Barcellos-Hoff, M.H.

    1996-02-01

    High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by {gamma} irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin inummoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation. 40 refs., 3 figs.

  7. Immunohistochemical evidence of rapid extracellular matrix remodeling after iron-particle irradiation of mouse mammary gland

    NASA Technical Reports Server (NTRS)

    Ehrhart, E. J.; Gillette, E. L.; Barcellos-Hoff, M. H.; Chaterjee, A. (Principal Investigator)

    1996-01-01

    High-LET radiation has unique physical and biological properties compared to sparsely ionizing radiation. Recent studies demonstrate that sparsely ionizing radiation rapidly alters the pattern of extracellular matrix expression in several tissues, but little is known about the effect of heavy-ion radiation. This study investigates densely ionizing radiation-induced changes in extracellular matrix localization in the mammary glands of adult female BALB/c mice after whole-body irradiation with 0.8 Gy 600 MeV iron particles. The basement membrane and interstitial extracellular matrix proteins of the mammary gland stroma were mapped with respect to time postirradiation using immunofluorescence. Collagen III was induced in the adipose stroma within 1 day, continued to increase through day 9 and was resolved by day 14. Immunoreactive tenascin was induced in the epithelium by day 1, was evident at the epithelial-stromal interface by day 5-9 and persisted as a condensed layer beneath the basement membrane through day 14. These findings parallel similar changes induced by gamma irradiation but demonstrate different onset and chronicity. In contrast, the integrity of epithelial basement membrane, which was unaffected by sparsely ionizing radiation, was disrupted by iron-particle irradiation. Laminin immunoreactivity was mildly irregular at 1 h postirradiation and showed discontinuities and thickening from days 1 to 9. Continuity was restored by day 14. Thus high-LET radiation, like sparsely ionizing radiation, induces rapid-remodeling of the stromal extracellular matrix but also appears to alter the integrity of the epithelial basement membrane, which is an important regulator of epithelial cell proliferation and differentiation.

  8. An Assay to Quantify Chemotactic Properties of Degradation Products from Extracellular Matrix

    PubMed Central

    Sicari, Brian M.; Zhang, Li; Londono, Ricardo; Badylak, Stephen F.

    2015-01-01

    The endogenous chemotaxis of cells toward sites of tissue injury and/or biomaterial implantation is an important component of the host response. Implanted biomaterials capable of recruiting host stem/progenitor cells to a site of interest may obviate challenges associated with cell transplantation. An assay for the identification and quantification of chemotaxis induced by surgically placed biologic scaffolds composed of extracellular matrix is described herein. PMID:24155230

  9. Differential expression of extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in avian tibial dyschondroplasia.

    PubMed

    Shahzad, Muhammad; Liu, Jingying; Gao, Jianfeng; Wang, Zhi; Zhang, Ding; Nabi, Fazul; Li, Kun; Li, Jiakui

    2015-01-01

    Tibial dyschondroplasia (TD) is an avian bone disorder of different aetiologies that may be associated with lameness. The disorder is characterized by focal disruption of endochondral bone formation, with a lack of matrix proteolysis and an accumulation of non-mineralized avascular cartilage. The aim of this study was to determine the expression of extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in normal, thiram-induced TD lesions and in the process of recovery from TD in broiler chickens. An extracellular matrix (ECM) degrading enzyme, matrix metalloproteinase-9 (MMP-9), was selected to investigate the effects of CD147 in the degradation of ECM. Gene expression was analysed by quantitative real-time polymerase chain reaction and protein levels by immunohistochemistry and western blotting. The birds were divided into three groups: thiram fed; recovery; and controls. Genes encoding CD147 and MMP-9 were down-regulated during the development of the disease, and were up-regulated during recovery. Western blotting also showed lower protein levels of CD147 in TD, which increased during the recovery phase associated with ECM degradation and growth plate repair. The findings of this study suggest that ECM has a crucial role in the occurrence of TD and that CD147 appears to play a pivotal role in matrix proteolysis in the chicken, similar to that in other species.

  10. FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells

    PubMed Central

    2011-01-01

    Background Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer. Methods We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses. Results We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated. Conclusion Cancer cell invasiveness can be affected by

  11. Extracellular matrix remodeling in wound healing of critical size defects in the mitral valve leaflet.

    PubMed

    Stephens, Elizabeth H; Nguyen, Tom C; Blazejewski, Jack G; Vekilov, Dragoslava P; Connell, Jennifer P; Itoh, Akinobu; Ingels, Neil B; Miller, D Craig; Grande-Allen, K Jane

    2016-07-01

    The details of valvular leaflet healing following valvuloplasty and leaflet perforation from endocarditis are poorly understood. In this study, the synthesis and turnover of valvular extracellular matrix due to healing of a critical sized wound was investigated. Twenty-nine sheep were randomized to either CTRL (n = 11) or HOLE (n = 18), in which a 2.8-4.8 mm diameter hole was punched in the posterior mitral leaflet. After 12 weeks, posterior leaflets were harvested and histologically stained to localize extracellular matrix components. Immunohistochemistry was also performed to assess matrix components and markers of matrix turnover. A semi-quantitative grading scale was used to quantify differences between HOLE and CTRL. After 12 weeks, the hole diameter was reduced by 71.3 ± 1.4 % (p < 0.001). Areas of remodeling surrounding the hole contained more activated cells, greater expression of proteoglycans, and markers of matrix turnover (prolyl 4-hydroxylase, metalloproteases, and lysyl oxidase, each p ≤ 0.025), along with fibrin accumulation. Two distinct remodeling regions were evident surrounding the hole, one directly bordering the hole rich in versican and hyaluronan and a second adjacent region with abundant collagen and elastic fiber turnover. The remodeling also caused reduced delineation between valve layers (p = 0.002), more diffuse staining of matrix components and markers of matrix turnover (p < 0.001), and disruption of the collagenous fibrosa. In conclusion, acute valve injury elicited distinct, heterogeneous alterations in valvular matrix composition and structure, resulting in partial wound closure. Because these changes could also affect leaflet mechanics and valve function, it will be important to determine their impact on healing wounds.

  12. Human Lung Cancer Cells Grown in an Ex Vivo 3D Lung Model Produce Matrix Metalloproteinases Not Produced in 2D Culture

    PubMed Central

    Mishra, Dhruva K.; Sakamoto, Jason H.; Thrall, Michael J.; Baird, Brandi N.; Blackmon, Shanda H.; Ferrari, Mauro; Kurie, Jonathan M.; Kim, Min P.

    2012-01-01

    We compared the growth of human lung cancer cells in an ex vivo three-dimensional (3D) lung model and 2D culture to determine which better mimics lung cancer growth in patients. A549 cells were grown in an ex vivo 3D lung model and in 2D culture for 15 days. We measured the size and formation of tumor nodules and counted the cells after 15 days. We also stained the tissue/cells for Ki-67, and Caspase-3. We measured matrix metalloproteinase (MMP) levels in the conditioned media and in blood plasma from patients with adenocarcinoma of the lung. Organized tumor nodules with intact vascular space formed in the ex vivo 3D lung model but not in 2D culture. Proliferation and apoptosis were greater in the ex vivo 3D lung model compared to the 2D culture. After 15 days, there were significantly more cells in the 2D culture than the 3D model. MMP-1, MMP-9, and MMP-10 production were significantly greater in the ex vivo 3D lung model. There was no production of MMP-9 in the 2D culture. The patient samples contained MMP-1, MMP-2, MMP-9, and MMP-10. The human lung cancer cells grown on ex vivo 3D model form perfusable nodules that grow over time. It also produced MMPs that were not produced in 2D culture but seen in human lung cancer patients. The ex vivo 3D lung model may more closely mimic the biology of human lung cancer development than the 2D culture. PMID:23028922

  13. Platelets and plasma stimulate sheep rotator cuff tendon tenocytes when cultured in an extracellular matrix scaffold.

    PubMed

    Kelly, Brian A; Proffen, Benedikt L; Haslauer, Carla M; Murray, Martha M

    2016-04-01

    The addition of platelet-rich plasma (PRP) to rotator cuff repair has not translated into improved outcomes after surgery. However, recent work stimulating ligament healing has demonstrated improved outcomes when PRP or whole blood is combined with an extracellular matrix carrier. The objective of this study was to evaluate the effect of three components of blood (plasma, platelets, and macrophages) on the in vitro activity of ovine rotator cuff cells cultured in an extracellular matrix environment. Tenocytes were obtained from six ovine infraspinatus tendons and cultured over 14 days in an extracellular matrix scaffold with the following additives: (1) plasma (PPP), (2) plasma and platelets (PAP), (3) plasma and macrophages (PPPM), (4) plasma, platelets and macrophages (PAPM), (5) phosphate buffered saline (PBS), and (6) PBS with macrophages (PBSM). Assays measuring cellular metabolism (AlamarBlue), proliferation (Quantitative DNA assay), synthesis of collagen and cytokines (SIRCOL, TNF-α and IL-10 ELISA, and MMP assay), and collagen gene expression (qPCR) were performed over the duration of the experiment, as well as histology at the conclusion. Plasma was found to stimulate cell attachment and spreading on the scaffold, as well as cellular proliferation. Platelets also stimulated cell proliferation, cellular metabolism, transition of cells to a myofibroblast phenotype, and contraction of the scaffolds. The addition of macrophages did not have any significant effect on the sheep rotator cuff cells in vitro. In vivo studies are needed to determine whether these changes in cellular function will translate into improved tendon healing.

  14. Quantitation and relative distribution of extracellular matrix in Staphylococcus epidermidis biofilm

    SciTech Connect

    Van Pett, K.; Schurman, D.J.; Smith, R.L. )

    1990-05-01

    The relationship between adherence of bacteria to foreign bodies and their deposition of extracellular matrix was examined on glass and suture material. To quantitate bacterial adherence, uptake of ({sup 3}H)thymidine into bacterial DNA was analyzed. Corresponding amounts of extracellular matrix were measured by a new technique using ({sup 14}C)glucose incorporation. This study shows that ({sup 14}C)glucose preferentially labeled bacterial strains in proportion to biofilm production. The ratio of {sup 3}H{sup 14}C in high biofilm producers was 0.9 and in low producers it was 3.7. Radioactive identification of organisms as high and low producers was confirmed by electron microscopy. The results presented here show that production and accumulation of biofilm over time is a stable characteristic in different strains of S. epidermidis. The use of ratios reflecting radiolabeling of bacteria and biofilm by ({sup 3}H)thymidine and ({sup 14}C)glucose, respectively, is a quantitative yet simple technique to assess extracellular matrix of different strains of S. epidermidis.

  15. Fast and efficient fully 3D PET image reconstruction using sparse system matrix factorization with GPU acceleration

    NASA Astrophysics Data System (ADS)

    Zhou, Jian; Qi, Jinyi

    2011-10-01

    Statistically based iterative image reconstruction has been widely used in positron emission tomography (PET) imaging. The quality of reconstructed images depends on the accuracy of the system matrix that defines the mapping from the image space to the data space. However, an accurate system matrix is often associated with high computation cost and huge storage requirement. In this paper, we present a method to address this problem using sparse matrix factorization and graphics processor unit (GPU) acceleration. We factor the accurate system matrix into three highly sparse matrices: a sinogram blurring matrix, a geometric projection matrix and an image blurring matrix. The geometrical projection matrix is precomputed based on a simple line integral model, while the sinogram and image blurring matrices are estimated from point-source measurements. The resulting factored system matrix has far less nonzero elements than the original system matrix, which substantially reduces the storage and computation cost. The smaller matrix size also allows an efficient implementation of the forward and backward projectors on a GPU, which often has a limited memory space. Our experimental studies show that the proposed method can dramatically reduce the computation cost of high-resolution iterative image reconstruction, while achieving better performance than existing factorization methods.

  16. Dynamic interactions between cells and their extracellular matrix mediate embryonic development.

    PubMed

    Goody, Michelle F; Henry, Clarissa A

    2010-06-01

    Cells and their surrounding extracellular matrix microenvironment interact throughout all stages of life. Understanding the continuously changing scope of cell-matrix interactions in vivo is crucial to garner insights into both congenital birth defects and disease progression. A current challenge in the field of developmental biology is to adapt in vitro tools and rapidly evolving imaging technology to study cell-matrix interactions in a complex 4-D environment. In this review, we highlight the dynamic modulation of cell-matrix interactions during development. We propose that individual cell-matrix adhesion proteins are best considered as complex proteins that can play multiple, often seemingly contradictory roles, depending upon the context of the microenvironment. In addition, cell-matrix proteins can also exert different short versus long term effects. It is thus important to consider cell behavior in light of the microenvironment because of the constant and dynamic reciprocal interactions occurring between them. Finally, we suggest that analysis of cell-matrix interactions at multiple levels (molecules, cells, tissues) in vivo is critical for an integrated understanding because different information can be acquired from all size scales. PMID:20108219

  17. Matrix Metalloproteinase 12-Deficiency Augments Extracellular Matrix Degrading Metalloproteinases and Attenuates IL-13–Dependent Fibrosis

    PubMed Central

    Madala, Satish K.; Pesce, John T.; Ramalingam, Thirumalai R.; Wilson, Mark S.; Minnicozzi, Samantha; Cheever, Allen W.; Thompson, Robert W.; Mentink-Kane, Margaret M.; Wynn, Thomas A.

    2011-01-01

    Infection with the parasitic helminth Schistosoma mansoni causes significant liver fibrosis and extracellular matrix (ECM) remodeling. Matrix metalloproteinases (MMP) are important regulators of the ECM by regulating cellular inflammation, extracellular matrix deposition, and tissue reorganization. MMP12 is a macrophage-secreted elastase that is highly induced in the liver and lung in response to S. mansoni eggs, confirmed by both DNA microarray and real-time PCR analysis. However, the function of MMP12 in chronic helminth-induced inflammation and fibrosis is unclear. In this study, we reveal that MMP12 acts as a potent inducer of inflammation and fibrosis after infection with the helminth parasite S. mansoni. Surprisingly, the reduction in liver and lung fibrosis in MMP12-deficient mice was not associated with significant changes in cytokine, chemokine, TGF-β1, or tissue inhibitors of matrix metalloproteinase expression. Instead, we observed marked increases in MMP2 and MMP13 expression, suggesting that Mmp12 was promoting fibrosis by limiting the expression of specific ECM-degrading MMPs. Interestingly, like MMP12, MMP13 expression was highly dependent on IL-13 and type II–IL-4 receptor signaling. However, in contrast to MMP12, expression of MMP13 was significantly suppressed by the endogenous IL-13 decoy receptor, IL-13Rα2. In the absence of MMP12, expression of IL-13Rα2 was significantly reduced, providing a possible explanation for the increased IL-13-driven MMP13 activity and reduced fibrosis. As such, these data suggest important counter-regulatory roles between MMP12 and ECM-degrading enzymes like MMP2, MMP9, and MMP13 in Th2 cytokine-driven fibrosis. PMID:20181883

  18. Development of an extracellular matrix delivery system for effective intramyocardial injection in ischemic tissue.

    PubMed

    Slaughter, Mark S; Soucy, Kevin G; Matheny, Robert G; Lewis, Beecher C; Hennick, Michael F; Choi, Young; Monreal, Gretel; Sobieski, Michael A; Giridharan, Guruprasad A; Koenig, Steven C

    2014-01-01

    Biomaterials with direct intramyocardial injection devices have been developed and are being investigated as a potential cardiac regenerative therapy for end-stage ischemic heart failure. Decellularized extracellular matrix (ECM) has been shown to improve cardiac function and attenuate or reverse pathologic remodeling cascades. CorMatrix Cardiovascular, Inc. has developed a porcine small intestinal submucosa-derived particulate extracellular matrix (P-ECM) and ECM Delivery System to provide uniform and controlled intramyocardial delivery of the injectable P-ECM material into infarcted regions. The CorMatrix ECM Delivery System is composed of a Multi-Needle P-ECM Syringe Assembly, Automated Injection Controller, and Tissue Depth Measurement System (portable ultrasound). Feasibility of the P-ECM delivery system was tested intraoperatively in a chronic ischemic heart failure bovine model (n = 11), and demonstrated the ability to control injection volume (0.1-1.0 ml) and depth of penetration (3-5 mm) under regulated injection pressure (150 psi CO2) into the ischemic region. Targeted intramyocardial delivery of P-ECM may improve efficacy and enable development of novel patient-specific therapy. PMID:25232775

  19. Extracellular Matrix Deposition in Engineered Micromass Cartilage Pellet Cultures: Measurements and Modelling.

    PubMed

    Lewis, Miranda C; MacArthur, Ben D; Tare, Rahul S; Oreffo, Richard O C; Please, Colin P

    2016-01-01

    This article explores possible mechanisms governing extracellular matrix deposition in engineered cartilaginous cell pellets. A theoretical investigation is carried out alongside an experimental study measuring proteoglycan and collagen volume fractions within murine chondrogenic (ATDC-5) cell pellets. The simple mathematical model, which adopts a nutrient-dependent proteoglycan production rate, successfully reproduces the periphery-dominated proteoglycan deposition, characteristic of the growth pattern observed experimentally within pellets after 21 days of culture. The results suggest that this inhomogeneous proteoglycan production is due to nutrient deficiencies at the pellet centre. Our model analysis further indicates that a spatially uniform distribution of proteoglycan matrix could be maintained by initiating the culture process with a smaller-sized pellet. Finally, possible extensions are put forward with an aim to improve the model predictions for the early behaviour, where different mechanisms appear to dominate the matrix production within the pellets.

  20. Extracellular Matrix Deposition in Engineered Micromass Cartilage Pellet Cultures: Measurements and Modelling

    PubMed Central

    Lewis, Miranda C.; MacArthur, Ben D.; Tare, Rahul S.; Oreffo, Richard O. C.; Please, Colin P.

    2016-01-01

    This article explores possible mechanisms governing extracellular matrix deposition in engineered cartilaginous cell pellets. A theoretical investigation is carried out alongside an experimental study measuring proteoglycan and collagen volume fractions within murine chondrogenic (ATDC-5) cell pellets. The simple mathematical model, which adopts a nutrient-dependent proteoglycan production rate, successfully reproduces the periphery-dominated proteoglycan deposition, characteristic of the growth pattern observed experimentally within pellets after 21 days of culture. The results suggest that this inhomogeneous proteoglycan production is due to nutrient deficiencies at the pellet centre. Our model analysis further indicates that a spatially uniform distribution of proteoglycan matrix could be maintained by initiating the culture process with a smaller-sized pellet. Finally, possible extensions are put forward with an aim to improve the model predictions for the early behaviour, where different mechanisms appear to dominate the matrix production within the pellets. PMID:26889833

  1. Extracellular matrix for repair of type IV laryngotracheo-esophageal cleft.

    PubMed

    Evans, Adele K; Kon, Neal D

    2015-12-01

    Type IV laryngotracheo-esophageal cleft (LTEC) extending to the level of the carina presents unique challenges to operative repair, particularly with respect to soft tissue durability. This is the first report of CorMatrix(®) extra-cellular matrix (ECM) material use as an interposition graft in a four-layered LTEC repair. At day seven post-operatively, there was epithelialization along the surface of the trachea. At 3 months, she was stable for tracheotomy. At 6 months, the posterior wall resembled completely native tissue. CorMatrix(®) ECM(®) use intra-operatively and post-operative outcome were both highly satisfactory. No adverse reaction was seen in this case through 12-month follow up. PMID:26616544

  2. Abnormal recruitment of extracellular matrix proteins by excess Notch3 ECD: a new pathomechanism in CADASIL.

    PubMed

    Monet-Leprêtre, Marie; Haddad, Iman; Baron-Menguy, Céline; Fouillot-Panchal, Maï; Riani, Meriem; Domenga-Denier, Valérie; Dussaule, Claire; Cognat, Emmanuel; Vinh, Joelle; Joutel, Anne

    2013-06-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the NOTCH3 receptor (Notch3(ECD)) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3(ECD)-containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel pathology. Herein, we hypothesized that excess Notch3(ECD) could recruit and sequester functionally important proteins within small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immunohistochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and artery samples demonstrated that mutant Notch3(ECD) accumulates in disulphide cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3(ECD)-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the formation of Notch3(ECD)-TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of both insoluble cross

  3. Host-Parasite Interaction: Parasite-Derived and -Induced Proteases That Degrade Human Extracellular Matrix

    PubMed Central

    Piña-Vázquez, Carolina; Reyes-López, Magda; Ortíz-Estrada, Guillermo; de la Garza, Mireya; Serrano-Luna, Jesús

    2012-01-01

    Parasitic protozoa are among the most important pathogens worldwide. Diseases such as malaria, leishmaniasis, amoebiasis, giardiasis, trichomoniasis, and trypanosomiasis affect millions of people. Humans are constantly threatened by infections caused by these pathogens. Parasites engage a plethora of surface and secreted molecules to attach to and enter mammalian cells. The secretion of lytic enzymes by parasites into host organs mediates critical interactions because of the invasion and destruction of interstitial tissues, enabling parasite migration to other sites within the hosts. Extracellular matrix is a complex, cross-linked structure that holds cells together in an organized assembly and that forms the basement membrane lining (basal lamina). The extracellular matrix represents a major barrier to parasites. Therefore, the evolution of mechanisms for connective-tissue degradation may be of great importance for parasite survival. Recent advances have been achieved in our understanding of the biochemistry and molecular biology of proteases from parasitic protozoa. The focus of this paper is to discuss the role of protozoan parasitic proteases in the degradation of host ECM proteins and the participation of these molecules as virulence factors. We divide the paper into two sections, extracellular and intracellular protozoa. PMID:22792442

  4. A novel extracellular role for tissue transglutaminase in matrix-bound VEGF-mediated angiogenesis

    PubMed Central

    Wang, Z; Perez, M; Caja, S; Melino, G; Johnson, T S; Lindfors, K; Griffin, M

    2013-01-01

    The importance of tissue transglutaminase (TG2) in angiogenesis is unclear and contradictory. Here we show that inhibition of extracellular TG2 protein crosslinking or downregulation of TG2 expression leads to inhibition of angiogenesis in cell culture, the aorta ring assay and in vivo models. In a human umbilical vein endothelial cell (HUVEC) co-culture model, inhibition of extracellular TG2 activity can halt the progression of angiogenesis, even when introduced after tubule formation has commenced and after addition of excess vascular endothelial growth factor (VEGF). In both cases, this leads to a significant reduction in tubule branching. Knockdown of TG2 by short hairpin (shRNA) results in inhibition of HUVEC migration and tubule formation, which can be restored by add back of wt TG2, but not by the transamidation-defective but GTP-binding mutant W241A. TG2 inhibition results in inhibition of fibronectin deposition in HUVEC monocultures with a parallel reduction in matrix-bound VEGFA, leading to a reduction in phosphorylated VEGF receptor 2 (VEGFR2) at Tyr1214 and its downstream effectors Akt and ERK1/2, and importantly its association with β1 integrin. We propose a mechanism for the involvement of matrix-bound VEGFA in angiogenesis that is dependent on extracellular TG2-related activity. PMID:24052076

  5. Active mechanical coupling between the nucleus, cytoskeleton and the extracellular matrix, and the implications for perinuclear actomyosin organization.

    PubMed

    Zemel, Assaf

    2015-03-28

    Experimental and theoretical studies have demonstrated that the polarization of actomyosin forces in the cytoskeleton of adherent cells is governed by local elastic stresses. Based on this phenomenon, and the established observation that the nucleus is mechanically connected to the extracellular matrix (ECM) via the cytoskeleton, we theoretically analyze here the active mechanical coupling between the nucleus, cytoskeleton and the ECM. The cell is modeled as an active spherical inclusion, containing a round nucleus at its center, and embedded in a 3D elastic matrix. We investigate three sources of cellular stress: spreading-induced stress, actomyosin contractility and chromatin entropic forces. Formulating the coupling of actomyosin contractility to the local stress we predict the consequences that the nucleus, cytoskeleton and ECM mechanical properties may have on the overall force-balance in the cell and the perinuclear acto-myosin polarization. We demonstrate that the presence of the nucleus induces symmetry breaking of the elastic stress that, we predict, elastically tends to orient actomyosin alignment tangentially around the nucleus; the softer the nucleus or the matrix, the stronger is the preference for tangential alignment. Spreading induced stresses may induce radial actomyosin alignment near stiff nuclei. In addition, we show that in regions of high actomyosin density myosin motors have an elastic tendency to orient tangentially as often occurs near the cell periphery. These conclusions highlight the role of the nucleus in the regulation of cytoskeleton organization and may provide new insight into the mechanics of stem cell differentiation involving few fold increase in nucleus stiffness. PMID:25652010

  6. Microenvironment complexity and matrix stiffness regulate breast cancer cell activity in a 3D in vitro model

    PubMed Central

    Cavo, Marta; Fato, Marco; Peñuela, Leonardo; Beltrame, Francesco; Raiteri, Roberto; Scaglione, Silvia

    2016-01-01

    Three-dimensional (3D) cell cultures represent fundamental tools for the comprehension of cellular phenomena both in normal and in pathological conditions. In particular, mechanical and chemical stimuli play a relevant role on cell fate, cancer onset and malignant evolution. Here, we use mechanically-tuned alginate hydrogels to study the role of substrate elasticity on breast adenocarcinoma cell activity. The hydrogel elastic modulus (E) was measured via atomic force microscopy (AFM) and a remarkable range (150–4000 kPa) was obtained. A breast cancer cell line, MCF-7, was seeded within the 3D gels, on standard Petri and alginate-coated dishes (2D controls). Cells showed dramatic morphological differences when cultured in 3D versus 2D, exhibiting a flat shape in both 2D conditions, while maintaining a circular, spheroid-organized (cluster) conformation within the gels, similar to those in vivo. Moreover, we observed a strict correlation between cell viability and substrate elasticity; in particular, the number of MCF-7 cells decreased constantly with increasing hydrogel elasticity. Remarkably, the highest cellular proliferation rate, associated with the formation of cell clusters, occurred at two weeks only in the softest hydrogels (E = 150–200 kPa), highlighting the need to adopt more realistic and a priori defined models for in vitro cancer studies. PMID:27734939

  7. ¹³C NMR-distance matrix descriptors: optimal abstract 3D space granularity for predicting estrogen binding.

    PubMed

    Slavov, Svetoslav H; Geesaman, Elizabeth L; Pearce, Bruce A; Schnackenberg, Laura K; Buzatu, Dan A; Wilkes, Jon G; Beger, Richard D

    2012-07-23

    An improved three-dimensional quantitative spectral data-activity relationship (3D-QSDAR) methodology was used to build and validate models relating the activity of 130 estrogen receptor binders to specific structural features. In 3D-QSDAR, each compound is represented by a unique fingerprint constructed from (13)C chemical shift pairs and associated interatomic distances. Grids of different granularity can be used to partition the abstract fingerprint space into congruent "bins" for which the optimal size was previously unexplored. For this purpose, the endocrine disruptor knowledge base data were used to generate 50 3D-QSDAR models with bins ranging in size from 2 ppm × 2 ppm × 0.5 Å to 20 ppm × 20 ppm × 2.5 Å, each of which was validated using 100 training/test set partitions. Best average predictivity in terms of R(2)test was achieved at 10 ppm ×10 ppm × Z Å (Z = 0.5, ..., 2.5 Å). It was hypothesized that this optimum depends on the chemical shifts' estimation error (±4.13 ppm) and the precision of the calculated interatomic distances. The highest ranked bins from partial least-squares weights were found to be associated with structural features known to be essential for binding to the estrogen receptor.

  8. Disturbances of T-cell interactions with endothelium and the extracellular matrix proteins in a patient with Takayasu arteritis.

    PubMed

    Imiela, J; Górski, A; Dybowska, B; Małecki, R; Nosarzewski, J

    1994-06-01

    T-cell interactions with endothelium and the extracellular matrix proteins were studied in vitro in a patient with Takayasu arteritis. Markedly enhanced spontaneous adhesiveness to cultured endothelium and collagen IV and fibronectin were found to be paralleled by abolished responses to the costimulating action of collagens I and IV and fibronectin. Those abnormalities were partly corrected by immunosuppressive therapy. Aberrant interactions of T cells with endothelium and the extracellular matrix proteins may underlay the pathogenesis of some forms of vasculitis. PMID:7911506

  9. Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix

    NASA Technical Reports Server (NTRS)

    Mooney, D. J.; Langer, R.; Ingber, D. E.

    1995-01-01

    This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In

  10. The dynamic sclera: extracellular matrix remodeling in normal ocular growth and myopia development.

    PubMed

    Harper, Angelica R; Summers, Jody A

    2015-04-01

    Myopia is a common ocular condition, characterized by excessive elongation of the ocular globe. The prevalence of myopia continues to increase, particularly among highly educated groups, now exceeding 80% in some groups. In parallel with the increased prevalence of myopia, are increases in associated blinding ocular conditions including glaucoma, retinal detachment and macular degeneration, making myopia a significant global health concern. The elongation of the eye is closely related to the biomechanical properties of the sclera, which in turn are largely dependent on the composition of the scleral extracellular matrix. Therefore an understanding of the cellular and extracellular events involved in the regulation of scleral growth and remodeling during childhood and young adulthood will provide future avenues for the treatment of myopia and its associated ocular complications.

  11. Extracellular matrix molecules, their receptors, and secreted proteases in synaptic plasticity.

    PubMed

    Wlodarczyk, Jakub; Mukhina, Irina; Kaczmarek, Leszek; Dityatev, Alexander

    2011-11-01

    Neural cells secrete diverse molecules, which accumulate in the extracellular space and form the extracellular matrix (ECM). Interactions between cells and the ECM are well recognized to play the crucial role in cell migration and guidance of growing axons, whereas formation of mature neural ECM in the form of perineuronal nets is believed to restrict certain forms of developmental plasticity. On the other hand, major components of perineuronal nets and other ECM molecules support induction of functional plasticity, the most studied form of which is long-term potentiation. Here, we review the underlying mechanisms by which ECM molecules, their receptors and remodeling proteases regulate the induction and maintenance of synaptic modifications. In particular, we highlight that activity-dependent secretion and activation of proteases leads to a local cleavage of the ECM and release of signaling proteolytic fragments. These molecules regulate transmitter receptor trafficking, actin cytoskeleton, growth of dendritic spines, and formation of dendritic filopodia.

  12. Casting a Net on Dendritic Spines: The Extracellular Matrix and its Receptors

    PubMed Central

    Dansie, Lorraine E.; Ethell, Iryna M.

    2011-01-01

    Dendritic spines are dynamic structures that accommodate the majority of excitatory synapses in the brain and are influenced by extracellular signals from presynaptic neurons, glial cells and the extracellular matrix (ECM). The ECM surrounds dendritic spines and extends into the synaptic cleft, maintaining synapse integrity as well as mediating trans-synaptic communications between neurons. Several scaffolding proteins and glycans that compose the ECM form a lattice-like network, which serves as an attractive ground for various secreted glycoproteins, lectins, growth factors and enzymes. ECM components can control dendritic spines through the interactions with their specific receptors or by influencing the functions of other synaptic proteins. In this review, we focus on ECM components and their receptors that regulate dendritic spine development and plasticity in the normal and diseased brain. PMID:21834084

  13. Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins, enzymes, and relationship to hypophosphatasia and X-linked hypophosphatemia.

    PubMed

    McKee, Marc D; Hoac, Betty; Addison, William N; Barros, Nilana M T; Millán, José L; Chaussain, Catherine

    2013-10-01

    As broadly demonstrated for the formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth - where calcium phosphate crystals are deposited and grow within an extracellular matrix - is essential for dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition in which teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibers (Sharpey's fibers) where they insert into tooth cementum and alveolar bone is also essential for the function of the tooth-suspensory apparatus in occlusion and mastication. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin-binding ligand N-linked glycoproteins and matrix vesicles. Amongst the enzymes important in regulating these mineralization determinants, two are discussed at length here, with clinical examples given, namely tissue-nonspecific alkaline phosphatase and phosphate-regulating gene with homologies to endopeptidases on the X chromosome. Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia and X-linked hypophosphatemia, respectively, where the levels of local and systemic circulating mineralization determinants are perturbed. In X-linked hypophosphatemia, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization-regulating small integrin-binding ligand N-linked glycoproteins, such as matrix extracellular phosphoglycoprotein and osteopontin, and the phosphorylated peptides proteolytically released from them, such as the acidic serine- and aspartate-rich-motif peptide, may accumulate locally to impair mineralization in this disease.

  14. Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration

    PubMed Central

    Zhang, Wei; Chen, Xiao-Ping; Zhang, Wan-Guang; Zhang, Feng; Xiang, Shuai; Dong, Han-Hua; Zhang, Lei

    2009-01-01

    AIM: To elucidate the interaction between non-parenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy (PH). METHODS: We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2-acetylaminofluorene (2-AAF)/PH rat model. RESULTS: By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic lobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells (HSCs), laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver lobule structures began to recover. CONCLUSION: Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions. PMID:19195056

  15. Semicarbazide-sensitive amine oxidase and extracellular matrix deposition by smooth-muscle cells

    NASA Technical Reports Server (NTRS)

    Langford, Shannon D.; Trent, Margaret B.; Boor, Paul J.

    2002-01-01

    We have recently reported in vivo disruption of collagen and elastin architecture within blood vessel walls resulting from the selective inhibition of the enzyme semicarbazide-sensitive amine oxidase (SSAO). This study further investigates the effects of SSAO inhibition on extracellular matrix deposition by smooth-muscle cells (SMCs) cultured from neonatal rat hearts. SMCs were characterized, SSAO activity was measured, and soluble and insoluble collagen and elastin in the extracellular matrix (ECM) were quantified. Cultured neonatal rat heart SMC exhibited a monotypic synthetic phenotype that likely represents a myofibroblast. Detectable levels of SSAO activity present throughout 30-d culture peaked at 7-14 d, coinciding with the production of ECM. The addition of enzyme inhibitors and alternate SSAO substrates (benzylamine) produced varied and, in some cases, marked changes in SSAO activity as well as in the composition of mature and soluble matrix components. Similar to our previous in vivo findings, in vitro SSAO inhibition produced aberrations in collagen and elastin deposition by heart SMC. Because changes in SSAO activity are associated with cardiovascular pathologic states, this enzyme may play a protective or modulating role by regulating ECM production during pathologic insult.

  16. Permeability characteristics of human endothelial monolayers seeded on different extracellular matrix proteins.

    PubMed Central

    Nooteboom, A; Hendriks, T; Ottehöller, I; van der Linden, C J

    2000-01-01

    OBJECTIVE: To investigate whether endothelial monolayer permeability changes induced by inflammatory mediators are affected by the extracellular matrix protein used for cell seeding. METHODS: Human umbilical venular endothelial cells (HUVEC) were grown to confluent monolayers on membranes coated with either collagen, fibronectin or gelatin. The permeability to albumin and dextran was then assessed, both under normal conditions and after treatment with tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide (LPS). RESULTS: With any of the three protein coatings, tight junctions were formed all over the monolayers. The permeability of the coated membranes to albumin and dextran was reduced strongly by confluent monolayers; the relative reduction was similar for the three matrix proteins used. Pre-incubation of the monolayers with either TNF-alpha or LPS increased permeability dose dependently. However, the relative increase due to either treatment was independent of the protein used for membrane coating. CONCLUSION: The extracellular matrix protein used for initial seeding of endothelial cultures plays a minor role in determining the permeability changes induced in HUVEC monolayers by inflammatory mediators. PMID:11200364

  17. In vitro influence of the extracellular matrix in myoepithelial cells stimulated by malignant conditioned medium.

    PubMed

    Martinez, Elizabeth F; Demasi, Ana Paula Dias; Napimoga, Marcelo Henrique; Arana-Chavez, Victor Elias; Altemani, Albina; de Araújo, Ney Soares; de Araújo, Vera Cavalcanti

    2012-02-01

    In order to investigate the role of myoepithelial cell and tumor microenvironment in salivary gland neoplasma, we have performed a study towards the effect of different extracellular matrix proteins (basement membrane matrix, type I collagen and fibronectin) on morphology and differentiation of benign myoepithelial cells from pleomorphic adenoma cultured with malignant cell culture medium from squamous cell carcinoma. We have also analyzed the expression of α-smooth muscle actin (α-SMA) and FGF-2 by immunofluorescence and qPCR. Our immunofluorescence results, supported by qPCR analysis, demonstrated that α-SMA and FGF-2 were upregulated in the benign myoepithelial cells from pleomorphic adenoma in all studied conditions on fibronectin substratum. However, the myoepithelial cells on fibronectin substratum did not alter their morphology under malignant conditioned medium stimulation and exhibited a stellate morphology and, occasionally focal adhesions with the substratum. In summary, our data demonstrated that the extracellular matrix exerts an important role in the morphology of the benign myoepithelial cells by the presence of focal adhesions and also inducing increase FGF-2 and α-SMA expression by these cells, especially in the fibronectin substratum.

  18. Reduced serum content and increased matrix stiffness promote the cardiac myofibroblast transition in 3D collagen matrices.

    PubMed Central

    Galie, Peter A.; Westfall, Margaret V.; Stegemann, Jan P.

    2011-01-01

    Introduction The fibroblast-myofibroblast transition is an important event in the development of cardiac fibrosis and scar formation initiated after myocardial ischemia. The goals of the present study were to better understand the contribution of environmental factors to this transition and determine whether myofibroblasts provide equally important feedback to the surrounding environment. Methods The influence of matrix stiffness and serum concentration on the myofibroblast transition was assessed by measuring message levels of a panel of cardiac fibroblast phenotype markers using quantitative rtPCR. Cell-mediated gel compaction measured the influence of environmental factors on cardiac fibroblast contractility. Immunohistochemistry characterized α-SMA expression and cell morphology, while static and dynamic compression testing evaluated the effect of the cell response on the mechanical properties of the cell-seeded collagen hydrogels. Results Both reduced serum content and increased matrix stiffness contributed to the myofibroblast transition, as indicated by contractile compaction of the gels, increased message levels of col3α1 and α-SMA, and a less stellate morphology. However, the effects of serum and matrix stiffness were not additive. Mechanical testing indicated the cell-seeded gels became less viscoelastic with time, and that reduced serum content also increased the initial elastic properties of the gel. Conclusions The results suggest that reduced serum and increased matrix stiffness promote the myofibroblast phenotype in the myocardium. This transition both enhances and is promoted by matrix stiffness, indicating the presence of positive feedback that may contribute to the pathogenesis of cardiac fibrosis. Summary Lower serum content and increased matrix stiffness accelerated the transition of cardiac fibroblasts seeded in collagen hydrogels to a myofibroblast phenotype, though their effects were not additive. Reduced serum also affected mechanical

  19. Contribution of the extracellular matrix to the viscoelastic behavior of the urinary bladder wall.

    PubMed

    Nagatomi, Jiro; Toosi, Kevin K; Chancellor, Michael B; Sacks, Michael S

    2008-10-01

    We previously reported that when the stress relaxation response of urinary bladder wall (UBW) tissue was analyzed using a single continuous reduced relaxation function (RRF), we observed non-uniformly distributed, time-dependent residuals (Ann Biomed Eng 32(10):1409-1419, 2004). We concluded that the single relaxation spectrum was inadequate and that a new viscoelastic model for bladder wall was necessary. In the present study, we report a new approach composed of independent RRFs for smooth muscle and the extracellular matrix components (ECM), connected through a stress-dependent recruitment function. In order to determine the RRF for the ECM component, biaxial stress relaxation experiments were first performed on decellularized extracellular matrix network of the bladder obtained from normal and spinal cord injured rats. While it was assumed that smooth muscle followed a single spectrum RRF, modeling the UBW ECM required a dual-Gaussian spectrum. Experimental results revealed that the ECM stress relaxation response was insensitive to the initial stress level. Thus, the average ECM RRF parameters were determined by fitting the average stress relaxation data. The resulting stress relaxation behavior of whole bladder tissue was modeled by combining the ECM RRF with the RRF for the smooth muscle component using an exponential recruitment function representing the recruitment of collagen fibers at higher stress levels. In summary, the present study demonstrated, for the first time, that stress relaxation response of bladder tissue can be better modeled when divided into the contributions of the extracellular matrix and smooth muscle components. This modeling approach is suitable for prediction of mechanical behaviors of the urinary bladder and other organs that exhibit rapid tissue remodeling (i.e., smooth muscle hypertrophy and altered ECM synthesis) under various pathological conditions.

  20. The contribution of the extracellular matrix to gravisensing in characean cells

    NASA Technical Reports Server (NTRS)

    Wayne, R.; Staves, M. P.; Leopold, A. C.

    1992-01-01

    The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing is sensitive to protease treatment; Proteinase K, thermolysin and collagenase but not trypsin, alpha-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.

  1. Influence of poly-N-acetylglucosamine in the extracellular matrix on N-chlorotaurine mediated killing of Staphylococcus epidermidis.

    PubMed

    Ammann, Christoph G; Fille, Manfred; Hausdorfer, Johann; Nogler, Michael; Nagl, Markus; Coraça-Huber, Débora C

    2014-07-01

    N-chlorotaurine (NCT) has recently been shown to have bactericidal activity against bacterial biofilm on metal discs (Coraca-Huber et al., 2014). In a biofilm, Staphylococcus epidermidis polymerizes poly-N-acetylglucosamine (PNAG) to form an extracellular matrix (ECM). Pseudomonas aeruginosa does not express this PNAG and has been shown to be highly susceptible to NCT. We compared the action of NCT on S. epidermidis 1457, a PNAG positive strain (SE1457) and S. epidermidis 1457- M10 an isogenic PNAG negative mutant (SE1457 M10). NCT-mediated killing was more effective and quicker on the PNAG negative strain SE1457 M10. Bacteria hidden in biofilms for prolonged periods of time were generally more susceptible than freshly formed biofilms. The differences in NCT-mediated killing might not be direct effects since NCT did not react with the monomeric N-acetylglucosamine, but might be explained by denser growth in the PNAG-containing biofilm produced by the wild type strain, which results in delayed penetration of NCT. The higher susceptibility of older biofilms to NCTmediated killing could be explained by more pronounced 3D architecture and subsequent larger surface area for interactions with NCT.

  2. In vitro elastogenesis: instructing human vascular smooth muscle cells to generate an elastic fiber-containing extracellular matrix scaffold.

    PubMed

    Hinderer, Svenja; Shena, Nian; Ringuette, Léa-Jeanne; Hansmann, Jan; Reinhardt, Dieter P; Brucker, Sara Y; Davis, Elaine C; Schenke-Layland, Katja

    2015-06-01

    Elastic fibers are essential for the proper function of organs including cardiovascular tissues such as heart valves and blood vessels. Although (tropo)elastin production in a tissue-engineered construct has previously been described, the assembly to functional elastic fibers in vitro using human cells has been highly challenging. In the present study, we seeded primary isolated human vascular smooth muscle cells (VSMCs) onto 3D electrospun scaffolds and exposed them to defined laminar shear stress using a customized bioreactor system. Increased elastin expression followed by elastin deposition onto the electrospun scaffolds, as well as on newly formed fibers, was observed after six days. Most interestingly, we identified the successful deposition of elastogenesis-associated proteins, including fibrillin-1 and -2, fibulin-4 and -5, fibronectin, elastin microfibril interface located protein 1 (EMILIN-1) and lysyl oxidase (LOX) within our engineered constructs. Ultrastructural analyses revealed a developing extracellular matrix (ECM) similar to native human fetal tissue, which is composed of collagens, microfibrils and elastin. To conclude, the combination of a novel dynamic flow bioreactor and an electrospun hybrid polymer scaffold allowed the production and assembly of an elastic fiber-containing ECM. PMID:25784676

  3. Of extracellular matrix, scaffolds, and signaling: Tissuearchitectureregulates development, homeostasis, and cancer

    SciTech Connect

    Nelson, Celeste M.; Bissell, Mina J.

    2006-03-09

    The microenvironment surrounding cells influences gene expression, such that a cell's behavior is largely determined by its interactions with the extracellular matrix, neighboring cells, and soluble cues released locally or by distant tissues. We describe the essential role of context and organ structure in directing mammary gland development and differentiated function, and in determining response to oncogenic insults including mutations. We expand on the concept of 'dynamic reciprocity' to present an integrated view of development, cancer, and aging, and posit that genes are like piano keys: while essential, it is the context that makes the music.

  4. Emerging Implications for Extracellular Matrix-Based Technologies in Vascularized Composite Allotransplantation

    PubMed Central

    Londono, Ricardo; Gorantla, Vijay S.; Badylak, Stephen F.

    2016-01-01

    Despite recent progress in vascularized composite allotransplantation (VCA), limitations including complex, high dose immunosuppression regimens, lifelong risk of toxicity from immunosuppressants, acute and most critically chronic graft rejection, and suboptimal nerve regeneration remain particularly challenging obstacles restricting clinical progress. When properly configured, customized, and implemented, biomaterials derived from the extracellular matrix (ECM) retain bioactive molecules and immunomodulatory properties that can promote stem cell migration, proliferation and differentiation, and constructive functional tissue remodeling. The present paper reviews the emerging implications of ECM-based technologies in VCA, including local immunomodulation, tissue repair, nerve regeneration, minimally invasive graft targeted drug delivery, stem cell transplantation, and other donor graft manipulation. PMID:26839554

  5. The Extracellular Matrix in Epithelial Ovarian Cancer – A Piece of a Puzzle

    PubMed Central

    Cho, Angela; Howell, Viive M.; Colvin, Emily K.

    2015-01-01

    Epithelial ovarian cancer is the fifth leading cause of cancer-related deaths in women and the most lethal gynecological malignancy. Extracellular matrix (ECM) is an integral component of both the normal and tumor microenvironment. ECM composition varies between tissues and is crucial for maintaining normal function and homeostasis. Dysregulation and aberrant deposition or loss of ECM components is implicated in ovarian cancer progression. The mechanisms by which tumor cells induce ECM remodeling to promote a malignant phenotype are yet to be elucidated. A thorough understanding of the role of the ECM in ovarian cancer is needed for the development of effective biomarkers and new therapies. PMID:26579497

  6. Suppression of ICE and Apoptosis in Mammary Epithelial Cells by Extracellular Matrix

    SciTech Connect

    Boudreau, Nancy; Sympson, C. J.; Werb, Zena; Bissell, Mina J.

    1994-12-01

    Apoptosis (programmed cell death) plays a major role in development and tissue regeneration. Basement membrane extracellular matrix (ECM), but not fibronectin or collagen, was shown to suppress apoptosis of mammary epithelial cells in tissue culture and in vivo. Apoptosis was induced by antibodies to beta 1 integrins or by overexpression of stromelysin-1, which degrades ECM. Expression of interleukin-1 beta converting enzyme (ICE) correlated with the loss of ECM, and inhibitors of ICE activity prevented apoptosis. These results suggest that ECM regulates apoptosis in mammary epithelial cells through an integrin-dependent negative regulation of ICE expression.

  7. Native Cardiac Extracellular Matrix Hydrogels for Cultivation of Human Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Freytes, Donald O; O’Neill, John D; Duan-Arnold, Yi; Wrona, Emily; Vunjak-Novakovic, Gordana

    2015-01-01

    Summary Biomaterial scaffolds made of native and synthetic materials are designed to serve as a structural and informational template for cell attachment and tissue formation. The use of native extracellular matrix (ECM) is of special interest for the culture of cardiac stem and progenitor cells due to the presence of intrinsic regulatory factors regulating cardiac function. We describe here how to obtain native ECM hydrogels from porcine hearts for the culture of human embryonic, induced pluripotent, and somatic stem cells for cardiac tissue engineering and regenerative medicine applications. PMID:25070328

  8. Induction of Tenogenic Differentiation Mediated by Extracellular Tendon Matrix and Short-Term Cyclic Stretching

    PubMed Central

    Plenge, Amelie; Heller, Sandra; Pfeiffer, Bastian; Kasper, Cornelia

    2016-01-01

    Tendon and ligament pathologies are still a therapeutic challenge, due to the difficulty in restoring the complex extracellular matrix architecture and biomechanical strength. While progress is being made in cell-based therapies and tissue engineering approaches, comprehensive understanding of the fate of progenitor cells in tendon healing is still lacking. The aim of this study was to investigate the effect of decellularized tendon matrix and moderate cyclic stretching as natural stimuli which could potentially direct tenogenic fate. Equine adipose-derived mesenchymal stromal cells (MSC) were seeded on decellularized tendon matrix scaffolds. Mechanical stimulation was applied in a custom-made cyclic strain bioreactor. Assessment was performed 4 h, 8 h, and 24 h following mechanical stimulation. Scaffold culture induced cell alignment and changes in expression of tendon-related genes, although cell viability was decreased compared to monolayer culture. Short mechanical stimulation periods enhanced most of the scaffold-induced effects. Collagen 1A2 expression levels were decreased, while collagen 3A1 and decorin levels were increased. Tenascin-C and scleraxis expression showed an initial decrease but had increased 24 h after stimulation. The results obtained suggest that decellularized tendon matrix, supported by cyclic stretching, can induce tenogenic differentiation and the synthesis of tendon components important for matrix remodeling.

  9. Induction of Tenogenic Differentiation Mediated by Extracellular Tendon Matrix and Short-Term Cyclic Stretching.

    PubMed

    Burk, Janina; Plenge, Amelie; Brehm, Walter; Heller, Sandra; Pfeiffer, Bastian; Kasper, Cornelia

    2016-01-01

    Tendon and ligament pathologies are still a therapeutic challenge, due to the difficulty in restoring the complex extracellular matrix architecture and biomechanical strength. While progress is being made in cell-based therapies and tissue engineering approaches, comprehensive understanding of the fate of progenitor cells in tendon healing is still lacking. The aim of this study was to investigate the effect of decellularized tendon matrix and moderate cyclic stretching as natural stimuli which could potentially direct tenogenic fate. Equine adipose-derived mesenchymal stromal cells (MSC) were seeded on decellularized tendon matrix scaffolds. Mechanical stimulation was applied in a custom-made cyclic strain bioreactor. Assessment was performed 4 h, 8 h, and 24 h following mechanical stimulation. Scaffold culture induced cell alignment and changes in expression of tendon-related genes, although cell viability was decreased compared to monolayer culture. Short mechanical stimulation periods enhanced most of the scaffold-induced effects. Collagen 1A2 expression levels were decreased, while collagen 3A1 and decorin levels were increased. Tenascin-C and scleraxis expression showed an initial decrease but had increased 24 h after stimulation. The results obtained suggest that decellularized tendon matrix, supported by cyclic stretching, can induce tenogenic differentiation and the synthesis of tendon components important for matrix remodeling. PMID:27630718

  10. Induction of Tenogenic Differentiation Mediated by Extracellular Tendon Matrix and Short-Term Cyclic Stretching

    PubMed Central

    Plenge, Amelie; Heller, Sandra; Pfeiffer, Bastian; Kasper, Cornelia

    2016-01-01

    Tendon and ligament pathologies are still a therapeutic challenge, due to the difficulty in restoring the complex extracellular matrix architecture and biomechanical strength. While progress is being made in cell-based therapies and tissue engineering approaches, comprehensive understanding of the fate of progenitor cells in tendon healing is still lacking. The aim of this study was to investigate the effect of decellularized tendon matrix and moderate cyclic stretching as natural stimuli which could potentially direct tenogenic fate. Equine adipose-derived mesenchymal stromal cells (MSC) were seeded on decellularized tendon matrix scaffolds. Mechanical stimulation was applied in a custom-made cyclic strain bioreactor. Assessment was performed 4 h, 8 h, and 24 h following mechanical stimulation. Scaffold culture induced cell alignment and changes in expression of tendon-related genes, although cell viability was decreased compared to monolayer culture. Short mechanical stimulation periods enhanced most of the scaffold-induced effects. Collagen 1A2 expression levels were decreased, while collagen 3A1 and decorin levels were increased. Tenascin-C and scleraxis expression showed an initial decrease but had increased 24 h after stimulation. The results obtained suggest that decellularized tendon matrix, supported by cyclic stretching, can induce tenogenic differentiation and the synthesis of tendon components important for matrix remodeling. PMID:27630718

  11. Teaching the Extracellular Matrix and Introducing Online Databases within a Multidisciplinary Course with i-Cell-MATRIX: A Student-Centered Approach

    ERIC Educational Resources Information Center

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

    2010-01-01

    The biochemistry and molecular biology of the extracellular matrix (ECM) is difficult to convey to students in a classroom setting in ways that capture their interest. The understanding of the matrix's roles in physiological and pathological conditions study will presumably be hampered by insufficient knowledge of its molecular structure.…

  12. Evidence for export of a muscle lectin from cytosol to extracellular matrix and for a novel secretory mechanism

    SciTech Connect

    Cooper, D.N.; Barondes, S.H. )

    1990-05-01

    A soluble lactose-binding lectin with subunit Mr of 14,500 is believed to function by interacting with extracellular glycoconjugates, because it has been detected extracellularly by immunohistochemistry. This localization has been questioned, however, since the lectin lacks a secretion signal sequence, which challenges the contention that it is secreted. We have demonstrated externalization of this lectin from C2 mouse muscle cells by both immunoprecipitation of metabolically labeled protein and immunohistochemical localization. We further show that externalization of the lectin is a developmentally regulated process that accompanies myoblast differentiation and that the lectin codistributes with laminin in myotube extracellular matrix. Immunohistochemical localization during intermediate stages of externalization suggests that the lectin becomes concentrated in evaginations of plasma membrane, which pinch off to form labile lectin-rich extracellular vesicles. This suggests a possible mechanism for lectin export from the cytosol to the extracellular matrix.

  13. Fibroblast-derived 3D matrix differentially regulates the growth and drug-responsiveness of human cancer cells

    PubMed Central

    Lamb, Acacia; Golemis, Erica A.; Cukierman, Edna

    2008-01-01

    Recent studies have emphasized the importance of cellular microenvironment in modulating cell growth and signaling. In vitro, collagen matrices, Matrigel, and other synthetic support systems have been used to simulate in vivo microenvironments, and epithelial cells grown in these matrices manifest significant differences in proliferation, differentiation, response to drugs, and other parameters. However, these substrates do not closely resemble the mesenchymal microenvironment that is typically associated with advanced carcinomas in vivo, which is produced to a large extent by fibroblasts. In this study, we have evaluated the ability of a fibroblast-derived three-dimensional matrix to regulate the growth of a panel of 11 human tumor epithelial cell lines. Although proliferative and morphological responses to three-dimensional cues segregated independently, general responsiveness to the matrix correlated with the ability of matrix to influence drug responses. Fibroblast-derived three-dimensional matrix increased β1-integrin-dependent survival of a subset of human cancer cell lines during taxol treatment, while it sensitized or minimally influenced survival of other cells. β1-integrin-dependent changes in cell resistance to taxol did not correlate with degree of modulation of FAK and Akt, implying additional signaling factors are involved. Based on these results, we propose these matrices potentially have value as in vitro drug screening platforms. PMID:18411046

  14. Nonlinear mechanical response of the extracellular matrix: learning from articular cartilage

    NASA Astrophysics Data System (ADS)

    Kearns, Sarah; Das, Moumita

    2015-03-01

    We study the mechanical structure-function relations in the extracellular matrix (ECM) with focus on nonlinear shear and compression response. As a model system, our study focuses on the ECM in articular cartilage tissue which has two major mechanobiological components: a network of the biopolymer collagen that acts as a stiff, reinforcing matrix, and a flexible aggrecan network that facilitates deformability. We model this system as a double network hydrogel made of interpenetrating networks of stiff and flexible biopolymers respectively. We study the linear and nonlinear mechanical response of the model ECM to shear and compression forces using a combination of rigidity percolation theory and energy minimization approaches. Our results may provide useful insights into the design principles of the ECM as well as biomimetic hydrogels that are mechanically robust and can, at the same time, easily adapt to cues in their surroundings.

  15. Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium.

    PubMed

    Chaudhuri, Ovijit; Koshy, Sandeep T; Branco da Cunha, Cristiana; Shin, Jae-Won; Verbeke, Catia S; Allison, Kimberly H; Mooney, David J

    2014-10-01

    In vitro models of normal mammary epithelium have correlated increased extracellular matrix (ECM) stiffness with malignant phenotypes. However, the role of increased stiffness in this transformation remains unclear because of difficulties in controlling ECM stiffness, composition and architecture independently. Here we demonstrate that interpenetrating networks of reconstituted basement membrane matrix and alginate can be used to modulate ECM stiffness independently of composition and architecture. We find that, in normal mammary epithelial cells, increasing ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. We also find that the combination of stiffness and composition is sensed through β4 integrin, Rac1, and the PI3K pathway, and suggest a mechanism in which an increase in ECM stiffness, without an increase in basement membrane ligands, prevents normal α6β4 integrin clustering into hemidesmosomes.

  16. Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium

    NASA Astrophysics Data System (ADS)

    Chaudhuri, Ovijit; Koshy, Sandeep T.; Branco da Cunha, Cristiana; Shin, Jae-Won; Verbeke, Catia S.; Allison, Kimberly H.; Mooney, David J.

    2014-10-01

    In vitro models of normal mammary epithelium have correlated increased extracellular matrix (ECM) stiffness with malignant phenotypes. However, the role of increased stiffness in this transformation remains unclear because of difficulties in controlling ECM stiffness, composition and architecture independently. Here we demonstrate that interpenetrating networks of reconstituted basement membrane matrix and alginate can be used to modulate ECM stiffness independently of composition and architecture. We find that, in normal mammary epithelial cells, increasing ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. We also find that the combination of stiffness and composition is sensed through β4 integrin, Rac1, and the PI3K pathway, and suggest a mechanism in which an increase in ECM stiffness, without an increase in basement membrane ligands, prevents normal α6β4 integrin clustering into hemidesmosomes.

  17. Cinematographic analysis of vascular smooth muscle cell interactions with extracellular matrix.

    PubMed

    Absher, M; Baldor, L

    1991-01-01

    The interactions of vascular smooth muscle cells with growth modulators and extracellular matrix molecules may play a role in the proliferation and migration of these cells after vascular injury and during the development of atherosclerosis. Time-lapse cinematographic techniques have been used to study cell division and migration of bovine carotid artery smooth muscle cells in response to matrix molecules consisting of solubilized basement membrane (Matrigel) and type I collagen. When cells were grown adjacent to Matrigel, both migration and cell proliferation were increased and interdivision time was shortened. Cells grown in Matrigel or in type I collagen had markedly reduced migration rates but interdivision time was not altered. Further, diffusible components of the Matrigel were found to stimulate proliferation of the smooth muscle cells.

  18. Bone regeneration with osteogenically enhanced mesenchymal stem cells and their extracellular matrix proteins.

    PubMed

    Clough, Bret H; McCarley, Matthew R; Krause, Ulf; Zeitouni, Suzanne; Froese, Jeremiah J; McNeill, Eoin P; Chaput, Christopher D; Sampson, H Wayne; Gregory, Carl A

    2015-01-01

    Although bone has remarkable regenerative capacity, about 10% of long bone fractures and 25% to 40% of vertebral fusion procedures fail to heal. In such instances, a scaffold is employed to bridge the lesion and accommodate osteoprogenitors. Although synthetic bone scaffolds mimic some of the characteristics of bone matrix, their effectiveness can vary because of biological incompatibility. Herein, we demonstrate that a composite prepared with osteogenically enhanced mesenchymal stem cells (OEhMSCs) and their extracellular matrix (ECM) has an unprecedented capacity for the repair of critical-sized defects of murine femora. Furthermore, OEhMSCs do not cause lymphocyte activation, and ECM/OEhMSC composites retain their in vivo efficacy after cryopreservation. Finally, we show that attachment to the ECM by OEhMSCs stimulates the production of osteogenic and angiogenic factors. These data demonstrate that composites of OEhMSCs and their ECM could be utilized in the place of autologous bone graft for complex orthopedic reconstructions.

  19. Human epidermal keratinocyte cell response on integrin-specific artificial extracellular matrix proteins.

    PubMed

    Tjin, Monica Suryana; Chua, Alvin Wen Choong; Ma, Dong Rui; Lee, Seng Teik; Fong, Eileen

    2014-08-01

    Cell-matrix interactions play critical roles in regulating cellular behavior in wound repair and regeneration of the human skin. In particular, human skin keratinocytes express several key integrins such as alpha5beta1, alpha3beta1, and alpha2beta1 for binding to the extracellular matrix (ECM) present in the basement membrane in uninjured skin. To mimic these key integrin-ECM interactions, artificial ECM (aECM) proteins containing functional domains derived from laminin 5, type IV collagen, fibronectin, and elastin are prepared. Human skin keratinocyte cell responses on the aECM proteins are specific to the cell-binding domain present in each construct. Keratinocyte attachment to the aECM protein substrates is also mediated by specific integrin-material interactions. In addition, the aECM proteins are able to support the proliferation of keratinocyte stem cells, demonstrating their promise for use in skin tissue engineering.

  20. Embryonic lung morphogenesis in organ culture: experimental evidence for a proteoglycan function in the extracellular matrix

    NASA Technical Reports Server (NTRS)

    Spooner, B. S.; Bassett, K. E.; Spooner, B. S. Jr

    1993-01-01

    The lung rudiment, isolated from mid-gestation (11 day) mouse embryos, can undergo morphogenesis in organ culture. Observation of living rudiments, in culture, reveals both growth and ongoing bronchiolar branching activity. To detect proteoglycan (PG) biosynthesis, and deposition in the extracellular matrix, rudiments were metabolically labeled with radioactive sulfate, then fixed, embedded, sectioned and processed for autoradiography. The sulfated glycosaminoglycan (GAG) types, composing the carbohydrate component of the proteoglycans, were evaluated by selective GAG degradative approaches that showed chondroitin sulfate PG principally associated with the interstitial matrix, and heparan sulfate PG principally associated with the basement membrane. Experiments using the proteoglycan biosynthesis disrupter, beta-xyloside, suggest that when chondroitin sulfate PG deposition into the ECM is perturbed, branching morphogenesis is compromised.

  1. [Theodor Huzella and the initiation of research on the interactions between cells and the extracellular matrix].

    PubMed

    Robert, Ladislas; Labat-Robert, Jacqueline; Michel Robert, Alexandre

    2012-01-01

    Interactions between cells and the surrounding "biomatrix", mediated by receptors as integrins or the elastin receptor is the most important topic in up to date research on connective tissues. Looking for the origin of this concept, one finds the pioneering work of Theodor Huzella, professor of histology-embryology at the Medical University of Budapest during pre-world war II decades. Using time-laps micro-cinematography in reflected light, he visualized the important role of connective tissue fibers, prepared in his laboratory, for the oriented migration of normal and malignant cells. His theoretical explanations, attributing an "active" role to the elasticity of the argyrophilic fibrous network in the coordination of cell societies, can now be reinterpreted in the light of recent work on the mechanotransduction of "messages" from the extracellular matrix to the cell inside. We propose a succinct review of Huzella's work and theories reinterpreted in the light of up-to-date knowledge on cell-matrix interactions.

  2. Response of endothelial cells to decellularized extracellular matrix deposited by bone marrow mesenchymal stem cells

    PubMed Central

    Xu, Yue; Yan, Mengdie; Gong, Yihong; Chen, Lei; Zhao, Feng; Zhang, Zhaoqiang

    2014-01-01

    Objective: Evaluate the behavior and function of human umbilical vein endothelial cells (HUVECs) on decellularized extracellular matrix (ECM) deposited by bone marrow mesenchymal stem cells (BMSCs). Methods: Prepared through chemical approach, decellularized ECM was characterized by use of immunofluorescence staining. The morphology, attachment, proliferation and migration of HUVECs cultured on six-well tissue culture plastic (TCP) and decellularized ECM were investigated. Results: Decellularized ECM was successfully prepared without three-dimensional architecture disruption. This biological scaffold is similar to nature vascular ECM, preserved various matrix proteins such as type I collagen, type III collagen and fibronection. HUVECs on decellularized ECM showed well attachment and regular arrangement. Decellularized ECM could also significantly enhance the migration and proliferation potential of HUVECs in contrast to TCP. Conclusion: Deposited by BMSCs, ECM can affect the behavior of endothelial cell and could be used as a promising material in tissue engineering. PMID:25663998

  3. Extracellular matrix proteins regulate epithelial-mesenchymal transition in mammary epithelial cells

    PubMed Central

    Chen, Qike K.; Lee, KangAe; Radisky, Derek C.; Nelson, Celeste M.

    2013-01-01

    Mouse mammary epithelial cells undergo transdifferentiation via epithelial-mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland. PMID:23660532

  4. Modulation of cardiac myocyte phenotype in vitro by the composition and orientation of the extracellular matrix.

    PubMed

    Simpson, D G; Terracio, L; Terracio, M; Price, R L; Turner, D C; Borg, T K

    1994-10-01

    Cellular phenotype is the result of a dynamic interaction between a cell's intrinsic genetic program and the morphogenetic signals that serve to modulate the extent to which that program is expressed. In the present study we have examined how morphogenetic information might be stored in the extracellular matrix (ECM) and communicated to the neonatal heart cell (NHC) by the cardiac alpha 1 beta 1 integrin molecule. A thin film of type I collagen (T1C) was prepared with a defined orientation. This was achieved by applying T1C to the peripheral edge of a 100 mm culture dish. The T1C was then drawn across the surface of the dish in a continuous stroke with a sterile cell scraper and allowed to polymerize. When NHCs were cultured on this substrate, they spread, as a population, along a common axis in parallel with the gel lattice and expressed an in vivo-like phenotype. Individual NHCs displayed an elongated, rod-like shape and disclosed parallel arrays of myofibrils. These phenotypic characteristics were maintained for at least 4 weeks in primary culture. The evolution of this tissue-like organizational pattern was dependent upon specific interactions between the NHCs and the collagen-based matrix that were mediated by the cardiac alpha 1 beta 1 integrin complex. This conclusion was supported by a variety of experimental results. Altering the tertiary structure of the matrix or blocking the extracellular domains of either the cardiac alpha 1 or beta 1 integrin chain inhibited the expression of the tissue-like pattern of organization. Neither cell-to-cell contact or contractile function were necessary to induce the formation of the rod-like cell shape. However, beating activity was necessary for the assembly of a well-differentiated myofibrillar apparatus. These data suggest that the cardiac alpha 1 beta 1 integrin complex serves to detect and transduce phenotypic information stored within the tertiary structure of the surrounding matrix.

  5. Modeling extracellular matrix (ECM) alterations in ovarian cancer by multiphoton excited fabrication of stromal models (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Campagnola, Paul J.; Ajeti, Visar; Lara, Jorge; Eliceiri, Kevin W.; Patankar, Mansh

    2016-04-01

    A profound remodeling of the extracellular matrix (ECM) occurs in human ovarian cancer but it unknown how this affects tumor growth, where this understanding could lead to better diagnostics and therapeutic approaches. We investigate the role of these ECM alterations by using multiphoton excited (MPE) polymerization to fabricate biomimetic models to investigate operative cell-matrix interactions in invasion/metastasis. First, we create nano/microstructured gradients mimicking the basal lamina to study adhesion/migration dynamics of ovarian cancer cells of differing metastatic potential. We find a strong haptotactic response that depends on both contact guidance and ECM binding cues. While we found enhanced migration for more invasive cells, the specifics of alignment and directed migration also depend on cell polarity. We further use MPE fabrication to create collagen scaffolds with complex, 3D submicron morphology. The stromal scaffold designs are derived directly from "blueprints" based on SHG images of normal, high risk, and malignant ovarian tissues. The models are seeded with different cancer cell lines and this allows decoupling of the roles of cell characteristics (metastatic potential) and ECM structure and composition (normal vs cancer) on adhesion/migration dynamics. We found the malignant stroma structure promotes enhanced migration and proliferation and also cytoskeletal alignment. Creating synthetic models based on fibers patterns further allows decoupling the topographic roles of the fibers themselves vs their alignment within the tissue. These models cannot be synthesized by other conventional fabrication methods and we suggest the MPE image-based fabrication method will enable a variety of studies in cancer biology.

  6. A matrix projection method for on line stable estimation of 1D and 3D shear building models

    NASA Astrophysics Data System (ADS)

    Angel García-Illescas, Miguel; Alvarez-Icaza, Luis

    2016-12-01

    An estimation method is presented that combines the use of recursive least squares, a matrix parameterized model, Gershgorin circles and tridiagonal matrices properties to allow the identification of stable shear building models in the presence of low excitation or low damping. The resultant scheme yields a significant reduction on the number of calculations involved, when compared with the standard vector parameterization based schemes. As real buildings are always open loop stable, the use of an stable shear building model for vibration control purposes allows the design of more robust control laws. Extensive simulation results are presented for cases of low excitation comparing the results of using or not this matrix projection method with different sets of initial conditions. Results indicate that the use of this projection method does not have an influence in the recovery of natural frequencies, however, it significantly improves the recovery of mode shapes.

  7. Reduction of Lysyl Hydroxylase 3 Causes Deleterious Changes in the Deposition and Organization of Extracellular Matrix*

    PubMed Central

    Risteli, Maija; Ruotsalainen, Heli; Salo, Antti M.; Sormunen, Raija; Sipilä, Laura; Baker, Naomi L.; Lamandé, Shireen R.; Vimpari-Kauppinen, Leena; Myllylä, Raili

    2009-01-01

    Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase, collagen galactosyltransferase, and glucosyltransferase (GGT) activities. We report here an important role for LH3 in the organization of the extracellular matrix (ECM) and cytoskeleton. Deposition of ECM was affected in heterozygous LH3 knock-out mouse embryonic fibroblasts (MEF+/−) and in skin fibroblasts collected from a member of a Finnish epidermolysis bullosa simplex (EBS) family known to be deficient in GGT activity. We show the GGT deficiency to be due to a transcriptional defect in one LH3 allele. The ECM abnormalities also lead to defects in the arrangement of the cytoskeleton in both cell lines. Ultrastructural abnormalities were observed in the skin of heterozygous LH3 knock-out mice indicating that even a moderate decrease in LH3 has deleterious consequences in vivo. The LH3 null allele in the EBS family member and the resulting abnormalities in the organization of the extracellular matrix, similar to those found in MEF+/−, may explain the correlation between the severity of the phenotype and the decrease in GGT activity reported in this family. PMID:19696018

  8. Amyloid β-Protein as a Substrate Interacts with Extracellular Matrix to Promote Neurite Outgrowth

    NASA Astrophysics Data System (ADS)

    Koo, Edward H.; Park, Lisa; Selkoe, Dennis J.

    1993-05-01

    Progressive deposition of amyloid β-protein (Aβ) in brain parenchyma and blood vessels is a characteristic feature of Alzheimer disease. Recent evidence suggests that addition of solubilized synthetic Aβ to medium may produce toxic or trophic effects on cultured hippocampal neurons. Because soluble Aβ may not accumulate in significant quantities in brain, we asked whether immobilized Aβ peptide as a substrate alters neurite outgrowth from cultured rat peripheral sensory neurons. This paradigm may closely mimic the conditions in Alzheimer disease brain tissue, in which neurites contact insoluble, extracellular aggregates of β-amyloid. We detected no detrimental effects of Aβ substrate on neurite outgrowth. Rather, Aβ in combination with low doses of laminin or fibronectin enhanced neurite out-growth from these neuronal explants. Our results suggest that insoluble Aβ in the cerebral neuropil may serve as a neurite-promoting matrix, perhaps explaining the apparent regenerative response of neurites observed around amyloid plaques in Alzheimer disease. Moreover, in concert with the recent discovery of Aβ production by cultured neurons, our data suggest that Aβ plays a normal physiological role in brain by complexing with the extracellular matrix.

  9. Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms.

    PubMed

    Martins, Margarida; Uppuluri, Priya; Thomas, Derek P; Cleary, Ian A; Henriques, Mariana; Lopez-Ribot, José L; Oliveira, Rosário

    2010-05-01

    DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.

  10. Marfan syndrome: defective synthesis, secretion, and extracellular matrix formation of fibrillin by cultured dermal fibroblasts.

    PubMed Central

    Milewicz, D M; Pyeritz, R E; Crawford, E S; Byers, P H

    1992-01-01

    We studied the synthesis, secretion, and aggregation into the extracellular matrix of fibrillin by dermal fibroblasts from 26 probands with the Marfan syndrome. Cells from seven probands synthesized approximately half the normal amount of fibrillin when compared with intrafamilial or unrelated controls. Cells from an additional seven probands synthesized a normal amount of fibrillin but secreted the protein less efficiently than control cells. Cells from a further eight probands synthesized and secreted normal amounts of fibrillin but the protein was poorly incorporated into extracellular matrix. Cells from the remaining four probands were indistinguishable from control cells in their synthesis and processing of fibrillin. Cells from 18 family members of 10 of the probands were also studied. Cells from affected individuals in the same family had the same biochemical defect and those from unaffected family members were indistinguishable from controls. These results indicate that mutations in the gene that encodes fibrillin are responsible for the Marfan syndrome in the majority of individuals (confirming recent immunohistochemical and genetic linkage studies) and that a variety of mutations can produce the phenotype associated with the syndrome. Images PMID:1729284

  11. Core-shell hydrogel beads with extracellular matrix for tumor spheroid formation

    PubMed Central

    Yu, L.; Grist, S. M.; Nasseri, S. S.; Ni, C.; Cheung, K. C.

    2015-01-01

    Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture. PMID:25945144

  12. Mesenchymal Remodeling during Palatal Shelf Elevation Revealed by Extracellular Matrix and F-Actin Expression Patterns.

    PubMed

    Chiquet, Matthias; Blumer, Susan; Angelini, Manuela; Mitsiadis, Thimios A; Katsaros, Christos

    2016-01-01

    During formation of the secondary palate in mammalian embryos, two vertically oriented palatal shelves rapidly elevate into a horizontal position above the tongue, meet at the midline, and fuse to form a single entity. Previous observations suggested that elevation occurs by a simple 90° rotation of the palatal shelves. More recent findings showed that the presumptive midline epithelial cells are not located at the tips of palatal shelves before elevation, but mostly toward their medial/lingual part. This implied extensive tissue remodeling during shelf elevation. Nevertheless, it is still not known how the shelf mesenchyme reorganizes during this process, and what mechanism drives it. To address this question, we mapped the distinct and restricted expression domains of certain extracellular matrix components within the developing palatal shelves. This procedure allowed to monitor movements of entire mesenchymal regions relative to each other during shelf elevation. Consistent with previous notions, our results confirm a flipping movement of the palatal shelves anteriorly, whereas extensive mesenchymal reorganization is observed more posteriorly. There, the entire lingual portion of the vertical shelves moves close to the midline after elevation, whereas the mesenchyme at the original tip of the shelves ends up ventrolaterally. Moreover, we observed that the mesenchymal cells of elevating palatal shelves substantially align their actin cytoskeleton, their extracellular matrix, and their nuclei in a ventral to medial direction. This indicates that, like in other morphogenetic processes, actin-dependent cell contractility is a major driving force for mesenchymal tissue remodeling during palatogenesis. PMID:27656150

  13. Mesenchymal Remodeling during Palatal Shelf Elevation Revealed by Extracellular Matrix and F-Actin Expression Patterns

    PubMed Central

    Chiquet, Matthias; Blumer, Susan; Angelini, Manuela; Mitsiadis, Thimios A.; Katsaros, Christos

    2016-01-01

    During formation of the secondary palate in mammalian embryos, two vertically oriented palatal shelves rapidly elevate into a horizontal position above the tongue, meet at the midline, and fuse to form a single entity. Previous observations suggested that elevation occurs by a simple 90° rotation of the palatal shelves. More recent findings showed that the presumptive midline epithelial cells are not located at the tips of palatal shelves before elevation, but mostly toward their medial/lingual part. This implied extensive tissue remodeling during shelf elevation. Nevertheless, it is still not known how the shelf mesenchyme reorganizes during this process, and what mechanism drives it. To address this question, we mapped the distinct and restricted expression domains of certain extracellular matrix components within the developing palatal shelves. This procedure allowed to monitor movements of entire mesenchymal regions relative to each other during shelf elevation. Consistent with previous notions, our results confirm a flipping movement of the palatal shelves anteriorly, whereas extensive mesenchymal reorganization is observed more posteriorly. There, the entire lingual portion of the vertical shelves moves close to the midline after elevation, whereas the mesenchyme at the original tip of the shelves ends up ventrolaterally. Moreover, we observed that the mesenchymal cells of elevating palatal shelves substantially align their actin cytoskeleton, their extracellular matrix, and their nuclei in a ventral to medial direction. This indicates that, like in other morphogenetic processes, actin-dependent cell contractility is a major driving force for mesenchymal tissue remodeling during palatogenesis. PMID:27656150

  14. Mesenchymal Remodeling during Palatal Shelf Elevation Revealed by Extracellular Matrix and F-Actin Expression Patterns

    PubMed Central

    Chiquet, Matthias; Blumer, Susan; Angelini, Manuela; Mitsiadis, Thimios A.; Katsaros, Christos

    2016-01-01

    During formation of the secondary palate in mammalian embryos, two vertically oriented palatal shelves rapidly elevate into a horizontal position above the tongue, meet at the midline, and fuse to form a single entity. Previous observations suggested that elevation occurs by a simple 90° rotation of the palatal shelves. More recent findings showed that the presumptive midline epithelial cells are not located at the tips of palatal shelves before elevation, but mostly toward their medial/lingual part. This implied extensive tissue remodeling during shelf elevation. Nevertheless, it is still not known how the shelf mesenchyme reorganizes during this process, and what mechanism drives it. To address this question, we mapped the distinct and restricted expression domains of certain extracellular matrix components within the developing palatal shelves. This procedure allowed to monitor movements of entire mesenchymal regions relative to each other during shelf elevation. Consistent with previous notions, our results confirm a flipping movement of the palatal shelves anteriorly, whereas extensive mesenchymal reorganization is observed more posteriorly. There, the entire lingual portion of the vertical shelves moves close to the midline after elevation, whereas the mesenchyme at the original tip of the shelves ends up ventrolaterally. Moreover, we observed that the mesenchymal cells of elevating palatal shelves substantially align their actin cytoskeleton, their extracellular matrix, and their nuclei in a ventral to medial direction. This indicates that, like in other morphogenetic processes, actin-dependent cell contractility is a major driving force for mesenchymal tissue remodeling during palatogenesis.

  15. Up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver

    PubMed Central

    Zhang, Shuai; Li, Tao-Sheng; Soyama, Akihiko; Tanaka, Takayuki; Yan, Chen; Sakai, Yusuke; Hidaka, Masaaki; Kinoshita, Ayaka; Natsuda, Koji; Fujii, Mio; Kugiyama, Tota; Baimakhanov, Zhassulan; Kuroki, Tamotsu; Gu, Weili; Eguchi, Susumu

    2016-01-01

    Although the healthy liver is known to have high regenerative potential, poor liver regeneration under pathological conditions remains a substantial problem. We investigated the key molecules that impair the regeneration of cholestatic liver. C57BL/6 mice were randomly subjected to partial hepatectomy and bile duct ligation (PH+BDL group, n = 16), partial hepatectomy only (PH group, n = 16), or sham operation (Sham group, n = 16). The liver sizes and histological findings were similar in the PH and sham groups 14 days after operation. However, compared with those in the sham group, the livers in mice in the PH+BDL group had a smaller size, a lower cell proliferative activity, and more fibrotic tissue 14 days after the operation, suggesting the insufficient regeneration of the cholestatic liver. Pathway-focused array analysis showed that many genes were up- or down-regulated over 1.5-fold in both PH+BDL and PH groups at 1, 3, 7, and 14 days after treatment. Interestingly, more genes that were functionally related to the extracellular matrix and inflammatory chemokines were found in the PH+BDL group than in the PH group at 7 and 14 days after treatment. Our data suggest that up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver. PMID:27226149

  16. Stirred flow bioreactor modulates chondrocyte growth and extracellular matrix biosynthesis in chitosan scaffolds.

    PubMed

    García Cruz, Dunia Mercedes; Salmerón-Sánchez, Manuel; Gómez-Ribelles, José Luis

    2012-09-01

    The aim of this study is to show the favorable effect of simple dynamic culture conditions on chondrogenesis of previously expanded human chondrocytes seeded in a macroporous scaffold with week cell-pore walls adhesion. We obtained enhanced chondrogenesis by the combination of chitosan porous supports with a double micro- and macro-pore structure and cell culture in a stirring bioreactor. Cell-scaffold constructs were cultured under static or mechanically stimulated conditions using an intermittent stirred flow bioreactor during 28 days. In static culture, the chondrocytes were homogeneously distributed throughout the scaffold pores; cells adhered to the scaffold pore walls, showed extended morphology and were able to proliferate. Immunofluorescense and biochemical assays showed abundant type I collagen deposition at day 28. However, the behavior of chondrocytes submitted to mechanical stimuli in the bioreactor was completely different. Mechanical loading influenced cell morphology and extracellular matrix composition. Under dynamic conditions, chondrocytes kept their characteristic phenotype and tended to form cell aggregates surrounded by a layer of the main components of the hyaline cartilage extracellular matrix, type II collagen, and aggrecan. An enhanced aggrecan and collagen type II production was observed in engineered cartilage constructs cultured under stirred flow compared with those cultured under static conditions. PMID:22529045

  17. Science of Hyaluronic Acid Beyond Filling: Fibroblasts and Their Response to the Extracellular Matrix.

    PubMed

    Landau, Marina; Fagien, Steven

    2015-11-01

    Loss of viscoelasticity is one of the primarily signs of skin aging, followed by appearance of visible wrinkles. Hyaluronic acid (HA)-based fillers are widely used to fill wrinkles and compensate for volume loss. Recent clinical observations demonstrate persistence of the filling effect longer than the biological availability of the filler. Stimulation of new collagen by cross-linked HA and up-regulation of elastin have been suggested as possible explanation to this observation and have been supported experimentally. Cross-linked HA substitutes for fragmented collagen in restoring extracellular matrix required for normal activity of fibroblasts, such as collagen and elastin production. To restore extracellular matrix efficiently, serial monthly treatments are required. Boosting of facial and nonfacial skin through fibroblast activation is a new indication for HA-based products. Injectable HA has also been recently registered in Europe as agents specific for the improvement of skin quality (Restylane Skinboosters). Further explanation of the possible mechanisms supported by long-term clinical examples is presented herein.

  18. Aortic Wall Extracellular Matrix Proteins Correlate with Syntax Score in Patients Undergoing Coronary Artery Bypass Surgery

    PubMed Central

    Chiong, Terri; Cheow, Esther S. H.; Woo, Chin C.; Lin, Xiao Y.; Khin, Lay W.; Lee, Chuen N.; Hartman, Mikael; Sze, Siu K.; Sorokin, Vitaly A.

    2016-01-01

    Aims: The SYNTAX score correlate with major cardiovascular events post-revascularization, although the histopathological basis is unclear. We aim to evaluate the association between syntax score and extracellular matrix histological characteristics of aortic punch tissue obtained during coronary artery bypass surgery (CABG). This analysis compares coronary artery bypass surgery patients with High and Low syntax score which were followed up for one year period. Methods and Results: Patients with High (score ≥ 33, (n=77)) and Low Syntax Scores (score ≤ 22, (n=71)) undergoing elective CABG were recruited prospectively. Baseline clinical characteristics and surgical risks were well matched. At 1 year, EMACCE (Sum of cardiovascular death, stroke, congestive cardiac failure, and limb, gut and myocardial ischemia) was significantly elevated in the High syntax group (P=0.022). Mass spectrometry (MS)-based quantitative iTRAQ proteomic results validated on independent cohort by immunohistochemistry (IHC) revealed that the High syntax group had significantly upraised Collagen I (P<0.0001) and Elastin (P<0.0001) content in ascending aortic wall. Conclusion: This study shows that aortic extracellular matrix (ECM) differ between High and Low syntax groups with up-regulation of Collagen I and Elastin level in High Syntax Score group. This identifies aortic punches collected during CABG as another biomarker source related with atherosclerosis severity and possible clinical outcome. PMID:27347220

  19. The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins.

    PubMed

    Lord, Megan S; Cheng, Bill; McCarthy, Simon J; Jung, MoonSun; Whitelock, John M

    2011-10-01

    Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by α(IIb)β(3) integrins, while α(2)β(1) integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of α(IIb)β(3) integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions.

  20. Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro.

    PubMed

    Alibardi, Lorenzo; Polazzi, Elisabetta

    2012-04-01

    The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.

  1. Bubaline Cholecyst Derived Extracellular Matrix for Reconstruction of Full Thickness Skin Wounds in Rats

    PubMed Central

    Shakya, Poonam; Sharma, A. K.; Kumar, Naveen; Vellachi, Remya; Mathew, Dayamon D.; Dubey, Prasoon; Singh, Kiranjeet; Shrivastava, Sonal; Shrivastava, Sameer; Maiti, S. K.; Hasan, Anwarul; Singh, K. P.

    2016-01-01

    An acellular cholecyst derived extracellular matrix (b-CEM) of bubaline origin was prepared using anionic biological detergent. Healing potential of b-CEM was compared with commercially available collagen sheet (b-CS) and open wound (C) in full thickness skin wounds in rats. Thirty-six clinically healthy adult Sprague Dawley rats of either sex were randomly divided into three equal groups. Under general anesthesia, a full thickness skin wound (20 × 20 mm2) was created on the dorsum of each rat. The defect in group I was kept as open wound and was taken as control. In group II, the defect was repaired with commercially available collagen sheet (b-CS). In group III, the defect was repaired with cholecyst derived extracellular matrix of bovine origin (b-CEM). Planimetry, wound contracture, and immunological and histological observations were carried out to evaluate healing process. Significantly (P < 0.05) increased wound contraction was observed in b-CEM (III) as compared to control (I) and b-CS (II) on day 21. Histologically, improved epithelization, neovascularization, fibroplasia, and best arranged collagen fibers were observed in b-CEM (III) as early as on postimplantation day 21. These findings indicate that b-CEM have potential for biomedical applications for full thickness skin wound repair in rats. PMID:27127678

  2. A Lipocalin-Derived Peptide Modulating Fibroblasts and Extracellular Matrix Proteins

    PubMed Central

    Carrijo-Carvalho, Linda Christian; Maria, Durvanei A.; Ventura, Janaina S.; Morais, Kátia L. P.; Melo, Robson L.; Rodrigues, Consuelo Junqueira; Chudzinski-Tavassi, Ana Marisa

    2012-01-01

    Lipocalin family members have been implicated in development, regeneration, and pathological processes, but their roles are unclear. Interestingly, these proteins are found abundant in the venom of the Lonomia obliqua caterpillar. Lipocalins are β-barrel proteins, which have three conserved motifs in their amino acid sequence. One of these motifs was shown to be a sequence signature involved in cell modulation. The aim of this study is to investigate the effects of a synthetic peptide comprising the lipocalin sequence motif in fibroblasts. This peptide suppressed caspase 3 activity and upregulated Bcl-2 and Ki-67, but did not interfere with GPCR calcium mobilization. Fibroblast responses also involved increased expression of proinflammatory mediators. Increase of extracellular matrix proteins, such as collagen, fibronectin, and tenascin, was observed. Increase in collagen content was also observed in vivo. Results indicate that modulation effects displayed by lipocalins through this sequence motif involve cell survival, extracellular matrix remodeling, and cytokine signaling. Such effects can be related to the lipocalin roles in disease, development, and tissue repair. PMID:22737165

  3. [Disorders of the extracellular matrix in epithelial-stromal and stromal corneal dystrophies].

    PubMed

    Varkoly, Gréta; Bencze, János; Módis, László; Hortobágyi, Tibor

    2016-08-01

    The human cornea is rich in extracellular matrix. The stroma constitutes the main thickness of the cornea, which consists of collagens and proteoglycans mainly. The epithelial-stromal and stromal dystrophies of the cornea are either autosomal dominant or recessive inherited disorders, which are unrelated to inflammation or trauma. The diseases can manifest in each layer of the cornea, but in most cases the corneal stroma is affected. Generally, they develop in childhood or young adulthood but the diagnosis is only possible when clinical signs (epithelial erosions, decreased visual acuity, photophobia) develop. The different protein aggregates (hyaline, amyloid, crystalline) deposited in the corneal layers result in mild or advanced corneal opacity and loss of the corneal transparency due to disorganisation of the extracellular matrix. In some of the corneal dystrophies the keratane sulphate proteoglycan looses its function which results in a loss of the regular interfibrillar spacing. Due to the severe corneal opacity patients may need corneal transplantation. Orv. Hetil., 2016, 157(33), 1299-1303. PMID:27523312

  4. Up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver.

    PubMed

    Zhang, Shuai; Li, Tao-Sheng; Soyama, Akihiko; Tanaka, Takayuki; Yan, Chen; Sakai, Yusuke; Hidaka, Masaaki; Kinoshita, Ayaka; Natsuda, Koji; Fujii, Mio; Kugiyama, Tota; Baimakhanov, Zhassulan; Kuroki, Tamotsu; Gu, Weili; Eguchi, Susumu

    2016-01-01

    Although the healthy liver is known to have high regenerative potential, poor liver regeneration under pathological conditions remains a substantial problem. We investigated the key molecules that impair the regeneration of cholestatic liver. C57BL/6 mice were randomly subjected to partial hepatectomy and bile duct ligation (PH+BDL group, n = 16), partial hepatectomy only (PH group, n = 16), or sham operation (Sham group, n = 16). The liver sizes and histological findings were similar in the PH and sham groups 14 days after operation. However, compared with those in the sham group, the livers in mice in the PH+BDL group had a smaller size, a lower cell proliferative activity, and more fibrotic tissue 14 days after the operation, suggesting the insufficient regeneration of the cholestatic liver. Pathway-focused array analysis showed that many genes were up- or down-regulated over 1.5-fold in both PH+BDL and PH groups at 1, 3, 7, and 14 days after treatment. Interestingly, more genes that were functionally related to the extracellular matrix and inflammatory chemokines were found in the PH+BDL group than in the PH group at 7 and 14 days after treatment. Our data suggest that up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver. PMID:27226149

  5. Up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver.

    PubMed

    Zhang, Shuai; Li, Tao-Sheng; Soyama, Akihiko; Tanaka, Takayuki; Yan, Chen; Sakai, Yusuke; Hidaka, Masaaki; Kinoshita, Ayaka; Natsuda, Koji; Fujii, Mio; Kugiyama, Tota; Baimakhanov, Zhassulan; Kuroki, Tamotsu; Gu, Weili; Eguchi, Susumu

    2016-01-01

    Although the healthy liver is known to have high regenerative potential, poor liver regeneration under pathological conditions remains a substantial problem. We investigated the key molecules that impair the regeneration of cholestatic liver. C57BL/6 mice were randomly subjected to partial hepatectomy and bile duct ligation (PH+BDL group, n = 16), partial hepatectomy only (PH group, n = 16), or sham operation (Sham group, n = 16). The liver sizes and histological findings were similar in the PH and sham groups 14 days after operation. However, compared with those in the sham group, the livers in mice in the PH+BDL group had a smaller size, a lower cell proliferative activity, and more fibrotic tissue 14 days after the operation, suggesting the insufficient regeneration of the cholestatic liver. Pathway-focused array analysis showed that many genes were up- or down-regulated over 1.5-fold in both PH+BDL and PH groups at 1, 3, 7, and 14 days after treatment. Interestingly, more genes that were functionally related to the extracellular matrix and inflammatory chemokines were found in the PH+BDL group than in the PH group at 7 and 14 days after treatment. Our data suggest that up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver.

  6. Extracellular matrix family proteins that are potential targets of Dd-STATa in Dictyostelium discoideum.

    PubMed

    Shimada, Nao; Nishio, Keiko; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

    2004-10-01

    Dd-STATa is a functional Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription) proteins, which is activated by cAMP and is thereby translocated into the nuclei of anterior tip cells of the prestalk region of the slug. By using in situ hybridization analyses, we found that the SLF308 cDNA clone, which contains the ecmF gene that encodes a putative extracellular matrix protein and is expressed in the anterior tip cells, was greatly down-regulated in the Dd-STATa-null mutant. Disruption of the ecmF gene, however, resulted in almost no phenotypic change. The absence of any obvious mutant phenotype in the ecmF-null mutant could be due to a redundancy of similar genes. In fact, a search of the Dictyostelium whole genome database demonstrates the existence of an additional 16 homologues, all of which contain a cellulose-binding module. Among these homologues, four genes show Dd-STATa-dependent expression, while the others are Dd-STATa-independent. We discuss the potential role of Dd-STATa in morphogenesis via its effect on the interaction between cellulose and these extracellular matrix family proteins.

  7. Response of Inflammatory Mediators, Extracellular Matrix Proteins and Stem and Progenitor Cells to Emphysema.

    PubMed

    Skurikhin, E G; Pakhomova, A V; Krupin, V A; Pershina, O V; Pan, E S; Ermolaeva, L A; Vaizova, O E; Rybalkina, O Yu; Dygai, A M

    2016-08-01

    Inflammation, extracellular matrix proteins (hydroxyproline, connective tissue growth factor, collagen, and fibronectin), stem and progenitor cells (multipotent mesenchymal stromal cells, Clara cells, angiogenesis, precursors, endothelial and epithelial cells) were studied in female C57Bl/6 mice with experimental elastase-induced emphysema. Diffuse emphysema reduced the number of endothelial (CD45(-)CD31(+)CD34(+)) and epithelial (CD45(-)CD117(+)CD49f(+)) cells, induced microcirculation disturbances, and decreased the area occupied by the connective tissue. Emphysematous changes in the lungs were accompanied by infiltration of the alveolar septa with macrophages and lymphocytes, increase in the serum and lung concentrations of transforming growth factor-β, IL-1β, IL-2, IL-5, IL-10, and IL-13, and lung concentration of IL-17. In the lungs, inflammation was associated with marked increase in the number of multipotent mesenchymal stromal cells CD90(+)CD73(+)CD106(+)CD44(+)) and Clara cells (CD45(-)CD34(-)CD31(-)Sca1(+)) and overexpression of extracellular matrix proteins (hydroxyproline, connective tissue growth factor, collagen, fibronectin) and Clara cells protein. On the other hand, elastase reduced the number of angiogenic precursor cells (CD45(-)CD117(+)Flk1(+)). PMID:27591877

  8. Science of Hyaluronic Acid Beyond Filling: Fibroblasts and Their Response to the Extracellular Matrix.

    PubMed

    Landau, Marina; Fagien, Steven

    2015-11-01

    Loss of viscoelasticity is one of the primarily signs of skin aging, followed by appearance of visible wrinkles. Hyaluronic acid (HA)-based fillers are widely used to fill wrinkles and compensate for volume loss. Recent clinical observations demonstrate persistence of the filling effect longer than the biological availability of the filler. Stimulation of new collagen by cross-linked HA and up-regulation of elastin have been suggested as possible explanation to this observation and have been supported experimentally. Cross-linked HA substitutes for fragmented collagen in restoring extracellular matrix required for normal activity of fibroblasts, such as collagen and elastin production. To restore extracellular matrix efficiently, serial monthly treatments are required. Boosting of facial and nonfacial skin through fibroblast activation is a new indication for HA-based products. Injectable HA has also been recently registered in Europe as agents specific for the improvement of skin quality (Restylane Skinboosters). Further explanation of the possible mechanisms supported by long-term clinical examples is presented herein. PMID:26441098

  9. Laser-Deposited In Situ TiC-Reinforced Nickel Matrix Composites: 3D Microstructure and Tribological Properties

    NASA Astrophysics Data System (ADS)

    Borkar, Tushar; Sosa, John; Hwang, Jun Yeon; Scharf, Thomas W.; Tiley, Jaimie; Fraser, Hamish; Banerjee, Rajarshi

    2014-06-01

    A new class of Ni-Ti-C-based metal-matrix composites has been developed using the laser-engineered net shaping™ process. These composites consist of an in situ formed and homogeneously distributed titanium carbide (TiC) phase reinforcing the nickel matrix. Additionally, by tailoring the Ti/C ratio in these composites, an additional graphitic phase can also be engineered into the microstructure. Serial-sectioning, followed by three-dimensional reconstruction of the microstructure in these composites, reveals homogeneously distributed primary and eutectic titanium carbide precipitates as well as a graphitic phase encompassing the primary carbides within the nickel matrix. The morphology and spatial distribution of these phases in three dimensions reveals that the eutectic carbides form a network linked by primary carbides or graphitic nodules at the nodes, which suggests interesting insights into the sequence of phase evolution. These three-phase Ni-TiC-C composites exhibit excellent tribological properties, in terms of an extremely low coefficient of friction while maintaining a relatively high hardness.

  10. Ubiquitylation functions in the calcium carbonate biomineralization in the extracellular matrix.

    PubMed

    Fang, Dong; Pan, Cong; Lin, Huijuan; Lin, Ya; Xu, Guangrui; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

    2012-01-01

    Mollusks shell formation is mediated by matrix proteins and many of these proteins have been identified and characterized. However, the mechanisms of protein control remain unknown. Here, we report the ubiquitylation of matrix proteins in the prismatic layer of the pearl oyster, Pinctada fucata. The presence of ubiquitylated proteins in the prismatic layer of the shell was detected with a combination of western blot and immunogold assays. The coupled ubiquitins were separated and identified by Edman degradation and liquid chromatography/mass spectrometry (LC/MS). Antibody injection in vivo resulted in large amounts of calcium carbonate randomly accumulating on the surface of the nacreous layer. These ubiquitylated proteins could bind to specific faces of calcite and aragonite, which are the two main mineral components of the shell. In the in vitro calcium carbonate crystallization assay, they could reduce the rate of calcium carbonate precipitation and induce the calcite formation. Furthermore, when the attached ubiquitins were removed, the functions of the EDTA-soluble matrix of the prismatic layer were changed. Their potency to inhibit precipitation of calcium carbonate was decreased and their influence on the morphology of calcium carbonate crystals was changed. Taken together, ubiquitylation is involved in shell formation. Although the ubiquitylation is supposed to be involved in every aspect of biophysical processes, our work connected the biomineralization-related proteins and the ubiquitylation mechanism in the extracellular matrix for the first time. This would promote our understanding of the shell biomineralization and the ubiquitylation processes.

  11. Comparison of Matrix Cracking in Melt-Infiltrated SiC/SiC Composites with 3D and 2D-Woven Orthogonal Architectures

    NASA Technical Reports Server (NTRS)

    Morscher, Gregory N.

    2003-01-01

    Acoustic emission techniques combined with microstructural observations were used to determine the dependence of through thickness matrix cracking (TTMC) on in-plane tensile stress for melt infiltrated Sylramic fiber-based SIC/SiC composite panels with various 3D-woven orthogonal fiber architectures. Results were compared with prior TTMC results from similar panels with 2D woven orthogonal architectures.Both data sets were analysed on the basis that the source for TTMC originated in the 90 degree or Z-fiber tows.

  12. Calcium diffusion enhanced after cleavage of negatively charged components of brain extracellular matrix by chondroitinase ABC

    PubMed Central

    Hrabětová, Sabina; Masri, Daniel; Tao, Lian; Xiao, Fanrong; Nicholson, Charles

    2009-01-01

    The concentration of extracellular calcium plays a critical role in synaptic transmission and neuronal excitability as well as other physiological processes. The time course and extent of local fluctuations in the concentration of this ion largely depend on its effective diffusion coefficient (D*) and it has been speculated that fixed negative charges on chondroitin sulphate proteoglycans (CSPGs) and other components of the extracellular matrix may influence calcium diffusion because it is a divalent cation. In this study we used ion-selective microelectrodes combined with pressure ejection or iontophoresis of ions from a micropipette to quantify diffusion characteristics of neocortex and hippocampus in rat brain slices. We show that D* for calcium is less than the value predicted from the behaviour of the monovalent cation tetramethylammonium (TMA), a commonly used diffusion probe, but D* for calcium increases in both brain regions after the slices are treated with chondroitinase ABC, an enzyme that predominantly cleaves chondroitin sulphate glycans. These results suggest that CSPGs do play a role in determining the local diffusion properties of calcium in brain tissue, most likely through electrostatic interactions mediating rapid equilibrium binding. In contrast, chondroitinase ABC does not affect either the TMA diffusion or the extracellular volume fraction, indicating that the enzyme does not alter the structure of the extracellular space and that the diffusion of small monovalent cations is not affected by CSPGs in the normal brain ionic milieu. Both calcium and CSPGs are known to have many distinct roles in brain physiology, including brain repair, and our study suggests they may be functionally coupled through calcium diffusion properties. PMID:19546165

  13. Efficient fully 3D list-mode TOF PET image reconstruction using a factorized system matrix with an image domain resolution model

    PubMed Central

    Zhou, Jian; Qi, Jinyi

    2014-01-01

    A factorized system matrix utilizing an image domain resolution model is attractive in fully 3D TOF PET image reconstruction using list-mode data. In this paper, we study a factored model based on sparse matrix factorization that is comprised primarily of a simplified geometrical projection matrix and an image blurring matrix. Beside the commonly-used Siddon's raytracer, we propose another more simplified geometrical projector based on the Bresenham's raytracer which further reduces the computational cost. We discuss in general how to obtain an image blurring matrix associated with a geometrical projector, and provide theoretical analysis that can be used to inspect the efficiency in model factorization. In simulation studies, we investigate the performance of the proposed sparse factorization model in terms of spatial resolution, noise properties and computational cost. The quantitative results reveal that the factorization model can be as efficient as a nonfactored model such as the analytical model while its computational cost can be much lower. In addition we conduct Monte Carlo simulations to identify the conditions under which the image resolution model can become more efficient in terms of image contrast recovery. We verify our observations using the provided theoretical analysis. The result offers a general guide to achieve optimal reconstruction performance based on a sparse factorization model with an only image domain resolution model. PMID:24434568

  14. Efficient fully 3D list-mode TOF PET image reconstruction using a factorized system matrix with an image domain resolution model

    NASA Astrophysics Data System (ADS)

    Zhou, Jian; Qi, Jinyi

    2014-02-01

    A factorized system matrix utilizing an image domain resolution model is attractive in fully 3D time-of-flight PET image reconstruction using list-mode data. In this paper, we study a factored model based on sparse matrix factorization that is comprised primarily of a simplified geometrical projection matrix and an image blurring matrix. Beside the commonly-used Siddon’s ray-tracer, we propose another more simplified geometrical projector based on the Bresenham’s ray-tracer which further reduces the computational cost. We discuss in general how to obtain an image blurring matrix associated with a geometrical projector, and provide theoretical analysis that can be used to inspect the efficiency in model factorization. In simulation studies, we investigate the performance of the proposed sparse factorization model in terms of spatial resolution, noise properties and computational cost. The quantitative results reveal that the factorization model can be as efficient as a non-factored model, while its computational cost can be much lower. In addition we conduct Monte Carlo simulations to identify the conditions under which the image resolution model can become more efficient in terms of image contrast recovery. We verify our observations using the provided theoretical analysis. The result offers a general guide to achieve the optimal reconstruction performance based on a sparse factorization model with an image domain resolution model.

  15. Collagen XII and XIV, New Partners of Cartilage Oligomeric Matrix Protein in the Skin Extracellular Matrix Suprastructure*

    PubMed Central

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R.; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-01-01

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone. PMID:22573329

  16. Matrilin-3 switches from anti- to pro-anabolic upon integration to the extracellular matrix.

    PubMed

    Vincourt, Jean-Baptiste; Etienne, Stéphanie; Grossin, Laurent; Cottet, Justine; Bantsimba-Malanda, Claudie; Netter, Patrick; Mainard, Didier; Libante, Virginie; Gillet, Pierre; Magdalou, Jacques

    2012-06-01

    The extracellular matrix (ECM) has long been viewed primarily as an organized network of solid-phase ligands for integrin receptors. During degenerative processes, such as osteoarthritis, the ECM undergoes deterioration, resulting in its remodeling and in the release of some of its components. Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. Here, to better understand the pathological and physiological functions of MATN3, its concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, however, MATN3 induced chondrocyte adhesion via a α5β1 integrin-dependent mechanism, AKT activation and favored surviva