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Sample records for aligned collagen fibrils

  1. In vitro collagen fibril alignment via incorporation of nanocrystalline cellulose.

    PubMed

    Rudisill, Stephen G; DiVito, Michael D; Hubel, Allison; Stein, Andreas

    2015-01-01

    This study demonstrates a method for producing ordered collagen fibrils on a similar length scale to those in the cornea, using a one-pot liquid-phase synthesis. The alignment persists throughout samples on the mm scale. The addition of nanocrystalline cellulose (NCC), a biocompatible and widely available material, to collagen prior to gelation causes the fibrils to align and achieve a narrow size distribution (36±8nm). The effects of NCC loading in the composites on microstructure, transparency and biocompatibility are studied by scanning electron microscopy, ultraviolet-visible spectroscopy and cell growth experiments. A 2% loading of NCC increases the transparency of collagen while producing an ordered microstructure. A mechanism is proposed for the ordering behavior on the basis of enhanced hydrogen bonding during collagen gel formation.

  2. Collagen fibril alignment and deformation during tensile strain of leather: a small-angle X-ray scattering study.

    PubMed

    Basil-Jones, Melissa M; Edmonds, Richard L; Norris, Gillian E; Haverkamp, Richard G

    2012-02-01

    The distribution and effect of applied strain on the collagen fibrils that make up leather may have an important bearing on the ultimate strength and other physical properties of the material. While sections of ovine and bovine leather were being subjected to tensile strain up to rupture, synchrotron-based small-angle X-ray scattering (SAXS) spectra were recorded edge-on to the leather at points from the corium to the grain. Measurements of both fibril orientation and collagen d spacing showed that, initially, the fibers reorient under strain, becoming more aligned. As the strain increases (5-10% strain), further fibril reorientation diminishes until, at 37% strain, the d spacing increases by up to 0.56%, indicating that significant tensile forces are being transmitted to individual fibrils. These changes, however, are not uniform through the cross-section of leather and differ between leathers of different strengths. The stresses are taken up more evenly through the leather cross-section in stronger leathers in comparison to weaker leathers, where stresses tended to be concentrated during strain. These observations contribute to our understanding of the internal strains and structural changes that take place in leather under stress.

  3. Collagen Fibrils: Nanoscale Ropes

    PubMed Central

    Bozec, Laurent; van der Heijden, Gert; Horton, Michael

    2007-01-01

    The formation of collagen fibrils from staggered repeats of individual molecules has become “accepted” wisdom. However, for over thirty years now, such a model has failed to resolve several structural and functional questions. In a novel approach, it was found, using atomic force microscopy, that tendon collagen fibrils are composed of subcomponents in a spiral disposition—that is, their structure is similar to that of macroscale ropes. Consequently, this arrangement was modeled and confirmed using elastic rod theory. This work provides new insight into collagen fibril structure and will have wide application—from the design of scaffolds for tissue engineering and a better understanding of pathogenesis of diseases of bone and tendon, to the conservation of irreplaceable parchment-based museum exhibits. PMID:17028135

  4. Collagen fibril diameter and leather strength.

    PubMed

    Wells, Hannah C; Edmonds, Richard L; Kirby, Nigel; Hawley, Adrian; Mudie, Stephen T; Haverkamp, Richard G

    2013-11-27

    The main structural component of leather and skin is type I collagen in the form of strong fibrils. Strength is an important property of leather, and the way in which collagen contributes to the strength is not fully understood. Synchrotron-based small angle X-ray scattering (SAXS) is used to measure the collagen fibril diameter of leather from a range of animals, including sheep and cattle, that had a range of tear strengths. SAXS data were fit to a cylinder model. The collagen fibril diameter and tear strength were found to be correlated in bovine leather (r(2) = 0.59; P = 0.009), with stronger leather having thicker fibrils. There was no correlation between orientation index, i.e., fibril alignment, and fibril diameter for this data set. Ovine leather showed no correlation between tear strength and fibril diameter, nor was there a correlation across a selection of other animal leathers. The findings presented here suggest that there may be a different structural motif in skin compared with tendon, particularly ovine skin or leather, in which the diameter of the individual fibrils contributes less to strength than fibril alignment does.

  5. Collagen fibril formation during development

    SciTech Connect

    Fleischmajer, R.; Perlish, J.S.; Timpl, R.; Olsen, B.R.

    1987-05-01

    Studies with embryonic skin and bone suggested that the aminopropeptide (AP) and carboxylpropeptide (CP) of type I pro-callagen (pro-col) play a role in fibril formation. Chick leg metatarsal tendons were studied by electron microscopy. AP and CP of type I pro-col were purified from chick leg tendons; antibodies developed in rabbits and purity tested by radioimmunoassays. Antibodies were used for immunofluorescence microscopy (IFM) and immunoblotting (IB). The peritendineum, consisting of thin 20-30 nm fibrils, revealed the AP of type I and type III procol. In the tendon area, collagen fibrils were arranged within small compartments and were of uniform diameter at 10d, 14d and 18d. However, beyond 21d, there was confluency of the compartments and a wide range of fibril diameters. IFM revealed fine streaks of collagen, staining with the AP of type I throughout the tendon. The CP was mainly intracellular with only a small amount present in the extracellular space. IB revealed procollagen, pN-collagen (AP+collagen) and pC-collagen, (CP+collagen) at all stages of development. Ratios of pN/pC collagen, determined by spectrophotometric scanning of autoradiographs, correlated well with the distribution of fibril diameter. This study suggests the hypothesis that AP initiates fibrillogenesis while CP may regulate additional fibril growth.

  6. 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.

  7. Type V collagen controls the initiation of collagen fibril assembly.

    PubMed

    Wenstrup, Richard J; Florer, Jane B; Brunskill, Eric W; Bell, Sheila M; Chervoneva, Inna; Birk, David E

    2004-12-17

    Vertebrate collagen fibrils are heterotypically composed of a quantitatively major and minor fibril collagen. In non-cartilaginous tissues, type I collagen accounts for the majority of the collagen mass, and collagen type V, the functions of which are poorly understood, is a minor component. Type V collagen has been implicated in the regulation of fibril diameter, and we reported recently preliminary evidence that type V collagen is required for collagen fibril nucleation (Wenstrup, R. J., Florer, J. B., Cole, W. G., Willing, M. C., and Birk, D. E. (2004) J. Cell. Biochem. 92, 113-124). The purpose of this study was to define the roles of type V collagen in the regulation of collagen fibrillogenesis and matrix assembly. Mouse embryos completely deficient in pro-alpha1(V) chains were created by homologous recombination. The col5a1-/- animals die in early embryogenesis, at approximately embryonic day 10. The type V collagen-deficient mice demonstrate a virtual lack of collagen fibril formation. In contrast, the col5a1+/- animals are viable. The reduced type V collagen content is associated with a 50% reduction in fibril number and dermal collagen content. In addition, relatively normal, cylindrical fibrils are assembled with a second population of large, structurally abnormal collagen fibrils. The structural properties of the abnormal matrix are decreased relative to the wild type control animals. These data indicate a central role for the evolutionary, ancient type V collagen in the regulation of fibrillogenesis. The complete dependence of fibril formation on type V collagen is indicative of the critical role of the latter in early fibril initiation. In addition, this fibril collagen is important in the determination of fibril structure and matrix organization. PMID:15383546

  8. Cell Alignment Driven by Mechanically Induced Collagen Fiber Alignment in Collagen/Alginate Coatings

    PubMed Central

    Chaubaroux, Christophe; Perrin-Schmitt, Fabienne; Senger, Bernard; Vidal, Loïc; Voegel, Jean-Claude; Schaaf, Pierre; Haikel, Youssef; Boulmedais, Fouzia; Lavalle, Philippe

    2015-01-01

    For many years it has been a major challenge to regenerate damaged tissues using synthetic or natural materials. To favor the healing processes after tendon, cornea, muscle, or brain injuries, aligned collagen-based architectures are of utmost interest. In this study, we define a novel aligned coating based on a collagen/alginate (COL/ALG) multilayer film. The coating exhibiting a nanofibrillar structure is cross-linked with genipin for stability in physiological conditions. By stretching COL/ALG-coated polydimethylsiloxane substrates, we developed a versatile method to align the collagen fibrils of the polymeric coating. Assays on cell morphology and alignment were performed to investigate the properties of these films. Microscopic assessments revealed that cells align with the stretched collagen fibrils of the coating. The degree of alignment is tuned by the stretching rate (i.e., the strain) of the COL/ALG-coated elastic substrate. Such coatings are of great interest for strategies that require aligned nanofibrillar biological material as a substrate for tissue engineering. PMID:25658028

  9. Tension tests on mammalian collagen fibrils.

    PubMed

    Liu, Yehe; Ballarini, Roberto; Eppell, Steven J

    2016-02-01

    A brief overview of isolated collagen fibril mechanics testing is followed by presentation of the first results testing fibrils isolated from load-bearing mammalian tendons using a microelectromechanical systems platform. The in vitro modulus (326 ± 112 MPa) and fracture stress (71 ± 23 MPa) are shown to be lower than previously measured on fibrils extracted from sea cucumber dermis and tested with the same technique. Scanning electron microscope images show the fibrils can fail with a mechanism that involves circumferential rupture, whereas the core of the fibril stays at least partially intact. PMID:26855757

  10. Study of Native Type I Collagen Fibrils

    NASA Astrophysics Data System (ADS)

    Heim, August

    2006-03-01

    Presented in this work is direct imaging and force microscopy of native, intact type I collagen fibrils extracted from the sea cucumber Cucumaria frondosa dermis with affiliated proteoglycan molecules. The prototypical collagen fibril structure is well conserved through higher mammalian species and presents a model for study of the mechanical properties of the primary individual components of the dermis and skeletal ligature. Common practice is to use reconstituted fibrils which lack the precise conformal structure and affiliated proteoglycans. We have performed force microscopy to probe the mechanical properties of native fibrils and extract the elastic modulus under natural conditions. This knowledge is combined transmission and atomic force imaging, in conjunction with applied computation models, to demonstrate an inherent semitubular structure of these fibrils.

  11. Viscoelastic properties of isolated collagen fibrils.

    PubMed

    Shen, Zhilei Liu; Kahn, Harold; Ballarini, Roberto; Eppell, Steven J

    2011-06-22

    Understanding the viscoelastic behavior of collagenous tissues with complex hierarchical structures requires knowledge of the properties at each structural level. Whole tissues have been studied extensively, but less is known about the mechanical behavior at the submicron, fibrillar level. Using a microelectromechanical systems platform, in vitro coupled creep and stress relaxation tests were performed on collagen fibrils isolated from the sea cucumber dermis. Stress-strain-time data indicate that isolated fibrils exhibit viscoelastic behavior that could be fitted using the Maxwell-Weichert model. The fibrils showed an elastic modulus of 123 ± 46 MPa. The time-dependent behavior was well fit using the two-time-constant Maxwell-Weichert model with a fast time response of 7 ± 2 s and a slow time response of 102 ± 5 s. The fibrillar relaxation time was smaller than literature values for tissue-level relaxation time, suggesting that tissue relaxation is dominated by noncollagenous components (e.g., proteoglycans). Each specimen was tested three times, and the only statistically significant difference found was that the elastic modulus is larger in the first test than in the subsequent two tests, indicating that viscous properties of collagen fibrils are not sensitive to the history of previous tests.

  12. Viscoelastic Properties of Isolated Collagen Fibrils

    PubMed Central

    Shen, Zhilei Liu; Kahn, Harold; Ballarini, Roberto; Eppell, Steven J.

    2011-01-01

    Understanding the viscoelastic behavior of collagenous tissues with complex hierarchical structures requires knowledge of the properties at each structural level. Whole tissues have been studied extensively, but less is known about the mechanical behavior at the submicron, fibrillar level. Using a microelectromechanical systems platform, in vitro coupled creep and stress relaxation tests were performed on collagen fibrils isolated from the sea cucumber dermis. Stress-strain-time data indicate that isolated fibrils exhibit viscoelastic behavior that could be fitted using the Maxwell-Weichert model. The fibrils showed an elastic modulus of 123 ± 46 MPa. The time-dependent behavior was well fit using the two-time-constant Maxwell-Weichert model with a fast time response of 7 ± 2 s and a slow time response of 102 ± 5 s. The fibrillar relaxation time was smaller than literature values for tissue-level relaxation time, suggesting that tissue relaxation is dominated by noncollagenous components (e.g., proteoglycans). Each specimen was tested three times, and the only statistically significant difference found was that the elastic modulus is larger in the first test than in the subsequent two tests, indicating that viscous properties of collagen fibrils are not sensitive to the history of previous tests. PMID:21689535

  13. Elastic Response of Crimped Collagen Fibrils

    NASA Technical Reports Server (NTRS)

    Freed, Alan D.; Doehring, Todd C.

    2005-01-01

    A physiologic constitutive expression is presented in algorithmic format for the elastic response of wavy collagen fibrils found in soft connective tissues. The model is based on the observation that crimped fibrils have a three-dimensional structure at the micrometer scale that we approximate as a helical spring. The symmetry of this waveform allows the force/displacement relationship derived from Castigliano's theorem to be solved in closed form. Model predictions are in good agreement with experimental observations for mitral-valve chordae tendineae

  14. Elastic model for crimped collagen fibrils

    NASA Technical Reports Server (NTRS)

    Freed, Alan D.; Doehring, Todd C.

    2005-01-01

    A physiologic constitutive expression is presented in algorithmic format for the nonlinear elastic response of wavy collagen fibrils found in soft connective tissues. The model is based on the observation that crimped fibrils in a fascicle have a three-dimensional structure at the micron scale that we approximate as a helical spring. The symmetry of this wave form allows the force/displacement relationship derived from Castigliano's theorem to be solved in closed form: all integrals become analytic. Model predictions are in good agreement with experimental observations for mitral-valve chordae tendinece.

  15. Collagen fibril arrangement and size distribution in monkey oral mucosa

    PubMed Central

    OTTANI, V.; FRANCHI, M.; DE PASQUALE, V.; LEONARDI, L.; MOROCUTTI, M.; RUGGERI, A.

    1998-01-01

    Collagen fibre organisation and fibril size were studied in the buccal gingival and hard palate mucosa of Macacus rhesus monkey. Light and electron microscopy analysis showed connective papillae exhibiting a similar inner structure in the different areas examined, but varying in distribution, shape and size. Moving from the deep to surface layers of the buccal gingival mucosa (free and attached portions), large collagen fibril bundles became smaller and progressively more wavy with decreasing collagen fibril diameter. This gradual diameter decrease did not occur in the hard palate mucosa (free portion, rugae and interrugal regions) where the fibril diameter remained constant. A link between collagen fibril diameter and mechanical function is discussed. PMID:9688498

  16. Uniform spatial distribution of collagen fibril radii within tendon implies local activation of pC-collagen at individual fibrils

    NASA Astrophysics Data System (ADS)

    Rutenberg, Andrew D.; Brown, Aidan I.; Kreplak, Laurent

    2016-08-01

    Collagen fibril cross-sectional radii show no systematic variation between the interior and the periphery of fibril bundles, indicating an effectively constant rate of collagen incorporation into fibrils throughout the bundle. Such spatially homogeneous incorporation constrains the extracellular diffusion of collagen precursors from sources at the bundle boundary to sinks at the growing fibrils. With a coarse-grained diffusion equation we determine stringent bounds, using parameters extracted from published experimental measurements of tendon development. From the lack of new fibril formation after birth, we further require that the concentration of diffusing precursors stays below the critical concentration for fibril nucleation. We find that the combination of the diffusive bound, which requires larger concentrations to ensure homogeneous fibril radii, and lack of nucleation, which requires lower concentrations, is only marginally consistent with fully processed collagen using conservative bounds. More realistic bounds may leave no consistent concentrations. Therefore, we propose that unprocessed pC-collagen diffuses from the bundle periphery followed by local C-proteinase activity and subsequent collagen incorporation at each fibril. We suggest that C-proteinase is localized within bundles, at fibril surfaces, during radial fibrillar growth. The much greater critical concentration of pC-collagen, as compared to fully processed collagen, then provides broad consistency between homogeneous fibril radii and the lack of fibril nucleation during fibril growth.

  17. Thermal Memory in Self-Assembled Collagen Fibril Networks

    PubMed Central

    de Wild, Martijn; Pomp, Wim; Koenderink, Gijsje H.

    2013-01-01

    Collagen fibrils form extracellular networks that regulate cell functions and provide mechanical strength to tissues. Collagen fibrillogenesis is an entropy-driven process promoted by warming and reversed by cooling. Here, we investigate the influence of noncovalent interactions mediated by the collagen triple helix on fibril stability. We measure the kinetics of cold-induced disassembly of fibrils formed from purified collagen I using turbimetry, probe the fibril morphology by atomic force microscopy, and measure the network connectivity by confocal microscopy and rheometry. We demonstrate that collagen fibrils disassemble by subunit release from their sides as well as their ends, with complex kinetics involving an initial fast release followed by a slow release. Surprisingly, the fibrils are gradually stabilized over time, leading to thermal memory. This dynamic stabilization may reflect structural plasticity of the collagen fibrils arising from their complex structure. In addition, we propose that the polymeric nature of collagen monomers may lead to slow kinetics of subunit desorption from the fibril surface. Dynamic stabilization of fibrils may be relevant in the initial stages of collagen assembly during embryogenesis, fibrosis, and wound healing. Moreover, our results are relevant for tissue repair and drug delivery applications, where it is crucial to control fibril stability. PMID:23823240

  18. An equilibrium double-twist model for the radial structure of collagen fibrils.

    PubMed

    Brown, Aidan I; Kreplak, Laurent; Rutenberg, Andrew D

    2014-11-14

    Mammalian tissues contain networks and ordered arrays of collagen fibrils originating from the periodic self-assembly of helical 300 nm long tropocollagen complexes. The fibril radius is typically between 25 to 250 nm, and tropocollagen at the surface appears to exhibit a characteristic twist-angle with respect to the fibril axis. Similar fibril radii and twist-angles at the surface are observed in vitro, suggesting that these features are controlled by a similar self-assembly process. In this work, we propose a physical mechanism of equilibrium radius control for collagen fibrils based on a radially varying double-twist alignment of tropocollagen within a collagen fibril. The free-energy of alignment is similar to that of liquid crystalline blue phases, and we employ an analytic Euler-Lagrange and numerical free energy minimization to determine the twist-angle between the molecular axis and the fibril axis along the radial direction. Competition between the different elastic energy components, together with a surface energy, determines the equilibrium radius and twist-angle at the fibril surface. A simplified model with a twist-angle that is linear with radius is a reasonable approximation in some parameter regimes, and explains a power-law dependence of radius and twist-angle at the surface as parameters are varied. Fibril radius and twist-angle at the surface corresponding to an equilibrium free-energy minimum are consistent with existing experimental measurements of collagen fibrils. Remarkably, in the experimental regime, all of our model parameters are important for controlling equilibrium structural parameters of collagen fibrils. PMID:25238208

  19. Structural investigations on native collagen type I fibrils using AFM

    SciTech Connect

    Strasser, Stefan; Zink, Albert; Janko, Marek; Heckl, Wolfgang M.; Thalhammer, Stefan . E-mail: stefan.thalhammer@gsf.de

    2007-03-02

    This study was carried out to determine the elastic properties of single collagen type I fibrils with the use of atomic force microscopy (AFM). Native collagen fibrils were formed by self-assembly in vitro characterized with the AFM. To confirm the inner assembly of the collagen fibrils, the AFM was used as a microdissection tool. Native collagen type I fibrils were dissected and the inner core uncovered. To determine the elastic properties of collagen fibrils the tip of the AFM was used as a nanoindentor by recording force-displacement curves. Measurements were done on the outer shell and in the core of the fibril. The structural investigations revealed the banding of the shell also in the core of native collagen fibrils. Nanoindentation experiments showed the same Young's modulus on the shell as well as in the core of the investigated native collagen fibrils. In addition, the measurements indicate a higher adhesion in the core of the collagen fibrils compared to the shell.

  20. Molecular structure and functional morphology of echinoderm collagen fibrils.

    PubMed

    Trotter, J A; Thurmond, F A; Koob, T J

    1994-03-01

    The collagenous tissues of echinoderms, which have the unique capacity to rapidly and reversibly alter their mechanical properties, resemble the collagenous tissues of other phyla in consisting of collagen fibrils in a nonfibrillar matrix. Knowledge of the composition and structure of their collagen fibrils and interfibrillar matrix is thus important for an understanding of the physiology of these tissues. In this report it is shown that the collagen molecules from the fibrils of the spine ligament of a sea-urchin and the deep dermis of a sea-cucumber are the same length as those from vertebrate fibrils and that they assemble into fibrils with the same repeat period and gap/overlap ratio as do those of vertebrate fibrils. The distributions of charged residues in echinoderm and vertebrate molecules are somewhat different, giving rise to segment-long-spacing crystallites and fibrils with different banding patterns. Compared to the vertebrate pattern, the banding pattern of echinoderm fibrils is characterized by greatly increased stain intensity in the c3 band and greatly reduced stain intensity in the a3 and b2 bands. The fibrils are spindle-shaped, possessing no constant-diameter region throughout their length. The shape of the fibrils is mechanically advantageous for their reinforcing role in a discontinuous fiber-composite material.

  1. Native collagen fibrils from echinoderms are molecularly bipolar.

    PubMed

    Thurmond, F A; Trotter, J A

    1994-01-01

    Collagen fibrils are generally assumed to be cylinders with uniform diameters (except possibly at their ends) and to be composed of molecules all of which have the same polarity. These assumptions have been largely untested because of the extreme difficulty associated with isolating entire native fibrils. Intact collagen fibrils are readily extracted from certain echinoderms, however, and we have therefore analyzed the molecular structure of these fibrils. Our electron microscopic analyses show the above assumptions to be false: echinoderm fibrils, which previously have been shown to be symmetrically spindle shaped, are also molecularly bipolar. Their constituent molecules have their N-termini oriented toward the nearest fibril end, and they are antiparallel in the fibril center. The shape and molecular arrangement of these fibrils have implications for fibrillogenesis.

  2. Capsaicin inhibits collagen fibril formation and increases the stability of collagen fibers.

    PubMed

    Perumal, Sathiamurthi; Dubey, Kriti; Badhwar, Rahul; George, Kodimattan Joseph; Sharma, Rakesh Kumar; Bagler, Ganesh; Madhan, Balaraman; Kar, Karunakar

    2015-02-01

    Capsaicin is a versatile plant product which has been ascribed several health benefits and anti-inflammatory and analgesic properties. We have investigated the effect of capsaicin on the molecular stability, self-assembly, and fibril stability of type-I collagen. It was found that capsaicin suppresses collagen fibril formation, increases the stability of collagen fibers in tendons, and has no effect on the molecular stability of collagen. Turbidity assay data show that capsaicin does not promote disassembly of collagen fibrils. However, capsaicin moderately protects collagen fibrils from enzymatic degradation. Computational studies revealed the functions of the aromatic group and amide region of capsaicin in the collagen-capsaicin interaction. The results may have significant implications for capsaicin-based therapeutics that target excess collagen accumulation-linked pathology, for example thrombosis, fibrosis, and sclerosis.

  3. Second harmonic generation quantitative measurements on collagen fibrils through correlation to electron microscopy

    NASA Astrophysics Data System (ADS)

    Bancelin, S.; Aimé, C.; Gusachenko, I.; Kowalczuk, L.; Latour, G.; Coradin, T.; Schanne-Klein, M.-C.

    2015-03-01

    Type I collagen is a major structural protein in mammals that shows highly structured macromolecular organizations specific to each tissue. This biopolymer is synthesized as triple helices, which self-assemble into fibrils (Ø =10-300 nm) and further form various 3D organization. In recent years, Second Harmonic Generation (SHG) microscopy has emerged as a powerful technique to probe in situ the fibrillar collagenous network within tissues. However, this optical technique cannot resolve most of the fibrils and is a coherent process, which has impeded quantitative measurements of the fibril diameter so far. In this study, we correlated SHG microscopy with Transmission Electron Microscopy to determine the sensitivity of SHG microscopy and to calibrate SHG signals as a function of the fibril diameter in reconstructed collagen gels. To that end, we synthetized isolated fibrils with various diameters and successfully imaged the very same fibrils with both techniques, down to 30 nm diameter. We observed that SHG signals scaled as the fourth power of the fibril diameter, as expected from analytical and numerical calculations. This calibration was then applied to diabetic rat cornea in which we successfully recovered the diameter of hyperglycemia-induced fibrils in the Descemet's membrane without having to resolve them. Finally we derived the first hyperpolarizability from a single collagen triple helix which validates the bottom-up approach used to calculate the non-linear response at the fibrillar scale and denotes a parallel alignment of triple helices within the fibrils. These results represent a major step towards quantitative SHG imaging of nm-sized collagen fibrils.

  4. Stress-strain experiments on individual collagen fibrils.

    PubMed

    Shen, Zhilei L; Dodge, Mohammad Reza; Kahn, Harold; Ballarini, Roberto; Eppell, Steven J

    2008-10-01

    Collagen, a molecule consisting of three braided protein helices, is the primary building block of many biological tissues including bone, tendon, cartilage, and skin. Staggered arrays of collagen molecules form fibrils, which arrange into higher-ordered structures such as fibers and fascicles. Because collagen plays a crucial role in determining the mechanical properties of these tissues, significant theoretical research is directed toward developing models of the stiffness, strength, and toughness of collagen molecules and fibrils. Experimental data to guide the development of these models, however, are sparse and limited to small strain response. Using a microelectromechanical systems platform to test partially hydrated collagen fibrils under uniaxial tension, we obtained quantitative, reproducible mechanical measurements of the stress-strain curve of type I collagen fibrils, with diameters ranging from 150-470 nm. The fibrils showed a small strain (epsilon < 0.09) modulus of 0.86 +/- 0.45 GPa. Fibrils tested to strains as high as 100% demonstrated strain softening (sigma(yield) = 0.22 +/- 0.14 GPa; epsilon(yield) = 0.21 +/- 0.13) and strain hardening, time-dependent recoverable residual strain, dehydration-induced embrittlement, and susceptibility to cyclic fatigue. The results suggest that the stress-strain behavior of collagen fibrils is dictated by global characteristic dimensions as well as internal structure. PMID:18641067

  5. The collagen fibril organization in human articular cartilage.

    PubMed Central

    Minns, R J; Steven, F S

    1977-01-01

    In this scanning electron microscopic study blocks of collagen fibrils were prepared from human articular cartilage, using two techinques which selectively removed either the proteoglycans alone, or both the proteoglycans and the collagen fibrils, of the non-calcified cartilage layer. Amino acid analysis of the fibrils confirmed the purity of the collagen after proteoglycan extraction. The cartilage was scanned in four different ways: (1) normal to the articular surface, (2) in superficial sections, (3) on surfaces of blocks which had been broken in planes parallel to artificial splits make by the insertion of a pin, and (4) on fracture surfaces which traversed the calcified cartilage and the subchondral bone. Five features of the organization of the collagen fibrils were specially noted: (1) Individual fibrils within the trabeculae joined to form small fibre bundles which became grouped into larger bundles at the calcified/uncalcified interface. (2) Fibrils in the deep and middle zones which, exhibiting the characteristic surface periodicity of collagen, were generally oriented towars the articular surface in large bundles approximately 55 micronm across. (3) In the superficial zone, fibrils ran parallel to the surface. (4) The surface fibrils had random orientation, even at the bases of empty lacunae vacated by chondrocytes during specimen preparation. (5) The collagen fibrils of the lacunar walls appeared to be thinner and more closely packed than thos between the lacunae. The fine collagen fibrils associated with the lacunar walls were frequently observed to pass through a large lacunar space, resulting in the formation of two or more compartments, each of which was presumably filled with a chondrocyte in the living cartilage. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 PMID:870478

  6. Deformation micromechanisms of collagen fibrils under uniaxial tension

    PubMed Central

    Tang, Yuye; Ballarini, Roberto; Buehler, Markus J.; Eppell, Steven J.

    2010-01-01

    Collagen, an essential building block of connective tissues, possesses useful mechanical properties due to its hierarchical structure. However, little is known about the mechanical properties of collagen fibril, an intermediate structure between the collagen molecule and connective tissue. Here, we report the results of systematic molecular dynamics simulations to probe the mechanical response of initially unflawed finite size collagen fibrils subjected to uniaxial tension. The observed deformation mechanisms, associated with rupture and sliding of tropocollagen molecules, are strongly influenced by fibril length, width and cross-linking density. Fibrils containing more than approximately 10 molecules along their length and across their width behave as representative volume elements and exhibit brittle fracture. Shorter fibrils experience a more graceful ductile-like failure. An analytical model is constructed and the results of the molecular modelling are used to find curve-fitted expressions for yield stress, yield strain and fracture strain as functions of fibril structural parameters. Our results for the first time elucidate the size dependence of mechanical failure properties of collagen fibrils. The associated molecular deformation mechanisms allow the full power of traditional material and structural engineering theory to be applied to our understanding of the normal and pathological mechanical behaviours of collagenous tissues under load. PMID:19897533

  7. Molecular packing in bone collagen fibrils prior to mineralization

    NASA Astrophysics Data System (ADS)

    Hsiao, Benjamin; Zhou, Hong-Wen; Burger, Christian; Chu, Benjamin; Glimcher, Melvin J.

    2012-02-01

    The three-dimensional packing of collagen molecules in bone collagen fibrils has been largely unknown because even in moderately mineralized bone tissues, the organic matrix structure is severely perturbed by the deposition of mineral crystals. During the past decades, the structure of tendon collagen (e.g. rat tail) --- a tissue that cannot mineralize in vivo, has been assumed to be representative for bone collagen fibrils. Small-angle X-ray diffraction analysis of the native, uncalcified intramuscular fish bone has revealed a new molecular packing scheme, significantly different from the quasi-hexagonal arrangement often found in tendons. The deduced structure in bone collagen fibrils indicates the presence of spatially discrete microfibrils, and an arrangement of intrafibrillar space to form ``channels'', which could accommodate crystals with dimensions typically found in bone apatite.

  8. Nanointerfacial strength between non-collagenous protein and collagen fibrils in antler bone

    PubMed Central

    Hang, Fei; Gupta, Himadri S.; Barber, Asa H.

    2014-01-01

    Antler bone displays considerable toughness through the use of a complex nanofibrous structure of mineralized collagen fibrils (MCFs) bound together by non-collagenous proteins (NCPs). While the NCP regions represent a small volume fraction relative to the MCFs, significant surface area is evolved upon failure of the nanointerfaces formed at NCP–collagen fibril boundaries. The mechanical properties of nanointerfaces between the MCFs are investigated directly in this work using an in situ atomic force microscopy technique to pull out individual fibrils from the NCP. Results show that the NCP–fibril interfaces in antler bone are weak, which highlights the propensity for interface failure at the nanoscale in antler bone and extensive fibril pullout observed at antler fracture surfaces. The adhesion between fibrils and NCP is additionally suggested as being rate dependent, with increasing interfacial strength and fracture energy observed when pullout velocity decreases. PMID:24352676

  9. Epitaxially Grown Collagen Fibrils Reveal Diversity in Contact Guidance Behavior among Cancer Cells

    PubMed Central

    2015-01-01

    Invasion of cancer cells into the surrounding tissue is an important step during cancer progression and is driven by cell migration. Cell migration can be random, but often it is directed by various cues such as aligned fibers composed of extracellular matrix (ECM), a process called contact guidance. During contact guidance, aligned fibers bias migration along the long axis of the fibers. These aligned fibers of ECM are commonly composed of type I collagen, an abundant structural protein around tumors. In this paper, we epitaxially grew several different patterns of organized type I collagen on mica and compared the morphology and contact guidance behavior of two invasive breast cancer cell lines (MDA-MB-231 and MTLn3 cells). Others have shown that these cells randomly migrate in qualitatively different ways. MDA-MB-231 cells exert large traction forces, tightly adhere to the ECM, and migrate with spindle-shaped morphology and thus adopt a mesenchymal mode of migration. MTLn3 cells exert small traction forces, loosely adhere to the ECM, and migrate with a more rounded morphology and thus adopt an amoeboid mode of migration. As the degree of alignment of type I collagen fibrils increases, cells become more elongated and engage in more directed contact guidance. MDA-MB-231 cells perceive the directional signal of highly aligned type I collagen fibrils with high fidelity, elongating to large extents and migrating directionally. Interestingly, behavior in MTLn3 cells differs. While highly aligned type I collagen fibril patterns facilitate spreading and random migration of MTLn3 cells, they do not support elongation or directed migration. Thus, different contact guidance cues bias cell migration differently and the fidelity of contact guidance is cell type dependent, suggesting that ECM alignment is a permissive cue for contact guidance, but requires a cell to have certain properties to interpret that cue. PMID:25531276

  10. Does the genetic type of collagen determine fibril structure

    SciTech Connect

    Eikenberry, E.; Brodsky, B.; Cassidy, K.

    1980-10-01

    A number of genetic types of collagen, all triple-helical but with significant variations in their amino acid sequences, have been found and the distribution of these genetic types is tissue specific. For example, tendon is composed only of type I collagen, while cartilage contains largely type II collagen. Skin contains a large amount of type I, but has a significant fraction, approx. 15%, of type III. Each of these types can form fibrils, but it is not known whether they form distinctive fibril structures that are important in determining tissue organization. We are using x-ray diffraction to analyze a variety of tissues with different collagen genetic types to compare the fibril structures and thus investigate whether genetic type is an important determinant of this structure.

  11. Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts

    PubMed Central

    1984-01-01

    The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments. PMID:6542105

  12. Measurement of the Mechanical Properties of Intact Collagen Fibrils

    NASA Astrophysics Data System (ADS)

    Mercedes, H.; Heim, A.; Matthews, W. G.; Koob, T.

    2006-03-01

    Motivated by the genetic disorder Ehlers-Danlos syndrome (EDS), in which proper collagen synthesis is interrupted, we are investigating the structural and mechanical properties of collagen fibrils. The fibrous glycoprotein collagen is the most abundant protein found in the human body and plays a key role in the extracellular matrix of the connective tissue, the properties of which are altered in EDS. We have selected as our model system the collagen fibrils of the sea cucumber dermis, a naturally mutable tissue. This system allows us to work with native fibrils which have their proteoglycan complement intact, something that is not possible with reconstituted mammalian collagen fibrils. Using atomic force microscopy, we measure, as a function of the concentration of divalent cations, the fibril diameter, its response to force loading, and the changes in its rigidity. Through these experiments, we will shed light on the mechanisms which control the properties of the sea cucumber dermis and hope to help explain the altered connective tissue extracellular matrix properties associated with EDS.

  13. Interpreting Second-Harmonic Generation Images of Collagen I Fibrils

    PubMed Central

    Williams, Rebecca M.; Zipfel, Warren R.; Webb, Watt W.

    2005-01-01

    Fibrillar collagen, being highly noncentrosymmetric, possesses a tremendous nonlinear susceptibility. As a result, second-harmonic generation (SHG) microscopy of collagen produces extremely bright and robust signals, providing an invaluable tool for imaging tissue structure with submicron resolution. Here we discuss fundamental principles governing SHG phase matching with the tightly focusing optics used in microscopy. Their application to collagen imaging yields several biophysical features characteristic of native collagen structure: SHG radiates from the shell of a collagen fibril, rather than from its bulk. This SHG shell may correspond to the supporting element of the fibril. Physiologically relevant changes in solution ionic strength alter the ratio of forward-to-backward propagating SHG, implying a resulting change in the SHG shell thickness. Fibrillogenesis can be resolved in immature tissue by directly imaging backward-propagating SHG. Such findings are crucial to the design and development of forthcoming diagnostic and research tools. PMID:15533922

  14. PRODUCTION OF HIGHLY-ALIGNED COLLAGEN LAMELLAE BY COMBINING SHEAR FORCE AND THIN-FILM CONFINEMENT

    PubMed Central

    Saeidi, Nima; Sander, Edward A.; Zareian, Ramin

    2012-01-01

    Load-bearing tissues owe their mechanical strength to their highly-anisotropic collagenous structure. To date, attempts to engineer mechanically strong connective tissue have failed mainly due to the lack of the ability to reproduce native collagen organization in constructs synthesized by cultured cells in vitro. The ability to influence the direction of the self-assembling collagen molecules and produce highly anisotropic structures has applications ranging from de novo engineering of complex tissues to the production of organized scaffolds for cell culture contact guidance. In this investigation we have used the simple technique of spin coating to produce highly-aligned arrays of collagen fibrils. By a simple modification of the method we have also successfully produced orthogonal collagen lamellae. Alternating collagen lamellae are frequently seen in load-bearing tissues such as cornea, annulus fibrosus, and cortical bone. Culturing of corneal fibroblasts onto aligned collagen shows that the cells adopt the organization of the fibrils. In this investigation, we observed the reversal of fibrillar growth direction or “hook” formation similar to those seen previously in a microfluidic shear-flow chamber. Although the results of this investigation clearly show that it is possible to produce small areas (O) 1 cm2 of collagen fibrils with enough alignment to guide fibroblasts, there is evidence that thin film instabilities are likely to be a significant barrier to producing organized collagen fibrils over larger areas. Successful application of this method to produce highly-controlled and organized collagenous structures will require the development of techniques to control thin film instability and will be the subject of the future work. PMID:21362500

  15. Viscoelastic behavior of discrete human collagen fibrils.

    PubMed

    Svensson, René B; Hassenkam, Tue; Hansen, Philip; Peter Magnusson, S

    2010-01-01

    Whole tendon and fibril bundles display viscoelastic behavior, but to the best of our knowledge this property has not been directly measured in single human tendon fibrils. In the present work an atomic force microscopy (AFM) approach was used for tensile testing of two human patellar tendon fibrils. Fibrils were obtained from intact human fascicles, without any pre-treatment besides frozen storage. In the dry state a single isolated fibril was anchored to a substrate using epoxy glue, and the end of the fibril was glued on to an AFM cantilever for tensile testing. In phosphate buffered saline, cyclic testing was performed in the pre-yield region at different strain rates, and the elastic response was determined by a stepwise stress relaxation test. The elastic stress-strain response corresponded to a second-order polynomial fit, while the viscous response showed a linear dependence on the strain. The slope of the viscous response showed a strain rate dependence corresponding to a power function of powers 0.242 and 0.168 for the two patellar tendon fibrils, respectively. In conclusion, the present work provides direct evidence of viscoelastic behavior at the single fibril level, which has not been previously measured. PMID:19878908

  16. A new model to simulate the elastic properties of mineralized collagen fibril

    SciTech Connect

    Yuan, F.; Stock, S.R.; Haeffner, D.R.; Almer, J.D.; Dunand, D.C.; Brinson, L.C.

    2012-05-02

    Bone, because of its hierarchical composite structure, exhibits an excellent combination of stiffness and toughness, which is due substantially to the structural order and deformation at the smaller length scales. Here, we focus on the mineralized collagen fibril, consisting of hydroxyapatite plates with nanometric dimensions aligned within a protein matrix, and emphasize the relationship between the structure and elastic properties of a mineralized collagen fibril. We create two- and three-dimensional representative volume elements to represent the structure of the fibril and evaluate the importance of the parameters defining its structure and properties of the constituent mineral and collagen phase. Elastic stiffnesses are calculated by the finite element method and compared with experimental data obtained by synchrotron X-ray diffraction. The computational results match the experimental data well, and provide insight into the role of the phases and morphology on the elastic deformation characteristics. Also, the effects of water, imperfections in the mineral phase and mineral content outside the mineralized collagen fibril upon its elastic properties are discussed.

  17. A new model to simulate the elastic properties of mineralized collagen fibril.

    SciTech Connect

    Yuan, F.; Stock, S.R.; Haeffner, D.R.; Almer, J.D.; Dunand , D.C.; Brinson, K.

    2011-01-01

    Bone, because of its hierarchical composite structure, exhibits an excellent combination of stiffness and toughness, which is due substantially to the structural order and deformation at the smaller length scales. Here, we focus on the mineralized collagen fibril, consisting of hydroxyapatite plates with nanometric dimensions aligned within a protein matrix, and emphasize the relationship between the structure and elastic properties of a mineralized collagen fibril. We create two- and three-dimensional representative volume elements to represent the structure of the fibril and evaluate the importance of the parameters defining its structure and properties of the constituent mineral and collagen phase. Elastic stiffnesses are calculated by the finite element method and compared with experimental data obtained by synchrotron X-ray diffraction. The computational results match the experimental data well, and provide insight into the role of the phases and morphology on the elastic deformation characteristics. Also, the effects of water, imperfections in the mineral phase and mineral content outside the mineralized collagen fibril upon its elastic properties are discussed.

  18. Mechanical Properties of Mineralized Collagen Fibrils As Influenced By Demineralization

    SciTech Connect

    Balooch, M.; Habelitz, S.; Kinney, J.H.; Marshall, S.J.; Marshall, G.W.

    2009-05-11

    Dentin and bone derive their mechanical properties from a complex arrangement of collagen type-I fibrils reinforced with nanocrystalline apatite mineral in extra- and intrafibrillar compartments. While mechanical properties have been determined for the bulk of the mineralized tissue, information on the mechanics of the individual fibril is limited. Here, atomic force microscopy was used on individual collagen fibrils to study structural and mechanical changes during acid etching. The characteristic 67 nm periodicity of gap zones was not observed on the mineralized fibril, but became apparent and increasingly pronounced with continuous demineralization. AFM-nanoindentation showed a decrease in modulus from 1.5 GPa to 50 MPa during acid etching of individual collagen fibrils and revealed that the modulus profile followed the axial periodicity. The nanomechanical data, Raman spectroscopy and SAXS support the hypothesis that intrafibrillar mineral etches at a substantially slower rate than the extrafibrillar mineral. These findings are relevant for understanding the biomechanics and design principles of calcified tissues derived from collagen matrices.

  19. Imaging and 3D morphological analysis of collagen fibrils.

    PubMed

    Altendorf, H; Decencière, E; Jeulin, D; De sa Peixoto, P; Deniset-Besseau, A; Angelini, E; Mosser, G; Schanne-Klein, M-C

    2012-08-01

    The recent booming of multiphoton imaging of collagen fibrils by means of second harmonic generation microscopy generates the need for the development and automation of quantitative methods for image analysis. Standard approaches sequentially analyse two-dimensional (2D) slices to gain knowledge on the spatial arrangement and dimension of the fibrils, whereas the reconstructed three-dimensional (3D) image yields better information about these characteristics. In this work, a 3D analysis method is proposed for second harmonic generation images of collagen fibrils, based on a recently developed 3D fibre quantification method. This analysis uses operators from mathematical morphology. The fibril structure is scanned with a directional distance transform. Inertia moments of the directional distances yield the main fibre orientation, corresponding to the main inertia axis. The collaboration of directional distances and fibre orientation delivers a geometrical estimate of the fibre radius. The results include local maps as well as global distribution of orientation and radius of the fibrils over the 3D image. They also bring a segmentation of the image into foreground and background, as well as a classification of the foreground pixels into the preferred orientations. This accurate determination of the spatial arrangement of the fibrils within a 3D data set will be most relevant in biomedical applications. It brings the possibility to monitor remodelling of collagen tissues upon a variety of injuries and to guide tissues engineering because biomimetic 3D organizations and density are requested for better integration of implants.

  20. Strain-Induced Alignment in Collagen Gels

    PubMed Central

    Vader, David; Kabla, Alexandre; Weitz, David; Mahadevan, Lakshminarayana

    2009-01-01

    Collagen is the most abundant extracellular-network-forming protein in animal biology and is important in both natural and artificial tissues, where it serves as a material of great mechanical versatility. This versatility arises from its almost unique ability to remodel under applied loads into anisotropic and inhomogeneous structures. To explore the origins of this property, we develop a set of analysis tools and a novel experimental setup that probes the mechanical response of fibrous networks in a geometry that mimics a typical deformation profile imposed by cells in vivo. We observe strong fiber alignment and densification as a function of applied strain for both uncrosslinked and crosslinked collagenous networks. This alignment is found to be irreversibly imprinted in uncrosslinked collagen networks, suggesting a simple mechanism for tissue organization at the microscale. However, crosslinked networks display similar fiber alignment and the same geometrical properties as uncrosslinked gels, but with full reversibility. Plasticity is therefore not required to align fibers. On the contrary, our data show that this effect is part of the fundamental non-linear properties of fibrous biological networks. PMID:19529768

  1. Nanoscale Swelling Heterogeneities in Type I Collagen Fibrils.

    PubMed

    Spitzner, Eike-Christian; Röper, Stephanie; Zerson, Mario; Bernstein, Anke; Magerle, Robert

    2015-06-23

    The distribution of water within the supramolecular structure of collagen fibrils is important for understanding their mechanical properties as well as the biomineralization processes in collagen-based tissues. We study the influence of water on the shape and the mechanical properties of reconstituted fibrils of type I collagen on the nanometer scale. Fibrils adsorbed on a silicon substrate were imaged with multiset point intermittent contact (MUSIC)-mode atomic force microscopy (AFM) in air at 28% relative humidity (RH) and in a hydrated state at 78% RH. Our data reveal the differences in the water uptake between the gap and overlap regions during swelling. This provides direct evidence for different amounts of bound and free water within the gap and overlap regions. In the dry state, the characteristic D-band pattern visible in AFM images is due to height corrugations along a fibril's axis. In the hydrated state, the fibril's surface is smooth and the D-band pattern reflects the different mechanical properties of the gap and overlap regions. PMID:25961780

  2. In vitro fracture testing of submicron diameter collagen fibril specimens.

    PubMed

    Shen, Zhilei Liu; Dodge, Mohammad Reza; Kahn, Harold; Ballarini, Roberto; Eppell, Steven J

    2010-09-22

    Mechanical testing of collagenous tissues at different length scales will provide improved understanding of the mechanical behavior of structures such as skin, tendon, and bone, and also guide the development of multiscale mechanical models. Using a microelectromechanical-systems (MEMS) platform, stress-strain response curves up to failure of type I collagen fibril specimens isolated from the dermis of sea cucumbers were obtained in vitro. A majority of the fibril specimens showed brittle fracture. Some displayed linear behavior up to failure, while others displayed some nonlinearity. The fibril specimens showed an elastic modulus of 470 ± 410 MPa, a fracture strength of 230 ± 160 MPa, and a fracture strain of 80% ± 44%. The fibril specimens displayed significantly lower elastic modulus in vitro than previously measured in air. Fracture strength/strain obtained in vitro and in air are both significantly larger than those obtained in vacuo, indicating that the difference arises from the lack of intrafibrillar water molecules produced by vacuum drying. Furthermore, fracture strength/strain of fibril specimens were different from those reported for collagenous tissues of higher hierarchical levels, indicating the importance of obtaining these properties at the fibrillar level for multiscale modeling.

  3. Collagen fibril aggregation-inhibitor from sea cucumber dermis.

    PubMed

    Trotter, J A; Lyons-Levy, G; Chino, K; Koob, T J; Keene, D R; Atkinson, M A

    1999-12-01

    Collagen fibrils from the dermis of the sea cucumber Cucumaria frondosa are aggregated in vitro by the dermal glycoprotein stiparin (Trotter et al., 1996). Under physiological ionic conditions stiparin appears to be both necessary and sufficient to cause fibrils to aggregate (Trotter et al., 1997). We report here the initial biochemical and biophysical characterization of a sulfated glycoprotein from C. frondosa dermis that binds stiparin and inhibits its fibril-aggregating activity. This inhibitory glycoprotein, which has been named 'stiparin-inhibitor,' has the highest negative charge density of all the macromolecules extracted from the dermis. SDS-PAGE reveals three approximately 31-kDa bands that stain with alcian blue but not with Coomassie blue. Analytical ultracentrifugation indicates a native molecular weight of 62 kDa. Transmission electron microscopy of rotary-shadowed molecules shows curved rods about 22 nm long. The glycoprotein does not bind collagen fibrils, but does bind stiparin with a 1:1 stoichiometry. The binding of stiparin-inhibitor to stiparin prevents the binding of stiparin to collagen fibrils. The carbohydrate moiety produced by papain-digestion of the glycoprotein retains all of its inhibitory activity. The carbohydrate moiety of the inhibitor is dominated by galactose and sulfate.

  4. In Vitro Fracture Testing of Submicron Diameter Collagen Fibril Specimens

    PubMed Central

    Shen, Zhilei Liu; Dodge, Mohammad Reza; Kahn, Harold; Ballarini, Roberto; Eppell, Steven J.

    2010-01-01

    Mechanical testing of collagenous tissues at different length scales will provide improved understanding of the mechanical behavior of structures such as skin, tendon, and bone, and also guide the development of multiscale mechanical models. Using a microelectromechanical-systems (MEMS) platform, stress-strain response curves up to failure of type I collagen fibril specimens isolated from the dermis of sea cucumbers were obtained in vitro. A majority of the fibril specimens showed brittle fracture. Some displayed linear behavior up to failure, while others displayed some nonlinearity. The fibril specimens showed an elastic modulus of 470 ± 410 MPa, a fracture strength of 230 ± 160 MPa, and a fracture strain of 80% ± 44%. The fibril specimens displayed significantly lower elastic modulus in vitro than previously measured in air. Fracture strength/strain obtained in vitro and in air are both significantly larger than those obtained in vacuo, indicating that the difference arises from the lack of intrafibrillar water molecules produced by vacuum drying. Furthermore, fracture strength/strain of fibril specimens were different from those reported for collagenous tissues of higher hierarchical levels, indicating the importance of obtaining these properties at the fibrillar level for multiscale modeling. PMID:20858445

  5. Collagen fibril surface displays a constellation of sites capable of promoting fibril assembly, stability, and hemostasis

    SciTech Connect

    Orgel, J.P.; Antipova, O.; Sagi, I.; Bitler, A.; Qiu, D.; Wang, R.; Xu, Y.; San Antonio, J.D.

    2011-12-14

    Fibrillar collagens form the structural basis of organs and tissues including the vasculature, bone, and tendon. They are also dynamic, organizational scaffolds that present binding and recognition sites for ligands, cells, and platelets. We interpret recently published X-ray diffraction findings and use atomic force microscopy data to illustrate the significance of new insights into the functional organization of the collagen fibril. These data indicate that collagen's most crucial functional domains localize primarily to the overlap region, comprising a constellation of sites we call the 'master control region.' Moreover, the collagen's most exposed aspect contains its most stable part - the C-terminal region that controls collagen assembly, cross-linking, and blood clotting. Hidden beneath the fibril surface exists a constellation of 'cryptic' sequences poised to promote hemostasis and cell - collagen interactions in tissue injury and regeneration. These findings begin to address several important, and previously unresolved, questions: How functional domains are organized in the fibril, which domains are accessible, and which require proteolysis or structural trauma to become exposed? Here we speculate as to how collagen fibrillar organization impacts molecular processes relating to tissue growth, development, and repair.

  6. Collagen fibril surface displays a constellation of sites capable of promoting fibril assembly, stability, and hemostasis.

    PubMed

    Orgel, J P R O; Antipova, O; Sagi, I; Bitler, A; Qiu, D; Wang, R; Xu, Y; San Antonio, J D

    2011-02-01

    Fibrillar collagens form the structural basis of organs and tissues including the vasculature, bone, and tendon. They are also dynamic, organizational scaffolds that present binding and recognition sites for ligands, cells, and platelets. We interpret recently published X-ray diffraction findings and use atomic force microscopy data to illustrate the significance of new insights into the functional organization of the collagen fibril. These data indicate that collagen's most crucial functional domains localize primarily to the overlap region, comprising a constellation of sites we call the "master control region." Moreover, the collagen's most exposed aspect contains its most stable part-the C-terminal region that controls collagen assembly, cross-linking, and blood clotting. Hidden beneath the fibril surface exists a constellation of "cryptic" sequences poised to promote hemostasis and cell-collagen interactions in tissue injury and regeneration. These findings begin to address several important, and previously unresolved, questions: How functional domains are organized in the fibril, which domains are accessible, and which require proteolysis or structural trauma to become exposed? Here we speculate as to how collagen fibrillar organization impacts molecular processes relating to tissue growth, development, and repair.

  7. Supramolecular Organization of Collagen Fibrils in Healthy and Osteoarthritic Human Knee and Hip Joint Cartilage

    PubMed Central

    Raiteri, Roberto; Loparic, Marko; Düggelin, Marcel; Mathys, Daniel; Friederich, Niklaus F.; Bruckner, Peter

    2016-01-01

    Cartilage matrix is a composite of discrete, but interacting suprastructures, i.e. cartilage fibers with microfibrillar or network-like aggregates and penetrating extrafibrillar proteoglycan matrix. The biomechanical function of the proteoglycan matrix and the collagen fibers are to absorb compressive and tensional loads, respectively. Here, we are focusing on the suprastructural organization of collagen fibrils and the degradation process of their hierarchical organized fiber architecture studied at high resolution at the authentic location within cartilage. We present electron micrographs of the collagenous cores of such fibers obtained by an improved protocol for scanning electron microscopy (SEM). Articular cartilages are permeated by small prototypic fibrils with a homogeneous diameter of 18 ± 5 nm that can align in their D-periodic pattern and merge into larger fibers by lateral association. Interestingly, these fibers have tissue-specific organizations in cartilage. They are twisted ropes in superficial regions of knee joints or assemble into parallel aligned cable-like structures in deeper regions of knee joint- or throughout hip joints articular cartilage. These novel observations contribute to an improved understanding of collagen fiber biogenesis, function, and homeostasis in hyaline cartilage. PMID:27780246

  8. Interfibrillar shear stress is the loading mechanism of collagen fibrils in tendon.

    PubMed

    Szczesny, Spencer E; Elliott, Dawn M

    2014-06-01

    Despite the critical role tendons play in transmitting loads throughout the musculoskeletal system, little is known about the microstructural mechanisms underlying their mechanical function. Of particular interest is whether collagen fibrils in tendon fascicles bear load independently or if load is transferred between fibrils through interfibrillar shear forces. We conducted multiscale experimental testing and developed a microstructural shear lag model to explicitly test whether interfibrillar shear load transfer is indeed the fibrillar loading mechanism in tendon. Experimental correlations between fascicle macroscale mechanics and microscale interfibrillar sliding suggest that fibrils are discontinuous and share load. Moreover, for the first time, we demonstrate that a shear lag model can replicate the fascicle macroscale mechanics as well as predict the microscale fibrillar deformations. Since interfibrillar shear stress is the fundamental loading mechanism assumed in the model, this result provides strong evidence that load is transferred between fibrils in tendon and possibly other aligned collagenous tissues. Conclusively establishing this fibrillar loading mechanism and identifying the involved structural components should help develop repair strategies for tissue degeneration and guide the design of tissue engineered replacements. PMID:24530560

  9. Agent-based modeling traction force mediated compaction of cell-populated collagen gels using physically realistic fibril mechanics.

    PubMed

    Reinhardt, James W; Gooch, Keith J

    2014-02-01

    Agent-based modeling was used to model collagen fibrils, composed of a string of nodes serially connected by links that act as Hookean springs. Bending mechanics are implemented as torsional springs that act upon each set of three serially connected nodes as a linear function of angular deflection about the central node. These fibrils were evaluated under conditions that simulated axial extension, simple three-point bending and an end-loaded cantilever. The deformation of fibrils under axial loading varied <0.001% from the analytical solution for linearly elastic fibrils. For fibrils between 100 μm and 200 μm in length experiencing small deflections, differences between simulated deflections and their analytical solutions were <1% for fibrils experiencing three-point bending and <7% for fibrils experiencing cantilever bending. When these new rules for fibril mechanics were introduced into a model that allowed for cross-linking of fibrils to form a network and the application of cell traction force, the fibrous network underwent macroscopic compaction and aligned between cells. Further, fibril density increased between cells to a greater extent than that observed macroscopically and appeared similar to matrical tracks that have been observed experimentally in cell-populated collagen gels. This behavior is consistent with observations in previous versions of the model that did not allow for the physically realistic simulation of fibril mechanics. The significance of the torsional spring constant value was then explored to determine its impact on remodeling of the simulated fibrous network. Although a stronger torsional spring constant reduced the degree of quantitative remodeling that occurred, the inclusion of torsional springs in the model was not necessary for the model to reproduce key qualitative aspects of remodeling, indicating that the presence of Hookean springs is essential for this behavior. These results suggest that traction force mediated matrix

  10. Effects of isopropanol on collagen fibrils in new parchment

    PubMed Central

    2012-01-01

    Background Isopropanol is widely used by conservators to relax the creases and folds of parchment artefacts. At present, little is known of the possible side effects of the chemical on parchments main structural component- collagen. This study uses X-ray Diffraction to investigate the effects of a range of isopropanol concentrations on the dimensions of the nanostructure of the collagen component of new parchment. Results It is found in this study that the packing features of the collagen molecules within the collagen fibril are altered by exposure to isopropanol. The results suggest that this chemical treatment can induce a loss of structural water from the collagen within parchment and thus a rearrangement of intermolecular bonding. This study also finds that the effects of isopropanol treatment are permanent to parchment artefacts and cannot be reversed with rehydration using deionised water. Conclusions This study has shown that isopropanol induces permanent changes to the packing features of collagen within parchment artefacts and has provided scientific evidence that its use to remove creases and folds on parchment artefacts will cause structural change that may contribute to long-term deterioration of parchment artefacts. This work provides valuable information that informs conservation practitioners regarding the use of isopropanol on parchment artefacts. PMID:22462769

  11. Large Deformation Mechanisms, Plasticity, and Failure of an Individual Collagen Fibril With Different Mineral Content.

    PubMed

    Depalle, Baptiste; Qin, Zhao; Shefelbine, Sandra J; Buehler, Markus J

    2016-02-01

    Mineralized collagen fibrils are composed of tropocollagen molecules and mineral crystals derived from hydroxyapatite to form a composite material that combines optimal properties of both constituents and exhibits incredible strength and toughness. Their complex hierarchical structure allows collagen fibrils to sustain large deformation without breaking. In this study, we report a mesoscale model of a single mineralized collagen fibril using a bottom-up approach. By conserving the three-dimensional structure and the entanglement of the molecules, we were able to construct finite-size fibril models that allowed us to explore the deformation mechanisms which govern their mechanical behavior under large deformation. We investigated the tensile behavior of a single collagen fibril with various intrafibrillar mineral content and found that a mineralized collagen fibril can present up to five different deformation mechanisms to dissipate energy. These mechanisms include molecular uncoiling, molecular stretching, mineral/collagen sliding, molecular slippage, and crystal dissociation. By multiplying its sources of energy dissipation and deformation mechanisms, a collagen fibril can reach impressive strength and toughness. Adding mineral into the collagen fibril can increase its strength up to 10 times and its toughness up to 35 times. Combining crosslinks with mineral makes the fibril stiffer but more brittle. We also found that a mineralized fibril reaches its maximum toughness to density and strength to density ratios for a mineral density of around 30%. This result, in good agreement with experimental observations, attests that bone tissue is optimized mechanically to remain lightweight but maintain strength and toughness. PMID:26866939

  12. Large Deformation Mechanisms, Plasticity, and Failure of an Individual Collagen Fibril With Different Mineral Content.

    PubMed

    Depalle, Baptiste; Qin, Zhao; Shefelbine, Sandra J; Buehler, Markus J

    2016-02-01

    Mineralized collagen fibrils are composed of tropocollagen molecules and mineral crystals derived from hydroxyapatite to form a composite material that combines optimal properties of both constituents and exhibits incredible strength and toughness. Their complex hierarchical structure allows collagen fibrils to sustain large deformation without breaking. In this study, we report a mesoscale model of a single mineralized collagen fibril using a bottom-up approach. By conserving the three-dimensional structure and the entanglement of the molecules, we were able to construct finite-size fibril models that allowed us to explore the deformation mechanisms which govern their mechanical behavior under large deformation. We investigated the tensile behavior of a single collagen fibril with various intrafibrillar mineral content and found that a mineralized collagen fibril can present up to five different deformation mechanisms to dissipate energy. These mechanisms include molecular uncoiling, molecular stretching, mineral/collagen sliding, molecular slippage, and crystal dissociation. By multiplying its sources of energy dissipation and deformation mechanisms, a collagen fibril can reach impressive strength and toughness. Adding mineral into the collagen fibril can increase its strength up to 10 times and its toughness up to 35 times. Combining crosslinks with mineral makes the fibril stiffer but more brittle. We also found that a mineralized fibril reaches its maximum toughness to density and strength to density ratios for a mineral density of around 30%. This result, in good agreement with experimental observations, attests that bone tissue is optimized mechanically to remain lightweight but maintain strength and toughness.

  13. Collagen assembly from acid solution to networks on solid surfaces and to fibrils

    NASA Astrophysics Data System (ADS)

    Bradt, Jens-Hilmar; Mertig, Michael; Winzer, Bettina; Thiele, Uwe; Pompe, Wolfgang

    1996-04-01

    Two different kinds of collagen assembly have been studied: the reconstitution of type I collagen to fibrils and the formation of 2D networks on surfaces. The kinetics of fibril assembly are influenced by polyaspartate, as measured turbidimetrically. Addition of polyaspartate increases the fibril diameter. The reconstituted fibrils are imaged by atomic force microscopy and scanning electron microscopy. The preparation of thin collagen films on highly oriented pyrolytic graphite leads to networks or tree like structures depending on the collagen concentration in the precursor. The results presented are of interest for the development of new bone-like implant materials and the covering of bone grafts with a biocompatible layer.

  14. Designed to Fail: A Novel Mode of Collagen Fibril Disruption and Its Relevance to Tissue Toughness

    PubMed Central

    Veres, Samuel P.; Lee, J. Michael

    2012-01-01

    Collagen fibrils are nanostructured biological cables essential to the structural integrity of many of our tissues. Consequently, understanding the structural basis of their robust mechanical properties is of great interest. Here we present what to our knowledge is a novel mode of collagen fibril disruption that provides new insights into both the structure and mechanics of native collagen fibrils. Using enzyme probes for denatured collagen and scanning electron microscopy, we show that mechanically overloading collagen fibrils from bovine tail tendons causes them to undergo a sequential, two-stage, selective molecular failure process. Denatured collagen molecules—meaning molecules with a reduced degree of time-averaged helicity compared to those packed in undamaged fibrils—were first created within kinks that developed at discrete, repeating locations along the length of fibrils. There, collagen denaturation within the kinks was concentrated within certain subfibrils. Additional denatured molecules were then created along the surface of some disrupted fibrils. The heterogeneity of the disruption within fibrils suggests that either mechanical load is not carried equally by a fibril's subcomponents or that the subcomponents do not possess homogenous mechanical properties. Meanwhile, the creation of denatured collagen molecules, which necessarily involves the energy intensive breaking of intramolecular hydrogen bonds, provides a physical basis for the toughness of collagen fibrils. PMID:22735538

  15. Candidate Cell and Matrix Interaction Domains on the Collagen Fibril, the Predominant Protein of Vertebrates

    SciTech Connect

    Sweeney, Shawn M.; Orgel, Joseph P.; Fertala, Andrzej; McAuliffe, Jon D.; Turner, Kevin R.; Di Lullo, Gloria A.; Chen, Steven; Antipova, Olga; Perumal, Shiamalee; Ala-Kokko, Leena; Forlinoi, Antonella; Cabral, Wayne A.; Barnes, Aileen M.; Marini, Joan C.; San Antonio, James D.

    2008-07-18

    Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The 'cell interaction domain' is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The 'matrix interaction domain' may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.

  16. Structure-mechanics relationships of collagen fibrils in the osteogenesis imperfecta mouse model.

    PubMed

    Andriotis, O G; Chang, S W; Vanleene, M; Howarth, P H; Davies, D E; Shefelbine, S J; Buehler, M J; Thurner, P J

    2015-10-01

    The collagen molecule, which is the building block of collagen fibrils, is a triple helix of two α1(I) chains and one α2(I) chain. However, in the severe mouse model of osteogenesis imperfecta (OIM), deletion of the COL1A2 gene results in the substitution of the α2(I) chain by one α1(I) chain. As this substitution severely impairs the structure and mechanics of collagen-rich tissues at the tissue and organ level, the main aim of this study was to investigate how the structure and mechanics are altered in OIM collagen fibrils. Comparing results from atomic force microscopy imaging and cantilever-based nanoindentation on collagen fibrils from OIM and wild-type (WT) animals, we found a 33% lower indentation modulus in OIM when air-dried (bound water present) and an almost fivefold higher indentation modulus in OIM collagen fibrils when fully hydrated (bound and unbound water present) in phosphate-buffered saline solution (PBS) compared with WT collagen fibrils. These mechanical changes were accompanied by an impaired swelling upon hydration within PBS. Our experimental and atomistic simulation results show how the structure and mechanics are altered at the individual collagen fibril level as a result of collagen gene mutation in OIM. We envisage that the combination of experimental and modelling approaches could allow mechanical phenotyping at the collagen fibril level of virtually any alteration of collagen structure or chemistry.

  17. Structure-mechanics relationships of collagen fibrils in the osteogenesis imperfecta mouse model.

    PubMed

    Andriotis, O G; Chang, S W; Vanleene, M; Howarth, P H; Davies, D E; Shefelbine, S J; Buehler, M J; Thurner, P J

    2015-10-01

    The collagen molecule, which is the building block of collagen fibrils, is a triple helix of two α1(I) chains and one α2(I) chain. However, in the severe mouse model of osteogenesis imperfecta (OIM), deletion of the COL1A2 gene results in the substitution of the α2(I) chain by one α1(I) chain. As this substitution severely impairs the structure and mechanics of collagen-rich tissues at the tissue and organ level, the main aim of this study was to investigate how the structure and mechanics are altered in OIM collagen fibrils. Comparing results from atomic force microscopy imaging and cantilever-based nanoindentation on collagen fibrils from OIM and wild-type (WT) animals, we found a 33% lower indentation modulus in OIM when air-dried (bound water present) and an almost fivefold higher indentation modulus in OIM collagen fibrils when fully hydrated (bound and unbound water present) in phosphate-buffered saline solution (PBS) compared with WT collagen fibrils. These mechanical changes were accompanied by an impaired swelling upon hydration within PBS. Our experimental and atomistic simulation results show how the structure and mechanics are altered at the individual collagen fibril level as a result of collagen gene mutation in OIM. We envisage that the combination of experimental and modelling approaches could allow mechanical phenotyping at the collagen fibril level of virtually any alteration of collagen structure or chemistry. PMID:26468064

  18. Structure–mechanics relationships of collagen fibrils in the osteogenesis imperfecta mouse model

    PubMed Central

    Andriotis, O. G.; Chang, S. W.; Vanleene, M.; Howarth, P. H.; Davies, D. E.; Shefelbine, S. J.; Buehler, M. J.; Thurner, P. J.

    2015-01-01

    The collagen molecule, which is the building block of collagen fibrils, is a triple helix of two α1(I) chains and one α2(I) chain. However, in the severe mouse model of osteogenesis imperfecta (OIM), deletion of the COL1A2 gene results in the substitution of the α2(I) chain by one α1(I) chain. As this substitution severely impairs the structure and mechanics of collagen-rich tissues at the tissue and organ level, the main aim of this study was to investigate how the structure and mechanics are altered in OIM collagen fibrils. Comparing results from atomic force microscopy imaging and cantilever-based nanoindentation on collagen fibrils from OIM and wild-type (WT) animals, we found a 33% lower indentation modulus in OIM when air-dried (bound water present) and an almost fivefold higher indentation modulus in OIM collagen fibrils when fully hydrated (bound and unbound water present) in phosphate-buffered saline solution (PBS) compared with WT collagen fibrils. These mechanical changes were accompanied by an impaired swelling upon hydration within PBS. Our experimental and atomistic simulation results show how the structure and mechanics are altered at the individual collagen fibril level as a result of collagen gene mutation in OIM. We envisage that the combination of experimental and modelling approaches could allow mechanical phenotyping at the collagen fibril level of virtually any alteration of collagen structure or chemistry. PMID:26468064

  19. Determination of collagen fibril structure and orientation in connective tissues by X-ray diffraction

    NASA Astrophysics Data System (ADS)

    Wilkinson, S. J.; Hukins, D. W. L.

    1999-08-01

    Elastic scattering of X-rays can provide the following information on the fibrous protein collagen: its molecular structure, the axial arrangement of rod-like collagen molecules in a fibril, the lateral arrangement of molecules within a fibril, and the orientation of fibrils within a biological tissue. The first part of the paper reviews the principles involved in deducing this information. The second part describes a new computer program for measuring the equatorial intensity distribution, that provides information on the lateral arrangement of molecules within a fibril, and the angular distribution of the equatorial peaks that provides information on the orientation of fibrils. Orientation of fibrils within a tissue is quantified by the orientation distribution function, g( φ), which represents the probability of finding a fibril oriented between φ and φ+ δφ. The application of the program is illustrated by measurement of g( φ) for the collagen fibrils in demineralised cortical bone from cow tibia.

  20. Tendon glycosaminoglycan proteoglycan sidechains promote collagen fibril sliding-AFM observations at the nanoscale.

    PubMed

    Rigozzi, S; Müller, R; Stemmer, A; Snedeker, J G

    2013-02-22

    The extracellular matrix of tendon is mainly composed of discontinuous Type-I collagen fibrils and small leucine rich proteoglycans (PG). Macroscopic tendon behaviors like stiffness and strength are determined by the ultrastructural arrangement of these components. When a tendon is submitted to load, the collagen fibrils both elongate and slide relative to their neighboring fibrils. The role of PG glycosaminoglycan (GAG) sidechains in mediating inter-fibril load sharing remains controversial, with competing structure-function theories suggesting that PGs may mechanically couple neighboring collagen fibrils (cross-linking them to facilitate fibril stretch) or alternatively isolating them (promoting fibril gliding). In this study, we sought to clarify the functional role of GAGs in tensile tendon mechanics by directly investigating the mechanical response of individual collagen fibrils within their collagen network in both native and GAG depleted tendons. A control group of Achilles tendons from adult mice was compared with tendons in which GAGs were enzymatically depleted using chondroitinase ABC. Tendons were loaded to specific target strains, chemically fixed under constant load, and later sectioned for morphological analysis by an atomic force microscope (AFM). Increases in periodic banding of the collagen fibrils (D-period) or decreases in fibril diameter was considered to be representative of collagen fibril elongation and the mechanical contribution of GAGs at the ultrascale was quantified on this basis. At high levels of applied tendon strain (10%), GAG depleted tendons showed increased collagen stretch (less fibril sliding). We conclude that the hydrophilic GAGs seem thus not to act as mechanical crosslinks but rather act to promote collagen fibril sliding under tension.

  1. He I Vector Magnetometry of Field-aligned Superpenumbral Fibrils

    NASA Astrophysics Data System (ADS)

    Schad, T. A.; Penn, M. J.; Lin, H.

    2013-05-01

    Atomic-level polarization and Zeeman effect diagnostics in the neutral helium triplet at 10830 Å in principle allow full vector magnetometry of fine-scaled chromospheric fibrils. We present high-resolution spectropolarimetric observations of superpenumbral fibrils in the He I triplet with sufficient polarimetric sensitivity to infer their full magnetic field geometry. He I observations from the Facility Infrared Spectropolarimeter are paired with high-resolution observations of the Hα 6563 Å and Ca II 8542 Å spectral lines from the Interferometric Bidimensional Spectrometer from the Dunn Solar Telescope in New Mexico. Linear and circular polarization signatures in the He I triplet are measured and described, as well as analyzed with the advanced inversion capability of the "Hanle and Zeeman Light" modeling code. Our analysis provides direct evidence for the often assumed field alignment of fibril structures. The projected angle of the fibrils and the inferred magnetic field geometry align within an error of ±10°. We describe changes in the inclination angle of these features that reflect their connectivity with the photospheric magnetic field. Evidence for an accelerated flow (~40 m s-2) along an individual fibril anchored at its endpoints in the strong sunspot and weaker plage in part supports the magnetic siphon flow mechanism's role in the inverse Evershed effect. However, the connectivity of the outer endpoint of many of the fibrils cannot be established.

  2. He I VECTOR MAGNETOMETRY OF FIELD-ALIGNED SUPERPENUMBRAL FIBRILS

    SciTech Connect

    Schad, T. A.; Penn, M. J.; Lin, H.

    2013-05-10

    Atomic-level polarization and Zeeman effect diagnostics in the neutral helium triplet at 10830 A in principle allow full vector magnetometry of fine-scaled chromospheric fibrils. We present high-resolution spectropolarimetric observations of superpenumbral fibrils in the He I triplet with sufficient polarimetric sensitivity to infer their full magnetic field geometry. He I observations from the Facility Infrared Spectropolarimeter are paired with high-resolution observations of the H{alpha} 6563 A and Ca II 8542 A spectral lines from the Interferometric Bidimensional Spectrometer from the Dunn Solar Telescope in New Mexico. Linear and circular polarization signatures in the He I triplet are measured and described, as well as analyzed with the advanced inversion capability of the ''Hanle and Zeeman Light'' modeling code. Our analysis provides direct evidence for the often assumed field alignment of fibril structures. The projected angle of the fibrils and the inferred magnetic field geometry align within an error of {+-}10 Degree-Sign . We describe changes in the inclination angle of these features that reflect their connectivity with the photospheric magnetic field. Evidence for an accelerated flow ({approx}40 m s{sup -2}) along an individual fibril anchored at its endpoints in the strong sunspot and weaker plage in part supports the magnetic siphon flow mechanism's role in the inverse Evershed effect. However, the connectivity of the outer endpoint of many of the fibrils cannot be established.

  3. Diabetes alters mechanical properties and collagen fiber re-alignment in multiple mouse tendons.

    PubMed

    Connizzo, Brianne K; Bhatt, Pankti R; Liechty, Kenneth W; Soslowsky, Louis J

    2014-09-01

    Tendons function to transfer load from muscle to bone through their complex composition and hierarchical structure, consisting mainly of type I collagen. Recent evidence suggests that type II diabetes may cause alterations in collagen structure, such as irregular fibril morphology and density, which could play a role in the mechanical function of tendons. Using the db/db mouse model of type II diabetes, the diabetic skin was found to have impaired biomechanical properties when compared to the non-diabetic group. The purpose of this study was to assess the effect of diabetes on biomechanics, collagen fiber re-alignment, and biochemistry in three functionally different tendons (Achilles, supraspinatus, patellar) using the db/db mouse model. Results showed that cross-sectional area and stiffness, but not modulus, were significantly reduced in all three tendons. However, the tendon response to load (transition strain, collagen fiber re-alignment) occurred earlier in the mechanical test, contrary to expectations. In addition, the patellar tendon had an altered response to diabetes when compared to the other two tendons, with no changes in fiber re-alignment and decreased collagen content at the midsubstance of the tendon. Overall, type II diabetes alters tendon mechanical properties and the dynamic response to load.

  4. Discerning the Subfibrillar Structure of Mineralized Collagen Fibrils: A Model for the Ultrastructure of Bone

    PubMed Central

    Li, Yuping; Aparicio, Conrado

    2013-01-01

    Biomineralization templated by organic molecules to produce inorganic-organic nanocomposites is a fascinating example of nature using bottom-up strategies at nanoscale to accomplish highly ordered multifunctional materials. One such nanocomposite is bone, composed primarily of hydroxyapatite (HA) nanocrystals that are embedded within collagen fibrils with their c-axes arranged roughly parallel to the long axis of the fibrils. Here we discern the ultra-structure of biomimetic mineralized collagen fibrils (MCFs) as consisting of bundles of subfibrils with approximately 10 nm diameter; each one with an organic-inorganic core-shell structure. Through an amorphous calcium phosphate precursor phase the HA nanocrystals were specifically grown along the longitudinal direction of the collagen microfibrils and encapsulated them within the crystal lattice. They intercalated throughout the collagen fibrils such that the mineral phase surrounded the surface of collagen microfibrils forming an interdigitated network. It appears that this arrangement of collagen microfibrils in collagen fibrils is responsible for the observed ultrastructure. Such a subfibrillar nanostructure in MCFs was identified in both synthetic and natural bone, suggesting this is the basic building block of collagen-based hard tissues. Insights into the ultrastructure of mineralized collagen fibrils have the potential to advance our understanding on the biomineralization principles and the relationship between bone’s structure and mechanical properties, including fracture toughness mechanisms. We anticipate that these principles from biological systems can be applied to the rational design of new nanocomposites with improved performance. PMID:24086763

  5. Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis

    SciTech Connect

    Perumal, Shiamalee; Antipova, Olga; Orgel, Joseph P.R.O.

    2008-06-24

    We describe the molecular structure of the collagen fibril and how it affects collagen proteolysis or 'collagenolysis.' The fibril-forming collagens are major components of all mammalian connective tissues, providing the structural and organizational framework for skin, blood vessels, bone, tendon, and other tissues. The triple helix of the collagen molecule is resistant to most proteinases, and the matrix metalloproteinases that do proteolyze collagen are affected by the architecture of collagen fibrils, which are notably more resistant to collagenolysis than lone collagen monomers. Until now, there has been no molecular explanation for this. Full or limited proteolysis of the collagen fibril is known to be a key process in normal growth, development, repair, and cell differentiation, and in cancerous tumor progression and heart disease. Peptide fragments generated by collagenolysis, and the conformation of exposed sites on the fibril as a result of limited proteolysis, regulate these processes and that of cellular attachment, but it is not known how or why. Using computational and molecular visualization methods, we found that the arrangement of collagen monomers in the fibril (its architecture) protects areas vulnerable to collagenolysis and strictly governs the process. This in turn affects the accessibility of a cell interaction site located near the cleavage region. Our observations suggest that the C-terminal telopeptide must be proteolyzed before collagenase can gain access to the cleavage site. Collagenase then binds to the substrate's 'interaction domain,' which facilitates the triple-helix unwinding/dissociation function of the enzyme before collagenolysis.

  6. Discerning the subfibrillar structure of mineralized collagen fibrils: a model for the ultrastructure of bone.

    PubMed

    Li, Yuping; Aparicio, Conrado

    2013-01-01

    Biomineralization templated by organic molecules to produce inorganic-organic nanocomposites is a fascinating example of nature using bottom-up strategies at nanoscale to accomplish highly ordered multifunctional materials. One such nanocomposite is bone, composed primarily of hydroxyapatite (HA) nanocrystals that are embedded within collagen fibrils with their c-axes arranged roughly parallel to the long axis of the fibrils. Here we discern the ultra-structure of biomimetic mineralized collagen fibrils (MCFs) as consisting of bundles of subfibrils with approximately 10 nm diameter; each one with an organic-inorganic core-shell structure. Through an amorphous calcium phosphate precursor phase the HA nanocrystals were specifically grown along the longitudinal direction of the collagen microfibrils and encapsulated them within the crystal lattice. They intercalated throughout the collagen fibrils such that the mineral phase surrounded the surface of collagen microfibrils forming an interdigitated network. It appears that this arrangement of collagen microfibrils in collagen fibrils is responsible for the observed ultrastructure. Such a subfibrillar nanostructure in MCFs was identified in both synthetic and natural bone, suggesting this is the basic building block of collagen-based hard tissues. Insights into the ultrastructure of mineralized collagen fibrils have the potential to advance our understanding on the biomineralization principles and the relationship between bone's structure and mechanical properties, including fracture toughness mechanisms. We anticipate that these principles from biological systems can be applied to the rational design of new nanocomposites with improved performance.

  7. Collagen fibril architecture, domain organization, and triple-helical conformation govern its proteolysis.

    PubMed

    Perumal, Shiamalee; Antipova, Olga; Orgel, Joseph P R O

    2008-02-26

    We describe the molecular structure of the collagen fibril and how it affects collagen proteolysis or "collagenolysis." The fibril-forming collagens are major components of all mammalian connective tissues, providing the structural and organizational framework for skin, blood vessels, bone, tendon, and other tissues. The triple helix of the collagen molecule is resistant to most proteinases, and the matrix metalloproteinases that do proteolyze collagen are affected by the architecture of collagen fibrils, which are notably more resistant to collagenolysis than lone collagen monomers. Until now, there has been no molecular explanation for this. Full or limited proteolysis of the collagen fibril is known to be a key process in normal growth, development, repair, and cell differentiation, and in cancerous tumor progression and heart disease. Peptide fragments generated by collagenolysis, and the conformation of exposed sites on the fibril as a result of limited proteolysis, regulate these processes and that of cellular attachment, but it is not known how or why. Using computational and molecular visualization methods, we found that the arrangement of collagen monomers in the fibril (its architecture) protects areas vulnerable to collagenolysis and strictly governs the process. This in turn affects the accessibility of a cell interaction site located near the cleavage region. Our observations suggest that the C-terminal telopeptide must be proteolyzed before collagenase can gain access to the cleavage site. Collagenase then binds to the substrate's "interaction domain," which facilitates the triple-helix unwinding/dissociation function of the enzyme before collagenolysis.

  8. Growth of sea cucumber collagen fibrils occurs at the tips and centers in a coordinated manner.

    PubMed

    Trotter, J A; Chapman, J A; Kadler, K E; Holmes, D F

    1998-12-18

    Collagen fibrils are the principle source of mechanical strength in the mutable dermis of the sea cucumber Cucumaria frondosa. To obtain information about the mechanism by which collagen molecules self-assemble into fibrils, we have isolated single intact fibrils with lengths in the range 14-444 microm. These fibrils have been studied by scanning transmission electron microscopy, yielding data that show how cross-sectional mass, and hence the number of molecules in the cross-section, depend on axial location. In an individual fibril, the two ends always display similar mass distributions. The two tips of each fibril must therefore maintain identity in shape and size throughout growth. The linear relationship between cross-sectional mass and distance from the adjacent end shows that a growing tip is (like the tip of a vertebrate collagen fibril) paraboloidal in shape. Comparison of data from many different fibrils, over a wide range of lengths, however, revealed that the paraboloidal tip becomes blunter as the fibril grows in length. In contrast to vertebrate fibrils, those from C. frondosa do not have a central shaft region of constant cross-sectional mass. Rather, the cross-sectional mass increases to a maximum in the center of each fibril. The maximum cross-sectional mass of the fibrils increases exponentially with increasing fibril length. The centrosymmetry, the paraboloidal shape of the tips, and the hyperbolic increase in maximum cross-sectional mass with fibril length, is evidence for a co-ordinated regulation of length and diameter, which differs from the kind of regulation that gives rise to collagen fibrils in vertebrates (chickens and mice).

  9. Large Deformation Mechanisms, Plasticity, and Failure of an Individual Collagen Fibril With Different Mineral Content

    PubMed Central

    Depalle, Baptiste; Qin, Zhao; Shefelbine, Sandra J

    2016-01-01

    ABSTRACT Mineralized collagen fibrils are composed of tropocollagen molecules and mineral crystals derived from hydroxyapatite to form a composite material that combines optimal properties of both constituents and exhibits incredible strength and toughness. Their complex hierarchical structure allows collagen fibrils to sustain large deformation without breaking. In this study, we report a mesoscale model of a single mineralized collagen fibril using a bottom‐up approach. By conserving the three‐dimensional structure and the entanglement of the molecules, we were able to construct finite‐size fibril models that allowed us to explore the deformation mechanisms which govern their mechanical behavior under large deformation. We investigated the tensile behavior of a single collagen fibril with various intrafibrillar mineral content and found that a mineralized collagen fibril can present up to five different deformation mechanisms to dissipate energy. These mechanisms include molecular uncoiling, molecular stretching, mineral/collagen sliding, molecular slippage, and crystal dissociation. By multiplying its sources of energy dissipation and deformation mechanisms, a collagen fibril can reach impressive strength and toughness. Adding mineral into the collagen fibril can increase its strength up to 10 times and its toughness up to 35 times. Combining crosslinks with mineral makes the fibril stiffer but more brittle. We also found that a mineralized fibril reaches its maximum toughness to density and strength to density ratios for a mineral density of around 30%. This result, in good agreement with experimental observations, attests that bone tissue is optimized mechanically to remain lightweight but maintain strength and toughness. © 2015 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR). PMID:26866939

  10. Structural changes in collagen fibrils across a mineralized interface revealed by cryo-TEM.

    PubMed

    Quan, Bryan D; Sone, Eli D

    2015-08-01

    The structure of the mineralized collagen fibril, which is the basic building block of mineralized connective tissues, is critical to its function. We use cryo-TEM to study collagen structure at a well-defined hard-soft tissue interface, across which collagen fibrils are continuous, in order to evaluate changes to collagen upon mineralization. To establish a basis for the analysis of collagen banding, we compared cryo-TEM images of rat-tail tendon collagen to a model based on the X-ray structure. While there is close correspondence of periodicity, differences in band intensity indicate fibril regions with high density but lacking order, providing new insight into collagen fibrillar structure. Across a mineralized interface, we show that mineralization results in an axial contraction of the fibril, concomitant with lateral expansion, and that this contraction occurs only in the more flexible gap region of the fibril. Nevertheless, the major features of the banding pattern are not significantly changed, indicating that the axial arrangement of molecules remains largely intact. These results suggest a mechanism by which collagen fibrils are able to accommodate large amounts of mineral without significant disruption of their molecular packing, leading to synergy of mechanical properties.

  11. The 3D structure of the collagen fibril network in human trabecular bone: relation to trabecular organization.

    PubMed

    Reznikov, Natalie; Chase, Hila; Brumfeld, Vlad; Shahar, Ron; Weiner, Steve

    2015-02-01

    Trabecular bone is morphologically and functionally different from compact bone at the tissue level, but both are composed of lamellae at the micrometer-scale level. We present a three-dimensional study of the collagenous network of human trabecular lamellar bone from the proximal femur using the FIB-SEM serial surface view method. The results are compared to human compact lamellar bone of the femoral shaft, studied by the same method. Both demineralized trabecular and compact lamellar bone display the same overall structural organization, namely the presence of ordered and disordered materials and the confinement of the canalicular network to the disordered material. However, in trabecular bone lamellae a significant proportion of the ordered collagen fibril arrays is aligned with the long axis of the trabecula and, unlike in compact bone, is not related to the anatomical axis of the whole femur. The remaining ordered collagen fibrils are offset from the axis of a trabecula either by about 30° or 70°. Interestingly, at the tissue scale of millimeters, the most abundant angles between any two connected trabeculae - the inter-trabecular angles - center around 30° and 70°. This implies that within a framework of interconnected trabeculae the same lamellar structure will always have a significant component of the fibrils aligned with the long axes of connected trabeculae. This structural complementarity at different hierarchical levels presumably reflects an adaptation of trabecular bone to function.

  12. Nanostructure of collagen fibrils in human nucleus pulposus and its correlation with macroscale tissue mechanics.

    PubMed

    Aladin, Darwesh M K; Cheung, Kenneth M C; Ngan, Alfonso H W; Chan, Danny; Leung, Victor Y L; Lim, Chwee Teck; Luk, Keith D K; Lu, William W

    2010-04-01

    Collagen fibrils are the main structural components of the nucleus pulposus tissue in the intervertebral discs. The structure-property relationship of the nucleus pulposus (NP) tissues is still unclear. We investigated the structure of individual collagen fibrils of the NP and evaluated its correlation with the bulk mechanical properties of the tissue. Collagen fibrils were extracted from the NP of discs retrieved from adolescents during scoliosis correction surgery, and the extracts were confirmed by SDS-PAGE. The diameters of the individual collagen fibrils were measured through atomic force microscopy, and the compressive mechanical properties of the tissues were evaluated by confined compression. The correlations between the nanoscale morphology of the collagen fibrils and the macroscale mechanical properties of the tissues were evaluated by linear regression. The SDS-PAGE results showed that the fibril extracts were largely composed of type II collagen. The mean diameter of the collagen fibrils was 92.1 +/- 26.54 nm; the mean swelling pressure and compressive modulus of the tissues were 6.15 +/- 4.3 kPa and 1.23 +/- 0.7 MPa, respectively. The mean fibril diameter had no linear correlation (R(2) = 0.30) with the swelling pressure of the tissues. However, it had a mild linear correlation with the compressive modulus (p = 0.023, R(2) = 0.68). This is the first study, to our knowledge, to evaluate the nanostructure of the individual collagen fibrils of the nucleus pulposus and its relationship with macroscale mechanical properties of the NP tissues.

  13. Determination of the elastic modulus of native collagen fibrils via radial indentation

    NASA Astrophysics Data System (ADS)

    Heim, August J.; Matthews, William G.; Koob, Thomas J.

    2006-10-01

    The authors studied the elastic response of single, native collagen fibrils extracted from tissues of the inner dermis of the sea cucumber, Cucumaria frondosa, via local nanoscale indentation with an atomic force microscope (AFM). AFM imaging of fibrils under ambient conditions are presented, demonstrating a peak-to-peak periodicity, the d band, of dehydrated, unfixed fibrils to be ˜64.5nm. Radial indentation experiments were performed, and the measured value for the reduced modulus is 1-2GPa.

  14. Influence of fibril taper on the function of collagen to reinforce extracellular matrix.

    PubMed

    Goh, K L; Meakin, J R; Aspden, R M; Hukins, D W L

    2005-09-22

    Collagen fibrils provide tensile reinforcement for extracellular matrix. In at least some tissues, the fibrils have a paraboloidal taper at their ends. The purpose of this paper is to determine the implications of this taper for the function of collagen fibrils. When a tissue is subjected to low mechanical forces, stress will be transferred to the fibrils elastically. This process was modelled using finite element analysis because there is no analytical theory for elastic stress transfer to a non-cylindrical fibril. When the tissue is subjected to higher mechanical forces, stress will be transferred plastically. This process was modelled analytically. For both elastic and plastic stress transfer, a paraboloidal taper leads to a more uniform distribution of axial tensile stress along the fibril than would be generated if it were cylindrical. The tapered fibril requires half the volume of collagen than a cylindrical fibril of the same length and the stress is shared more evenly along its length. It is also less likely to fracture than a cylindrical fibril of the same length in a tissue subjected to the same mechanical force.

  15. Nonmuscle myosin II powered transport of newly formed collagen fibrils at the plasma membrane

    PubMed Central

    Kalson, Nicholas S.; Starborg, Tobias; Lu, Yinhui; Mironov, Aleksandr; Humphries, Sally M.; Holmes, David F.; Kadler, Karl E.

    2013-01-01

    Collagen fibrils can exceed thousands of microns in length and are therefore the longest, largest, and most size-pleomorphic protein polymers in vertebrates; thus, knowing how cells transport collagen fibrils is essential for a more complete understanding of protein transport and its role in tissue morphogenesis. Here, we identified newly formed collagen fibrils being transported at the surface of embryonic tendon cells in vivo by using serial block face-scanning electron microscopy of the cell-matrix interface. Newly formed fibrils ranged in length from ∼1 to ∼30 µm. The shortest (1–10 µm) occurred in intracellular fibricarriers; the longest (∼30 µm) occurred in plasma membrane fibripositors. Fibrils and fibripositors were reduced in numbers when collagen secretion was blocked. ImmunoEM showed the absence of lysosomal-associated membrane protein 2 on fibricarriers and fibripositors and there was no effect of leupeptin on fibricarrier or fibripositor number and size, suggesting that fibricarriers and fibripositors are not part of a fibril degradation pathway. Blebbistatin decreased fibricarrier number and increased fibripositor length; thus, nonmuscle myosin II (NMII) powers the transport of these compartments. Inhibition of dynamin-dependent endocytosis with dynasore blocked fibricarrier formation and caused accumulation of fibrils in fibripositors. Data from fluid-phase HRP electron tomography showed that fibricarriers could originate at the plasma membrane. We propose that NMII-powered transport of newly formed collagen fibrils at the plasma membrane is fundamental to the development of collagen fibril-rich tissues. A NMII-dependent cell-force model is presented as the basis for the creation and dynamics of fibripositor structures. PMID:24248360

  16. Nanoscale characterization of the biomechanical properties of collagen fibrils in the sclera

    SciTech Connect

    Papi, M.; Paoletti, P.; Geraghty, B.; Akhtar, R.

    2014-03-10

    We apply the PeakForce Quantitative Nanomechanical Property Mapping (PFQNM) atomic force microscopy mode for the investigation of regional variations in the nanomechanical properties of porcine sclera. We examine variations in the collagen fibril diameter, adhesion, elastic modulus and dissipation in the posterior, equatorial and anterior regions of the sclera. The mean fibril diameter, elastic modulus and dissipation increased from the posterior to the anterior region. Collagen fibril diameter correlated linearly with elastic modulus. Our data matches the known macroscopic mechanical behavior of the sclera. We propose that PFQNM has significant potential in ocular biomechanics and biophysics research.

  17. Second harmonic generation imaging of collagen fibrils in cornea and sclera

    NASA Astrophysics Data System (ADS)

    Han, Meng; Giese, Günter; Bille, Josef F.

    2005-07-01

    Collagen, as the most abundant protein in the human body, determines the unique physiological and optical properties of the connective tissues including cornea and sclera. The ultrastructure of collagen, which conventionally can only be resolved by electron microscopy, now can be probed by optical second harmonic generation (SHG) imaging. SHG imaging revealed that corneal collagen fibrils are regularly packed as a polycrystalline lattice, accounting for the transparency of cornea. In contrast, scleral fibrils possess inhomogeneous, tubelike structures with thin hard shells, maintaining the high stiffness and elasticity of the sclera.

  18. Characterization of type I collagen fibril formation using thioflavin T fluorescent dye.

    PubMed

    Morimoto, Koichi; Kawabata, Kazuya; Kunii, Saori; Hamano, Kaori; Saito, Takuya; Tonomura, Ben'ichiro

    2009-05-01

    Collagen is composed of fibrils that are formed by self-assembly of smaller units, monomers which are triple-helical polypeptide. However, the mechanism of fibril formation at the level of individual molecules has remained to be clarified. We found that the fluorescence of thioflavin T, which has been widely used as a specific dye for amyloid fibrils, also increased by binding with fibrils of atelocollagen prepared from yellowfin tuna skin. There was a linear correlation between the fluorescence increase and the amount of atelocollagen within a collagen concentration range of 0-0.15 mg/ml at pH 6.5 with 50 microM thioflavin T. In contrast, neither actinidain-processed collagen that keeps monomeric nature nor heat-denatured collagen could cause the fluorescence increase of thioflavin T at all. The relationship between the fluorescence increase and thioflavin T concentration was fit to a theoretical binary binding curve. An apparent dissociation constant, K(d), and a maximal fluorescence increase, DeltaF(max), were calculated at various pHs. The values of K(d) and DeltaF(max) were dependent on pH (K(d) was 9.4 microM at pH 6.5). The present finding demonstrates that thioflavin T specifically binds to collagen fibrils and may be used as a sensitive tool for the study of collagen structure. PMID:19204013

  19. Automated quantification of aligned collagen for human breast carcinoma prognosis

    PubMed Central

    Bredfeldt, Jeremy S.; Liu, Yuming; Conklin, Matthew W.; Keely, Patricia J.; Mackie, Thomas R.; Eliceiri, Kevin W.

    2014-01-01

    Background: Mortality in cancer patients is directly attributable to the ability of cancer cells to metastasize to distant sites from the primary tumor. This migration of tumor cells begins with a remodeling of the local tumor microenvironment, including changes to the extracellular matrix and the recruitment of stromal cells, both of which facilitate invasion of tumor cells into the bloodstream. In breast cancer, it has been proposed that the alignment of collagen fibers surrounding tumor epithelial cells can serve as a quantitative image-based biomarker for survival of invasive ductal carcinoma patients. Specific types of collagen alignment have been identified for their prognostic value and now these tumor associated collagen signatures (TACS) are central to several clinical specimen imaging trials. Here, we implement the semi-automated acquisition and analysis of this TACS candidate biomarker and demonstrate a protocol that will allow consistent scoring to be performed throughout large patient cohorts. Methods: Using large field of view high resolution microscopy techniques, image processing and supervised learning methods, we are able to quantify and score features of collagen fiber alignment with respect to adjacent tumor-stromal boundaries. Results: Our semi-automated technique produced scores that have statistically significant correlation with scores generated by a panel of three human observers. In addition, our system generated classification scores that accurately predicted survival in a cohort of 196 breast cancer patients. Feature rank analysis reveals that TACS positive fibers are more well-aligned with each other, are of generally lower density, and terminate within or near groups of epithelial cells at larger angles of interaction. Conclusion: These results demonstrate the utility of a supervised learning protocol for streamlining the analysis of collagen alignment with respect to tumor stromal boundaries. PMID:25250186

  20. Micromechanical analysis of native and cross-linked collagen type I fibrils supports the existence of microfibrils.

    PubMed

    Yang, L; van der Werf, K O; Dijkstra, P J; Feijen, J; Bennink, M L

    2012-02-01

    The mechanical properties of individual collagen fibrils of approximately 200 nm in diameter were determined using a slightly adapted AFM system. Single collagen fibrils immersed in PBS buffer were attached between an AFM cantilever and a glass surface to perform tensile tests at different strain rates and stress relaxation measurements. The stress-strain behavior of collagen fibrils immersed in PBS buffer comprises a toe region up to a stress of 5 MPa, followed by the heel and linear region at higher stresses. Hysteresis and strain-rate dependent stress-strain behavior of collagen fibrils were observed, which suggest that single collagen fibrils have viscoelastic properties. The stress relaxation process of individual collagen fibrils could be best fitted using a two-term Prony series. Furthermore, the influence of different cross-linking agents on the mechanical properties of single collagen fibrils was investigated. Based on these results, we propose that sliding of microfibrils with respect to each other plays a role in the viscoelastic behavior of collagen fibrils in addition to the sliding of collagen molecules with respect to each other. Our finding provides a better insight into the relationship between the structure and mechanical properties of collagen and the micro-mechanical behavior of tissues. PMID:22301184

  1. Bowstring Stretching and Quantitative Imaging of Single Collagen Fibrils via Atomic Force Microscopy

    PubMed Central

    Quigley, Andrew S.; Veres, Samuel P.; Kreplak, Laurent

    2016-01-01

    Collagen is the primary structural protein in animals. Serving as nanoscale biological ropes, collagen fibrils are responsible for providing strength to a variety of connective tissues such as tendon, skin, and bone. Understanding structure-function relationships in collagenous tissues requires the ability to conduct a variety of mechanical experiments on single collagen fibrils. Though significant advances have been made, certain tests are not possible using the techniques currently available. In this report we present a new atomic force microscopy (AFM) based method for tensile manipulation and subsequent nanoscale structural assessment of single collagen fibrils. While the method documented here cannot currently capture force data during loading, it offers the great advantage of allowing structural assessment after subrupture loading. To demonstrate the utility of this technique, we describe the results of 23 tensile experiments in which collagen fibrils were loaded to varying levels of strain and subsequently imaged in both the hydrated and dehydrated states. We show that following a dehydration-rehydration cycle (necessary for sample preparation), fibrils experience an increase in height and decrease in radial modulus in response to one loading-unloading cycle to strain <5%. This change is not altered by a second cycle to strain >5%. In fibril segments that ruptured during their second loading cycle, we show that the fibril structure is affected away from the rupture site in the form of discrete permanent deformations. By comparing the severity of select damage sites in both hydrated and dehydrated conditions, we demonstrate that dehydration masks damage features, leading to an underestimate of the degree of structural disruption. Overall, the method shows promise as a powerful tool for the investigation of structure-function relationships in nanoscale fibrous materials. PMID:27598334

  2. Bowstring Stretching and Quantitative Imaging of Single Collagen Fibrils via Atomic Force Microscopy.

    PubMed

    Quigley, Andrew S; Veres, Samuel P; Kreplak, Laurent

    2016-01-01

    Collagen is the primary structural protein in animals. Serving as nanoscale biological ropes, collagen fibrils are responsible for providing strength to a variety of connective tissues such as tendon, skin, and bone. Understanding structure-function relationships in collagenous tissues requires the ability to conduct a variety of mechanical experiments on single collagen fibrils. Though significant advances have been made, certain tests are not possible using the techniques currently available. In this report we present a new atomic force microscopy (AFM) based method for tensile manipulation and subsequent nanoscale structural assessment of single collagen fibrils. While the method documented here cannot currently capture force data during loading, it offers the great advantage of allowing structural assessment after subrupture loading. To demonstrate the utility of this technique, we describe the results of 23 tensile experiments in which collagen fibrils were loaded to varying levels of strain and subsequently imaged in both the hydrated and dehydrated states. We show that following a dehydration-rehydration cycle (necessary for sample preparation), fibrils experience an increase in height and decrease in radial modulus in response to one loading-unloading cycle to strain <5%. This change is not altered by a second cycle to strain >5%. In fibril segments that ruptured during their second loading cycle, we show that the fibril structure is affected away from the rupture site in the form of discrete permanent deformations. By comparing the severity of select damage sites in both hydrated and dehydrated conditions, we demonstrate that dehydration masks damage features, leading to an underestimate of the degree of structural disruption. Overall, the method shows promise as a powerful tool for the investigation of structure-function relationships in nanoscale fibrous materials. PMID:27598334

  3. Decorin Core Protein (Decoron) Shape Complements Collagen Fibril Surface Structure and Mediates Its Binding

    SciTech Connect

    Orgel, Joseph P.R.O.; Eid, Aya; Antipova, Olga; Bella, Jordi; Scott, John E.

    2010-02-11

    Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e{sub 1} bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

  4. Decorin core protein (decoron) shape complements collagen fibril surface structure and mediates its binding.

    PubMed

    Orgel, Joseph P R O; Eid, Aya; Antipova, Olga; Bella, Jordi; Scott, John E

    2009-01-01

    Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e(1) bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e(1) bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

  5. Generation of Spatially Aligned Collagen Fiber Networks through Microtransfer Molding

    PubMed Central

    Naik, Nisarga; Caves, Jeffrey

    2013-01-01

    The unique biomechanical properties of native tissue are governed by the organization and composition of integrated collagen and elastin networks. We report an approach for fabricating spatially aligned, fiber-reinforced composites (FRC) with adjustable collagen fiber dimensions, layouts, and distribution within an elastin-like protein matrix yielding a biocomposite with controllable mechanical responses. Microtransfer molding is employed for the fabrication of hollow and solid collagen fibers with straight or crimped fiber geometries. Collagen fibers (width: 2 – 50 μm, thickness: 300 nm – 3 μm) exhibit a Young’s modulus of 126 ± 61 MPa and an ultimate tensile strength (UTS) of 7 ± 3.2 MPa. As fiber networks within composite structures, straight fiber layouts display orthotropic responses with Young’s modulus ranging from 0.95 ± 0.35 to 10.4 ± 0.5 MPa and tensile strength from 0.22 ± 0.08 to 0.87 ± 0.5 MPa with increasing fraction of collagen fibers (1–10% v/v). In contrast, composites based on crimped fiber layouts exhibit strain-dependent stiffness with an increase in Young’s modulus from 0.7 ± 0.14 MPa to 3.15 ± 0.49 MPa, at a specific transition strain. Through controlling the microstructure of engineered collagen fiber networks, a facile means has been established to control macroscale mechanical responses of composite protein-based materials. PMID:24039146

  6. Generation of spatially aligned collagen fiber networks through microtransfer molding.

    PubMed

    Naik, Nisarga; Caves, Jeffrey; Chaikof, Elliot L; Allen, Mark G

    2014-03-01

    The unique biomechanical properties of native tissue are governed by the organization and composition of integrated collagen and elastin networks. An approach for fabricating spatially aligned, fiber-reinforced composites with adjustable collagen fiber dimensions, layouts, and distribution within an elastin-like protein matrix yielding a biocomposite with controllable mechanical responses is reported. Microtransfer molding is employed for the fabrication of hollow and solid collagen fibers with straight or crimped fiber geometries. Collagen fibers (width: 2-50 μm, thickness: 300 nm to 3 μm) exhibit a Young's modulus of 126 ± 61 MPa and an ultimate tensile strength of 7 ± 3.2 MPa. As fiber networks within composite structures, straight fiber layouts display orthotropic responses with Young's modulus ranging from 0.95 ± 0.35 to 10.4 ± 0.5 MPa and tensile strength from 0.22 ± 0.08 to 0.87 ± 0.5 MPa with increasing fraction of collagen fibers (1-10%, v/v). In contrast, composites based on crimped fiber layouts exhibit strain-dependent stiffness with an increase in Young's modulus from 0.7 ± 0.14 MPa to 3.15 ± 0.49 MPa, at a specific transition strain. Through controlling the microstructure of engineered collagen fiber networks, a facile means is established to control macroscale mechanical responses of composite protein-based materials. PMID:24039146

  7. Intracellular collagen fibrils: evidence of an intracellular source from experiments with tendon fibroblasts and fibroblastic tumour cells.

    PubMed Central

    Michna, H

    1988-01-01

    This study was designed to substantiate one or both of the two hypotheses for the explanation of intracellular collagen fibrils in collagen-producing cells. The more obvious is the phagocytosis of extracellular collagen fibrils by the cell and the other is a form of autophagocytosis of newly synthesised collagenous products. Information was collected on fibroblasts from murine tendons after exercise and simultaneously stimulating collagen synthesis by treatment with an anabolic steroid hormone. Moreover, in vivo and in vitro fibroblastic tumour cells which demonstrate enhanced protein synthesis were also treated with the anabolic steroid. The findings of intracellular collagen fibrils in tendon fibroblasts and the sarcoma cells after experimentally stimulating collagen synthesis are discussed in the light of the hypothesis that the findings may represent steps of autophagocytosis of newly synthesised collagenous products in the absence of a control mechanism to remove collagenous products which cannot be secreted. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:3225213

  8. Nanoscale characterization of isolated individual type I collagen fibrils: polarization and piezoelectricity

    NASA Astrophysics Data System (ADS)

    Minary-Jolandan, Majid; Yu, Min-Feng

    2009-02-01

    Piezoresponse force microscopy was applied to directly study individual type I collagen fibrils with diameters of ~100 nm isolated from bovine Achilles tendon. It was revealed that single collagen fibrils behave predominantly as shear piezoelectric materials with a piezoelectric coefficient on the order of 1 pm V-1, and have unipolar axial polarization throughout their entire length. It was estimated that, under reasonable shear load conditions, the fibrils were capable of generating an electric potential up to tens of millivolts. The result substantiates the nanoscale origin of piezoelectricity in bone and tendons, and implies also the potential importance of the shear load-transfer mechanism, which has been the principle basis of the nanoscale mechanics model of collagen, in mechanoelectric transduction in bone.

  9. Effect of structural change of collagen fibrils on the durability of dentin bonding.

    PubMed

    Yang, Bin; Adelung, Rainer; Ludwig, Klaus; Bössmann, Klaus; Pashley, David H; Kern, Matthias

    2005-08-01

    This study investigates the effect of structural changes of collagen fibrils on the bonding durability of a total etch luting resin (Super-Bond C&B) and a self-etching luting resin (Panavia F 2.0) to dentin. An atomic force microscope (AFM) was used to observe structural changes of intact dentin collagen fibrils after acidic conditionings of two bonding systems. After 90 d water storage and 15,000 thermal cycles (TC) as artificial aging, micro-tensile bond strength (microTBS) was utilized to evaluate the bonding durability of the two bonding systems to dentin. microTBS after 1 d or 90 d water storage without TC were separately measured in control groups. A cross-banding periodicity of about 67 nm along collagen fibrils was seen on demineralized intertubular dentin surfaces in AFM images. For both luting resins, thermal cycling decreased (p < 0.05) microTBS of 1 d and 90 d, compared to controls. Scanning electron microscope and transmission electron microscopic examinations revealed that the top and bottom of hybrid layer (HL) were weak links in the bonding interface over time. The results suggest that the top of HL contains disorganized collagen fibrils from the smear layer which degrade over time. AFM results indicate that the demineralized intact collagen fibrils beneath the smear layer were not denatured during acidic conditioning. However, these collagen fibrils may be structurally unstable due to poor infiltration by resin or loss of resin protection within the HL over time, reducing the long-term microTBS. This process was accelerated by thermal fatigue cycling.

  10. Characterization of collagen fibrils after equine suspensory ligament injury: an ultrastructural and biochemical approach.

    PubMed

    Shikh Alsook, M K; Gabriel, A; Salouci, M; Piret, J; Alzamel, N; Moula, N; Denoix, J-M; Antoine, N; Baise, E

    2015-04-01

    Suspensory ligament (SL) injuries are an important cause of lameness in horses. The mechanical properties of connective tissue in normal and pathological ligaments are mainly related to fibril morphology, as well as collagen content and types. The purpose of this study was to evaluate, using biochemical and ultrastructural approaches, the alterations in collagen fibrils after injury. Eight Warmblood horses with visible signs of injury in only one forelimb SL were selected and specimens were examined by transmission electron microscope (TEM). Collagen types I, III and V were purified by differential salt precipitation after collagen extraction with acetic acid containing pepsin. TEM revealed abnormal organization as well as alterations in the diameter and shape of fibrils after SL injury. The bands corresponding to types I, III and V collagen were assessed by densitometry after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis indicated that the proportions of type III and type V collagen were higher (P < 0.001) in damaged tissues compared with normal tissues with a mean increase of 20.9% and 17.3%, respectively. Concurrently, a decrease (P < 0.001) in type I collagen within damaged tissues was recorded with a mean decrease of 15.2%. These alterations could be the hallmark of a decrease in the tissue quality and mechanical properties of the ligament. The findings provide new insight for subsequent research on tissue regeneration that may lead to the development of future treatment strategies for SL injury.

  11. Techniques to assess bone ultrastructure organization: orientation and arrangement of mineralized collagen fibrils

    PubMed Central

    Georgiadis, Marios; Müller, Ralph; Schneider, Philipp

    2016-01-01

    Bone's remarkable mechanical properties are a result of its hierarchical structure. The mineralized collagen fibrils, made up of collagen fibrils and crystal platelets, are bone's building blocks at an ultrastructural level. The organization of bone's ultrastructure with respect to the orientation and arrangement of mineralized collagen fibrils has been the matter of numerous studies based on a variety of imaging techniques in the past decades. These techniques either exploit physical principles, such as polarization, diffraction or scattering to examine bone ultrastructure orientation and arrangement, or directly image the fibrils at the sub-micrometre scale. They make use of diverse probes such as visible light, X-rays and electrons at different scales, from centimetres down to nanometres. They allow imaging of bone sections or surfaces in two dimensions or investigating bone tissue truly in three dimensions, in vivo or ex vivo, and sometimes in combination with in situ mechanical experiments. The purpose of this review is to summarize and discuss this broad range of imaging techniques and the different modalities of their use, in order to discuss their advantages and limitations for the assessment of bone ultrastructure organization with respect to the orientation and arrangement of mineralized collagen fibrils. PMID:27335222

  12. Nano-mechanical properties of individual mineralized collagen fibrils from bone tissue.

    PubMed

    Hang, Fei; Barber, Asa H

    2011-04-01

    Mineralized collagen fibrils (MCFs) are distinct building blocks for bone material and perform an important mechanical function. A novel experimental technique using combined atomic force microscopy and scanning electron microscopy is used to manipulate and measure the mechanical properties of individual MCFs from antler, which is a representative bone tissue. The recorded stress-strain response of individual MCFs under tension shows an initial linear deformation region for all fibrils, followed by inhomogeneous deformation above a critical strain. This inhomogeneous deformation is indicative of fibrils exhibiting either yield or strain hardening and suggests possible mineral compositional changes within each fibril. A phenomenological model is used to describe the fibril nano-mechanical behaviour. PMID:20961895

  13. Second harmonic generation imaging of the collagen in myocardium for atrial fibrillation diagnosis

    NASA Astrophysics Data System (ADS)

    Tsai, Ming-Rung; Chiou, Yu-We; Sun, Chi-Kuang

    2009-02-01

    Myocardial fibrosis, a common sequela of cardiac hypertrophy, has been shown to be associated with arrhythmias in experimental models. Some research has indicated that myocardial fibrosis plays an important role in predisposing patients to atrial fibrillation. Second harmonic generation (SHG) is an optically nonlinear coherent process to image the collagen network. In this presentation, we observe the SHG images of the collagen matrix in atrial myocardium and we analyzed of collagen fibers arrangement by using Fourier-transform analysis. Moreover, comparing the SHG images of the collagen fibers in atrial myocardium between normal sinus rhythm (NSR) and atrial fibrillation (AF), our result indicated that it is possible to realize the relation between myocardial fibrosis and AF.

  14. Poisson's ratio of collagen fibrils measured by small angle X-ray scattering of strained bovine pericardium

    SciTech Connect

    Wells, Hannah C.; Sizeland, Katie H.; Kayed, Hanan R.; Haverkamp, Richard G.; Kirby, Nigel; Hawley, Adrian; Mudie, Stephen T.

    2015-01-28

    Type I collagen is the main structural component of skin, tendons, and skin products, such as leather. Understanding the mechanical performance of collagen fibrils is important for understanding the mechanical performance of the tissues that they make up, while the mechanical properties of bulk tissue are well characterized, less is known about the mechanical behavior of individual collagen fibrils. In this study, bovine pericardium is subjected to strain while small angle X-ray scattering (SAXS) patterns are recorded using synchrotron radiation. The change in d-spacing, which is a measure of fibril extension, and the change in fibril diameter are determined from SAXS. The tissue is strained 0.25 (25%) with a corresponding strain in the collagen fibrils of 0.045 observed. The ratio of collagen fibril width contraction to length extension, or the Poisson's ratio, is 2.1 ± 0.7 for a tissue strain from 0 to 0.25. This Poisson's ratio indicates that the volume of individual collagen fibrils decreases with increasing strain, which is quite unlike most engineering materials. This high Poisson's ratio of individual fibrils may contribute to high Poisson's ratio observed for tissues, contributing to some of the remarkable properties of collagen-based materials.

  15. Poisson's ratio of collagen fibrils measured by small angle X-ray scattering of strained bovine pericardium

    NASA Astrophysics Data System (ADS)

    Wells, Hannah C.; Sizeland, Katie H.; Kayed, Hanan R.; Kirby, Nigel; Hawley, Adrian; Mudie, Stephen T.; Haverkamp, Richard G.

    2015-01-01

    Type I collagen is the main structural component of skin, tendons, and skin products, such as leather. Understanding the mechanical performance of collagen fibrils is important for understanding the mechanical performance of the tissues that they make up, while the mechanical properties of bulk tissue are well characterized, less is known about the mechanical behavior of individual collagen fibrils. In this study, bovine pericardium is subjected to strain while small angle X-ray scattering (SAXS) patterns are recorded using synchrotron radiation. The change in d-spacing, which is a measure of fibril extension, and the change in fibril diameter are determined from SAXS. The tissue is strained 0.25 (25%) with a corresponding strain in the collagen fibrils of 0.045 observed. The ratio of collagen fibril width contraction to length extension, or the Poisson's ratio, is 2.1 ± 0.7 for a tissue strain from 0 to 0.25. This Poisson's ratio indicates that the volume of individual collagen fibrils decreases with increasing strain, which is quite unlike most engineering materials. This high Poisson's ratio of individual fibrils may contribute to high Poisson's ratio observed for tissues, contributing to some of the remarkable properties of collagen-based materials.

  16. Rapid oriented fibril formation of fish scale collagen facilitates early osteoblastic differentiation of human mesenchymal stem cells.

    PubMed

    Matsumoto, Rena; Uemura, Toshimasa; Xu, Zhefeng; Yamaguchi, Isamu; Ikoma, Toshiyuki; Tanaka, Junzo

    2015-08-01

    We studied the effect of fibril formation of fish scale collagen on the osteoblastic differentiation of human mesenchymal stem cells (hMSCs). We found that hMSCs adhered easily to tilapia scale collagen, which remarkably accelerated the early stage of osteoblastic differentiation in hMSCs during in vitro cell culture. Osteoblastic markers such as ALP activity, osteopontin, and bone morphogenetic protein 2 were markedly upregulated when the hMSCs were cultured on a tilapia collagen surface, especially in the early osteoblastic differentiation stage. We hypothesized that this phenomenon occurs due to specific fibril formation of tilapia collagen. Thus, we examined the time course of collagen fibril formation using high-speed atomic force microscopy. Moreover, to elucidate the effect of the orientation of fibril formation on the differentiation of hMSCs, we measured ALP activity of hMSCs cultured on two types of tilapia scale collagen membranes with different degrees of fibril formation. The ALP activity in hMSCs cultured on a fibrous collagen membrane was significantly higher than on a non-fibrous collagen membrane even before adding osteoblastic differentiation medium. These results showed that the degree of the fibril formation of tilapia collagen was essential for the osteoblastic differentiation of hMSCs.

  17. High-speed atomic force microscopy reveals strongly polarized movement of clostridial collagenase along collagen fibrils

    PubMed Central

    Watanabe-Nakayama, Takahiro; Itami, Masahiro; Kodera, Noriyuki; Ando, Toshio; Konno, Hiroki

    2016-01-01

    Bacterial collagenases involved in donor infection are widely applied in many fields due to their high activity and specificity; however, little is known regarding the mechanisms by which bacterial collagenases degrade insoluble collagen in host tissues. Using high-speed atomic force microscopy, we simultaneously visualized the hierarchical structure of collagen fibrils and the movement of a representative bacterial collagenase, Clostridium histolyticum type I collagenase (ColG), to determine the relationship between collagen structure and collagenase movement. Notably, ColG moved ~14.5 nm toward the collagen N terminus in ~3.8 s in a manner dependent on a catalytic zinc ion. While ColG was engaged, collagen molecules were not only degraded but also occasionally rearranged to thicken neighboring collagen fibrils. Importantly, we found a similarity of relationship between the enzyme-substrate interface structure and enzyme migration in collagen-collagenase and DNA-nuclease systems, which share a helical substrate structure, suggesting a common strategy in enzyme evolution. PMID:27373458

  18. Cross-linking connectivity in bone collagen fibrils: the COOH-terminal locus of free aldehyde

    NASA Technical Reports Server (NTRS)

    Otsubo, K.; Katz, E. P.; Mechanic, G. L.; Yamauchi, M.

    1992-01-01

    Quantitative analyses of the chemical state of the 16c residue of the alpha 1 chain of bone collagen were performed on samples from fetal (4-6-month embryo) and mature (2-3 year old) bovine animals. All of this residue could be accounted for in terms of three chemical states, in relative amounts which depended upon the age of the animal. Most of the residue was incorporated into either bifunctional or trifunctional cross-links. Some of it, however, was present as free aldehyde, and the content increased with maturation. This was established by isolating and characterizing the aldehyde-containing peptides generated by tryptic digestion of NaB3H4-reduced mature bone collagen. We have concluded that the connectivity of COOH-terminal cross-linking in bone collagen fibrils changes with maturation in the following way: at first, each 16c residue in each of the two alpha 1 chains of the collagen molecule is incorporated into a sheet-like pattern of intermolecular iminium cross-links, which stabilizes the young, nonmineralized fibril as a whole. In time, some of these labile cross-links maturate into pyridinoline while others dissociate back to their precursor form. The latter is likely due to changes in the molecular packing brought about by the mineralization of the collagen fibrils. The resultant reduction in cross-linking connectivity may provide a mechanism for enhancing certain mechanical characteristics of the skeleton of a mature animal.

  19. Characteristics and Young's Modulus of Collagen Fibrils from Expanded Skin Using Anisotropic Controlled Rate Self-Inflating Tissue Expander.

    PubMed

    Manssor, Nur Aini S; Radzi, Zamri; Yahya, Noor Azlin; Mohamad Yusof, Loqman; Hariri, Firdaus; Khairuddin, Nurul Hayah; Abu Kasim, Noor Hayaty; Czernuszka, Jan T

    2016-01-01

    Mechanical properties of expanded skin tissue are different from normal skin, which is dependent mainly on the structural and functional integrity of dermal collagen fibrils. In the present study, mechanical properties and surface topography of both expanded and nonexpanded skin collagen fibrils were evaluated. Anisotropic controlled rate self-inflating tissue expanders were placed beneath the skin of sheep's forelimbs. The tissue expanders gradually increased in height and reached equilibrium in 2 weeks. They were left in situ for another 2 weeks before explantation. Expanded and normal skin samples were surgically harvested from the sheep (n = 5). Young's modulus and surface topography of collagen fibrils were measured using an atomic force microscope. A surface topographic scan showed organized hierarchical structural levels: collagen molecules, fibrils and fibers. No significant difference was detected for the D-banding pattern: 63.5 ± 2.6 nm (normal skin) and 63.7 ± 2.7 nm (expanded skin). Fibrils from expanded tissues consisted of loosely packed collagen fibrils and the width of the fibrils was significantly narrower compared to those from normal skin: 153.9 ± 25.3 and 106.7 ± 28.5 nm, respectively. Young's modulus of the collagen fibrils in the expanded and normal skin was not statistically significant: 46.5 ± 19.4 and 35.2 ± 27.0 MPa, respectively. In conclusion, the anisotropic controlled rate self-inflating tissue expander produced a loosely packed collagen network and the fibrils exhibited similar D-banding characteristics as the control group in a sheep model. However, the fibrils from the expanded skin were significantly narrower. The stiffness of the fibrils from the expanded skin was higher but it was not statistically different. PMID:26836267

  20. The use of reflection anisotropy spectroscopy to assess the alignment of collagen

    NASA Astrophysics Data System (ADS)

    Schofield, A. L.; Smith, C. I.; Kearns, V. R.; Martin, D. S.; Farrell, T.; Weightman, P.; Williams, R. L.

    2011-08-01

    The alignment of collagen fibres in tissue has a major influence on their mechanical properties. This study investigated the ability of reflection anisotropy spectroscopy (RAS) to determine the degree of alignment of collagen fibres deposited onto surfaces and secreted by mouse fibroblast cells in vitro. Aligned nanofibres of polytetrafluoroethylene were deposited on glass coverslips using a simple friction transfer method. These linear parallel nanofibres were used as topographical cues to orientate and align L929 fibroblasts and their deposited collagen. The strength of the RAS signal was demonstrated to correlate with the degree of collagen alignment. Immunochemical staining and atomic force microscopy were used to visualize the topography of the fibres and confirm that the RAS signal was as a result of collagen fibres. Collagen deposited onto glass coverslips from a solution that had been subjected to dialysis that caused 'nanofibrillar' collagen to form also resulted in a strong RAS signal whereas collagen adsorbed from a simple solution of collagen in which collagen fibres are not formed resulted in no RAS signal. It was concluded that the RAS signal could be used to determine the degree of alignment of collagen and that this could have a potential application in the assessment of collagen orientation in tissue repair.

  1. Physical evidence for a glue holding mineralized collagen fibrils together in bone*

    NASA Astrophysics Data System (ADS)

    Hansma, P.

    2005-03-01

    Evidence from Atomic Force Microscope indentation, pulling and imaging, and macroscopic testing and enzymatic digestion, suggests that collagen fibrils and mineral plates are not the only components of bone with mechanical roles. A ``glue'' appears to bind mineralized collagen fibrils together. Order of magnitude calculations show that less than 1% by weight of this ``glue'' profoundly affects bone fracture resistance, as it involves a remarkable natural toughening and strengthening system: sacrificial bonds and hidden length. This system dissipates large amounts of work against entropic forces while stretching out the hidden length that is exposed when sacrificial bonds break. This appears to occur when mineralized collagen fibrils are torn apart or slid against each other during bone fracture. In bone, this system depends on multivalent positive ions such as calcium ions, which allows us to follow its influence up to macroscopic fracture testing levels. Many bone matrix proteoglycans and glycoproteins have negatively charged groups at physiological pHs that could be bound together into sacrificial bonds by multivalent positive ions, and are thus natural candidates for this ``glue.'' We cannot rule out a possible involvement of nonfibrillar collagen. Precisely which candidates are involved is yet to be determined. *NSF MRL DMR00-80034, NIH GM65354, NASA BiMAT URETI NCC-1-02037 (00000532), Veeco, USARL ARO DAAD19-03-D-0004

  2. Glycated collagen decreased endothelial cell fibronectin alignment in response to cyclic stretch via interruption of actin alignment.

    PubMed

    Figueroa, Dannielle S; Kemeny, Steven F; Clyne, Alisa Morss

    2014-10-01

    Hyperglycemia is a defining characteristic of diabetes, and uncontrolled blood glucose in diabetes is associated with accelerated cardiovascular disease. Chronic hyperglycemia glycates extracellular matrix (ECM) collagen, which can lead to endothelial cell dysfunction. In healthy conditions, endothelial cells respond to mechanical stimuli such as cyclic stretch (CS) by aligning their actin cytoskeleton. Other cell types, specifically fibroblasts, align their ECM in response to CS. We previously demonstrated that glycated collagen inhibits endothelial cell actin alignment in response to CS. The aim of this study was to determine the effect of glycated collagen on ECM remodeling and protein alignment in response to stretch. Porcine aortic endothelial cells (PAEC) seeded on native or glycated collagen coated elastic substrates were exposed to 10% CS. Cells on native collagen aligned subcellular fibronectin fibers in response to stretch, whereas cells on glycated collagen did not. The loss of fibronectin alignment was due to inhibited actin alignment in response to CS, since fibronectin alignment did not occur in cells on native collagen when actin alignment was inhibited with cytochalasin. Further, while ECM protein content did not change in cells on native or glycated collagen in response to CS, degradation activity decreased in cells on glycated collagen. Matrix metalloproteinase 2 (MMP-2) and membrane-associated type 1 matrix metalloproteinase (MT1-MMP) protein levels decreased, and therefore MMP-2 activity also decreased. These MMP changes may relate to c-Jun N-terminal kinase (Jnk) phosphorylation inhibition with CS, which has previously been linked to focal adhesion kinase (FAK). These data demonstrate the importance of endothelial cell actin tension in remodeling and aligning matrix proteins in response to mechanical stimuli, which is critical to vascular remodeling in health and disease.

  3. Influence of the mineral staggering on the elastic properties of the mineralized collagen fibril in lamellar bone.

    PubMed

    Vercher-Martínez, Ana; Giner, Eugenio; Arango, Camila; Fuenmayor, F Javier

    2015-02-01

    In this work, a three-dimensional finite element model of the staggered distribution of the mineral within the mineralized collagen fibril has been developed to characterize the lamellar bone elastic behavior at the sub-micro length scale. Minerals have been assumed to be embedded in a collagen matrix, and different degrees of mineralization have been considered allowing the growth of platelet-shaped minerals both in the axial and the transverse directions of the fibril, through the variation of the lateral space between platelets. We provide numerical values and trends for all the elastic constants of the mineralized collagen fibril as a function of the volume fraction of mineral. In our results, we verify the high influence of the mineral overlapping on the mechanical response of the fibril and we highlight that the lateral distance between crystals is relevant to the mechanical behavior of the fibril and not only the mineral overlapping in the axial direction.

  4. The relation between collagen fibril kinematics and mechanical properties in the mitral valve anterior leaflet.

    PubMed

    Liao, Jun; Yang, Lin; Grashow, Jonathan; Sacks, Michael S

    2007-02-01

    We have recently demonstrated that the mitral valve anterior leaflet (MVAL) exhibited minimal hysteresis, no strain rate sensitivity, stress relaxation but not creep (Grashow et al., 2006, Ann Biomed Eng., 34(2), pp. 315-325; Grashow et al., 2006, Ann Biomed. Eng., 34(10), pp. 1509-1518). However, the underlying structural basis for this unique quasi-elastic mechanical behavior is presently unknown. As collagen is the major structural component of the MVAL, we investigated the relation between collagen fibril kinematics (rotation and stretch) and tissue-level mechanical properties in the MVAL under biaxial loading using small angle X-ray scattering. A novel device was developed and utilized to perform simultaneous measurements of tissue level forces and strain under a planar biaxial loading state. Collagen fibril D-period strain (epsilonD) and the fibrillar angular distribution were measured under equibiaxial tension, creep, and stress relaxation to a peak tension of 90 N/m. Results indicated that, under equibiaxial tension, collagen fibril straining did not initiate until the end of the nonlinear region of the tissue-level stress-strain curve. At higher tissue tension levels, epsilonD increased linearly with increasing tension. Changes in the angular distribution of the collagen fibrils mainly occurred in the tissue toe region. Using epsilonD, the tangent modulus of collagen fibrils was estimated to be 95.5+/-25.5 MPa, which was approximately 27 times higher than the tissue tensile tangent modulus of 3.58+/-1.83 MPa. In creep tests performed at 90 N/m equibiaxial tension for 60 min, both tissue strain and epsilonD remained constant with no observable changes over the test length. In contrast, in stress relaxation tests performed for 90 min epsilonD was found to rapidly decrease in the first 10 min followed by a slower decay rate for the remainder of the test. Using a single exponential model, the time constant for the reduction in collagen fibril strain was 8

  5. The Relation Between Collagen Fibril Kinematics and Mechanical Properties in the Mitral Valve Anterior Leaflet

    SciTech Connect

    Liao,J.; Yang, L.; Grashow, J.; Sacks, M.

    2007-01-01

    We have recently demonstrated that the mitral valve anterior leaflet (MVAL) exhibited minimal hysteresis, no strain rate sensitivity, stress relaxation but not creep (Grashow et al., 2006, Ann Biomed Eng., 34(2), pp. 315-325; Grashow et al., 2006, Ann Biomed. Eng., 34(10), pp. 1509-1518). However, the underlying structural basis for this unique quasi-elastic mechanical behavior is presently unknown. As collagen is the major structural component of the MVAL, we investigated the relation between collagen fibril kinematics (rotation and stretch) and tissue-level mechanical properties in the MVAL under biaxial loading using small angle X-ray scattering. A novel device was developed and utilized to perform simultaneous measurements of tissue level forces and strain under a planar biaxial loading state. Collagen fibril D-period strain ({epsilon}{sub D}) and the fibrillar angular distribution were measured under equibiaxial tension, creep, and stress relaxation to a peak tension of 90 N/m. Results indicated that, under equibiaxial tension, collagen fibril straining did not initiate until the end of the nonlinear region of the tissue-level stress-strain curve. At higher tissue tension levels, {epsilon}{sub D} increased linearly with increasing tension. Changes in the angular distribution of the collagen fibrils mainly occurred in the tissue toe region. Using {epsilon}{sub D}, the tangent modulus of collagen fibrils was estimated to be 95.5{+-}25.5 MPa, which was {approx}27 times higher than the tissue tensile tangent modulus of 3.58{+-}1.83 MPa. In creep tests performed at 90 N/m equibiaxial tension for 60 min, both tissue strain and D remained constant with no observable changes over the test length. In contrast, in stress relaxation tests performed for 90 min {epsilon}{sub D} was found to rapidly decrease in the first 10 min followed by a slower decay rate for the remainder of the test. Using a single exponential model, the time constant for the reduction in collagen

  6. Influence of telopeptides, fibrils and crosslinking on physicochemical properties of type I collagen films.

    PubMed

    Walton, Robin S; Brand, David D; Czernuszka, Jan T

    2010-02-01

    Type I collagen is widely used in various different forms for research and commercial applications. Different forms of collagen may be classified according to their source, extraction method, crosslinking and resultant ultrastructure. In this study, afibrillar and reconstituted fibrillar films, derived from acid soluble and pepsin digested Type I collagen, were analysed using Lateral Force Microscopy (LFM), Fourier Transform Infra-Red Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC) and enzymatic stability assays to asses the influence of telopeptides, fibrils and crosslinking. LFM proved to be a useful technique to confirm an afibrillar/fibrillar ultrastructure and to elucidate fibril diameters. FTIR has proved insensitive to ultrastructural differences involving telopeptides and fibrils. DSC results showed a significant increase in T(d) for crosslinked samples (+22-28 degrees C), and demonstrated that the thermal behaviour of hydrated, afibrillar films is more akin to reconstituted fibrillar films than monomeric solutions. The enzymatic stability assay has provided new evidence to show that afibrillar films of Type I collagen can be significantly more resistant to collagenase (by up to 3.5 times), than reconstituted fibrillar films, as a direct consequence of the different spatial arrangement of collagen molecules. A novel mechanism for this phenomenon is proposed and discussed. Additionally, the presence of telopeptide regions in afibrillar tropocollagen samples has been shown to increase resistance to collagenase by greater than 3.5 times compared to counterpart afibrillar atelocollagen samples. One-factor ANOVA analysis, with Fisher's LSD post-hoc test, confirms these key findings to be of statistical significance (P < 0.05). The profound physicochemical effects of collagen ultrastructure demonstrated in this study reiterates the need for comprehensive materials disclosure and classification when using these biomaterials.

  7. Effect of ultrasonication on the fibril-formation and gel properties of collagen from grass carp skin.

    PubMed

    Jiang, Ying; Wang, Haibo; Deng, Mingxia; Wang, Zhongwen; Zhang, Juntao; Wang, Haiyin; Zhang, Hanjun

    2016-02-01

    Controlling the fibril-formation process of collagen in vitro to fabricate novel biomaterials is a new area in the field of collagen research. This study aimed to determine the effect of ultrasonication on collagen fibril formation and the properties of the resulting collagen gels. Native collagen, extracted from the skin of grass carp, self-assembled under ultrasonic conditions (at different ultrasonic power and duration). The self-assembly kinetics, fibrillar morphology, and physical and cell growth-promoting properties of the collagen gels were analyzed and compared. The results showed that the self-assembly rate of collagen was increased by ultrasonication at the nucleation stage. The resulting fibrils exhibited smaller diameters and D-periodicity lengths than that of the untreated collagen samples (p<0.05). The viscoelasticity and textural properties of collagen gels also changed after ultrasonication at the nucleation stage. Texture profile analysis and cell proliferation assays showed that ultrasonication produced softer collagen gel colloids, which were more suitable for cell proliferation than the untreated collagen gels.

  8. Effect of loading on the organization of the collagen fibril network in juvenile equine articular cartilage.

    PubMed

    Brama, Pieter A J; Holopainen, Jaakko; van Weeren, P René; Firth, Elwyn C; Helminen, Heikki J; Hyttinen, Mika M

    2009-09-01

    We investigated the effects of exercise-induced loading on the collagen network of equine articular cartilage. Collagen fibril architecture at a site (1) subjected to intermittent high-intensity loading was compared with that of an adjacent site (2) sustaining continuous low-level load. From horses exposed to forced exercise (CONDEX group) or not (PASTEX group), the spatial parallelism of fibrils and the orientation angle between fibrils and the surface at depths 9 microm apart through cartilage from surface to tidemark were determined using polarized light microscopy, and expressed as parallelism index (PI) and orientation index (OI). PI was significantly higher in site 2 than 1 in CONDEX and PASTEX groups. PI was significantly higher in forced exercised horses at site 2 but not site 1. OI was significantly greater (more perpendicular to the surface) in the superficial and deep cartilage of site 2 than 1 in both CONDEX and PASTEX groups. Superficial zone OI was higher in exercised horses at site 1 but not at site 2. Exercise increased collagen parallelism and affected orientation. The site differences in OI indicate that Benninghoff's classic predominantly perpendicular arcades appear not to be a consistent architectural feature, but adapt to local forces sustained.

  9. Modeling of bovine type-I collagen fibrils: interaction with pickling and retanning agents.

    PubMed

    Bulo, Rosa E; Siggel, Lorenz; Molnar, Ferenc; Weiss, Horst

    2007-02-12

    Bovine Type I collagen was investigated, building on a large scale computer model of a collagen fibril in water, and focusing on two stages of the leather manufacturing process. The effects of different salts (NaCl, CaCl(2), and Na(2)SO(4)) on the swelling behavior of collagen at low pH (the pickling process) were studied. The salts suppress the swelling of the fibrils at low pH and we find specific stabilizing influences for CaCl(2) and Na(2)SO(4), due to weak Ca(2+)/Cl(-) and strong SO(4) (2-)/lysine/arginine interactions, respectively. Using state-of-the-art sampling techniques, such as the metadynamics algorithm, to allow an efficient exploration of configuration space, we were able to investigate the effect of polyacrylate and poly(methyl acrylate) - two polymeric retanning agents - on the fibril. Both polymers interact with the ammonium groups on the surface, but polyacrylate shows significantly stronger interactions. We suggest that it is this stronger interaction that contributes to the reduced suitability of PAA as a tanning agent. PMID:17295396

  10. Homogenized stiffness matrices for mineralized collagen fibrils and lamellar bone using unit cell finite element models.

    PubMed

    Vercher, Ana; Giner, Eugenio; Arango, Camila; Tarancón, José E; Fuenmayor, F Javier

    2014-04-01

    Mineralized collagen fibrils have been usually analyzed like a two-phase composite material where crystals are considered as platelets that constitute the reinforcement phase. Different models have been used to describe the elastic behavior of the material. In this work, it is shown that when Halpin-Tsai equations are applied to estimate elastic constants from typical constituent properties, not all crystal dimensions yield a model that satisfy thermodynamic restrictions. We provide the ranges of platelet dimensions that lead to positive definite stiffness matrices. On the other hand, a finite element model of a mineralized collagen fibril unit cell under periodic boundary conditions is analyzed. By applying six canonical load cases, homogenized stiffness matrices are numerically calculated. Results show a monoclinic behavior of the mineralized collagen fibril. In addition, a 5-layer lamellar structure is also considered where crystals rotate in adjacent layers of a lamella. The stiffness matrix of each layer is calculated applying Lekhnitskii transformations, and a new finite element model under periodic boundary conditions is analyzed to calculate the homogenized 3D anisotropic stiffness matrix of a unit cell of lamellar bone. Results are compared with the rule-of-mixtures showing in general good agreement. PMID:23793930

  11. Homogenized stiffness matrices for mineralized collagen fibrils and lamellar bone using unit cell finite element models.

    PubMed

    Vercher, Ana; Giner, Eugenio; Arango, Camila; Tarancón, José E; Fuenmayor, F Javier

    2014-04-01

    Mineralized collagen fibrils have been usually analyzed like a two-phase composite material where crystals are considered as platelets that constitute the reinforcement phase. Different models have been used to describe the elastic behavior of the material. In this work, it is shown that when Halpin-Tsai equations are applied to estimate elastic constants from typical constituent properties, not all crystal dimensions yield a model that satisfy thermodynamic restrictions. We provide the ranges of platelet dimensions that lead to positive definite stiffness matrices. On the other hand, a finite element model of a mineralized collagen fibril unit cell under periodic boundary conditions is analyzed. By applying six canonical load cases, homogenized stiffness matrices are numerically calculated. Results show a monoclinic behavior of the mineralized collagen fibril. In addition, a 5-layer lamellar structure is also considered where crystals rotate in adjacent layers of a lamella. The stiffness matrix of each layer is calculated applying Lekhnitskii transformations, and a new finite element model under periodic boundary conditions is analyzed to calculate the homogenized 3D anisotropic stiffness matrix of a unit cell of lamellar bone. Results are compared with the rule-of-mixtures showing in general good agreement.

  12. Age dependent differences in collagen alignment of glutaraldehyde fixed bovine pericardium.

    PubMed

    Sizeland, Katie H; Wells, Hannah C; Higgins, John; Cunanan, Crystal M; Kirby, Nigel; Hawley, Adrian; Mudie, Stephen T; Haverkamp, Richard G

    2014-01-01

    Bovine pericardium is used for heart valve leaflet replacement where the strength and thinness are critical properties. Pericardium from neonatal animals (4-7 days old) is advantageously thinner and is considered as an alternative to that from adult animals. Here, the structures of adult and neonatal bovine pericardium tissues fixed with glutaraldehyde are characterized by synchrotron-based small angle X-ray scattering (SAXS) and compared with the mechanical properties of these materials. Significant differences are observed between adult and neonatal tissue. The glutaraldehyde fixed neonatal tissue has a higher modulus of elasticity (83.7 MPa) than adult pericardium (33.5 MPa) and a higher normalised ultimate tensile strength (32.9 MPa) than adult pericardium (19.1 MPa). Measured edge on to the tissue, the collagen in neonatal pericardium is significantly more aligned (orientation index (OI) 0.78) than that in adult pericardium (OI 0.62). There is no difference in the fibril diameter between neonatal and adult pericardium. It is shown that high alignment in the plane of the tissue provides the mechanism for the increased strength of the neonatal material. The superior strength of neonatal compared with adult tissue supports the use of neonatal bovine pericardium in heterografts. PMID:25295250

  13. Age Dependent Differences in Collagen Alignment of Glutaraldehyde Fixed Bovine Pericardium

    PubMed Central

    Sizeland, Katie H.; Wells, Hannah C.; Higgins, John; Cunanan, Crystal M.; Kirby, Nigel; Hawley, Adrian; Mudie, Stephen T.; Haverkamp, Richard G.

    2014-01-01

    Bovine pericardium is used for heart valve leaflet replacement where the strength and thinness are critical properties. Pericardium from neonatal animals (4–7 days old) is advantageously thinner and is considered as an alternative to that from adult animals. Here, the structures of adult and neonatal bovine pericardium tissues fixed with glutaraldehyde are characterized by synchrotron-based small angle X-ray scattering (SAXS) and compared with the mechanical properties of these materials. Significant differences are observed between adult and neonatal tissue. The glutaraldehyde fixed neonatal tissue has a higher modulus of elasticity (83.7 MPa) than adult pericardium (33.5 MPa) and a higher normalised ultimate tensile strength (32.9 MPa) than adult pericardium (19.1 MPa). Measured edge on to the tissue, the collagen in neonatal pericardium is significantly more aligned (orientation index (OI) 0.78) than that in adult pericardium (OI 0.62). There is no difference in the fibril diameter between neonatal and adult pericardium. It is shown that high alignment in the plane of the tissue provides the mechanism for the increased strength of the neonatal material. The superior strength of neonatal compared with adult tissue supports the use of neonatal bovine pericardium in heterografts. PMID:25295250

  14. Tectorins crosslink type II collagen fibrils and connect the tectorial membrane to the spiral limbus.

    PubMed

    Andrade, Leonardo R; Salles, Felipe T; Grati, M'hamed; Manor, Uri; Kachar, Bechara

    2016-05-01

    All inner ear organs possess extracellular matrix appendices over the sensory epithelia that are crucial for their proper function. The tectorial membrane (TM) is a gelatinous acellular membrane located above the hearing sensory epithelium and is composed mostly of type II collagen, and α and β tectorins. TM molecules self-assemble in the endolymph fluid environment, interacting medially with the spiral limbus and distally with the outer hair cell stereocilia. Here, we used immunogold labeling in freeze-substituted mouse cochleae to assess the fine localization of both tectorins in distinct TM regions. We observed that the TM adheres to the spiral limbus through a dense thin matrix enriched in α- and β-tectorin, both likely bound to the membranes of interdental cells. Freeze-etching images revealed that type II collagen fibrils were crosslinked by short thin filaments (4±1.5nm, width), resembling another collagen type protein, or chains of globular elements (15±3.2nm, diameter). Gold-particles for both tectorins also localized adjacent to the type II collagen fibrils, suggesting that these globules might be composed essentially of α- and β-tectorins. Finally, the presence of gold-particles at the TM lower side suggests that the outer hair cell stereocilia membrane has a molecular partner to tectorins, probably stereocilin, allowing the physical connection between the TM and the organ of Corti. PMID:26806019

  15. Small-Angle X-ray Scattering Study of Intramuscular Fish Bone: Collagen Fibril Superstructure Determined from Equidistant Meridional Reflections

    SciTech Connect

    Burger,C.; Zhou, H.; Sics, I.; Hsiao, B.; Chu, B.; Graham, L.; Glimcher, M.

    2008-01-01

    New insights into the bone collagen fibril superstructure have been obtained by novel small-angle X-ray scattering analysis. The analysis was carried out on the small-angle equidistant meridional reflections resulting from the periodic structure of collagen fibrils in their axial direction. Conventional two-dimensional analysis is difficult because of the large discrepancy of longitudinal and lateral length scales for individual fibrils, as well as their preferred orientation. The new approach represents an unapproximated analysis of the equidistant meridional reflections, which takes the exact separation of preferred orientation and fibril size effects into account. The analytical results (e.g. axial period, fibril diameter etc.) agree well with the parameters obtained from transmission electron microscopy.

  16. Collagen density and alignment in responsive and resistant trastuzumab-treated breast cancer xenografts

    NASA Astrophysics Data System (ADS)

    Walsh, Alex J.; Cook, Rebecca S.; Lee, Jae H.; Arteaga, Carlos L.; Skala, Melissa C.

    2015-02-01

    Tumor collagen characteristics influence tumor malignancy, invasion, and metastasis. This study investigates the effects of trastuzumab (Tz) on the collagen of Tz-responsive (BT474) and Tz-resistant (HR6) breast cancer xenografts. Collagen content was assessed by in vivo second harmonic generation (SHG) imaging and histological trichrome staining of tumor sections. Collagen SHG imaging of control BT474 and HR6 tumors demonstrated increased collagen density after 14 days of treatment (p<0.05). Trichrome staining revealed decreased collagen in Tz-treated BT474 and HR6 tumors at 2, 5, and 14 days of treatment, suggesting that Tz affects the tumor microenvironment independent of epithelial cell response. Additionally, collagen alignment analysis revealed significantly less aligned collagen in the Tz-treated BT474 tumors at day 14 compared with control BT474 tumors. There was no correlation between SHG endpoints (collagen density and alignment) and trichrome staining (p>0.05), consistent with the physically distinctive nature of these measurements. There was also no correlation between tumor size and collagen endpoints (p>0.05). These results identify changes within the collagen compartment of the tumor microenvironment following Tz treatment, which are independent from the tumor cell response to Tz, and demonstrate that intravital collagen SHG imaging is capable of measuring dynamic changes in tumor microenvironment following treatment that complements trichrome staining.

  17. Modeling the collagen fibril network of biological tissues as a nonlinearly elastic material using a continuous volume fraction distribution function

    PubMed Central

    Shirazi, Reza; Vena, Pasquale; Sah, Robert L.; Klisch, Stephen M.

    2012-01-01

    Despite distinct mechanical functions, biological soft tissues have a common microstructure in which a ground matrix is reinforced by a collagen fibril network. The microstructural properties of the collagen network contribute to continuum mechanical tissue properties that are strongly anisotropic with tensile-compressive asymmetry. In this study, a novel approach based on a continuous distribution of collagen fibril volume fractions is developed to model fibril reinforced soft tissues as a nonlinearly elastic and anisotropic material. Compared with other approaches that use a normalized number of fibrils for the definition of the distribution function, this representation is based on a distribution parameter (i.e. volume fraction) that is commonly measured experimentally while also incorporating pre-stress of the collagen fibril network in a tissue natural configuration. After motivating the form of the collagen strain energy function, examples are provided for two volume fraction distribution functions. Consequently, collagen second-Piola Kirchhoff stress and elasticity tensors are derived, first in general form and then specifically for a model that may be used for immature bovine articular cartilage. It is shown that the proposed strain energy is a convex function of the deformation gradient tensor and, thus, is suitable for the formation of a polyconvex tissue strain energy function. PMID:23390357

  18. Influence of cross-link structure, density and mechanical properties in the mesoscale deformation mechanisms of collagen fibrils.

    PubMed

    Depalle, Baptiste; Qin, Zhao; Shefelbine, Sandra J; Buehler, Markus J

    2015-12-01

    Collagen is a ubiquitous protein with remarkable mechanical properties. It is highly elastic, shows large fracture strength and enables substantial energy dissipation during deformation. Most of the connective tissue in humans consists of collagen fibrils composed of a staggered array of tropocollagen molecules, which are connected by intermolecular cross-links. In this study, we report a three-dimensional coarse-grained model of collagen and analyze the influence of enzymatic cross-links on the mechanics of collagen fibrils. Two representatives immature and mature cross-links are implemented in the mesoscale model using a bottom-up approach. By varying the number, type and mechanical properties of cross-links in the fibrils and performing tensile test on the models, we systematically investigate the deformation mechanisms of cross-linked collagen fibrils. We find that cross-linked fibrils exhibit a three phase behavior, which agrees closer with experimental results than what was obtained using previous models. The fibril mechanical response is characterized by: (i) an initial elastic deformation corresponding to the collagen molecule uncoiling, (ii) a linear regime dominated by molecule sliding and (iii) the second stiffer elastic regime related to the stretching of the backbone of the tropocollagen molecules until the fibril ruptures. Our results suggest that both cross-link density and type dictate the stiffness of large deformation regime by increasing the number of interconnected molecules while cross-links mechanical properties determine the failure strain and strength of the fibril. These findings reveal that cross-links play an essential role in creating an interconnected fibrillar material of tunable toughness and strength.

  19. Influence of cross-link structure, density and mechanical properties in the mesoscale deformation mechanisms of collagen fibrils

    PubMed Central

    Depalle, Baptiste; Qin, Zhao; Shefelbine, Sandra J.; Buehler, Markus J.

    2015-01-01

    Collagen is a ubiquitous protein with remarkable mechanical properties. It is highly elastic, shows large fracture strength and enables substantial energy dissipation during deformation. Most of the connective tissue in humans consists of collagen fibrils composed of a staggered array of tropocollagen molecules, which are connected by intermolecular cross-links. In this study, we report a three-dimensional coarse-grained model of collagen and analyze the influence of enzymatic cross-links on the mechanics of collagen fibrils. Two representatives immature and mature cross-links are implemented in the mesoscale model using a bottom-up approach. By varying the number, type and mechanical properties of cross-links in the fibrils and performing tensile test on the models, we systematically investigate the deformation mechanisms of cross-linked collagen fibrils. We find that cross-linked fibrils exhibit a three phase behavior, which agrees closer with experimental results than what was obtained using previous models. The fibril mechanical response is characterized by: (i) an initial elastic deformation corresponding to the collagen molecule uncoiling, (ii) a linear regime dominated by molecule sliding and (iii) the second stiffer elastic regime related to the stretching of the backbone of the tropocollagen molecules until the fibril ruptures. Our results suggest that both cross-link density and type dictate the stiffness of large deformation regime by increasing the number of interconnected molecules while cross-links mechanical properties determine the failure strain and strength of the fibril. These findings reveal that cross-links play an essential role in creating an interconnected fibrillar material of tunable toughness and strength. PMID:25153614

  20. Superficial Collagen Fibril Modulus and Pericellular Fixed Charge Density Modulate Chondrocyte Volumetric Behaviour in Early Osteoarthritis

    PubMed Central

    Turunen, Siru M.; Han, Sang Kuy; Herzog, Walter; Korhonen, Rami K.

    2013-01-01

    The aim of this study was to investigate if the experimentally detected altered chondrocyte volumetric behavior in early osteoarthritis can be explained by changes in the extracellular and pericellular matrix properties of cartilage. Based on our own experimental tests and the literature, the structural and mechanical parameters for normal and osteoarthritic cartilage were implemented into a multiscale fibril-reinforced poroelastic swelling model. Model simulations were compared with experimentally observed cell volume changes in mechanically loaded cartilage, obtained from anterior cruciate ligament transected rabbit knees. We found that the cell volume increased by 7% in the osteoarthritic cartilage model following mechanical loading of the tissue. In contrast, the cell volume decreased by 4% in normal cartilage model. These findings were consistent with the experimental results. Increased local transversal tissue strain due to the reduced collagen fibril stiffness accompanied with the reduced fixed charge density of the pericellular matrix could increase the cell volume up to 12%. These findings suggest that the increase in the cell volume in mechanically loaded osteoarthritic cartilage is primarily explained by the reduction in the pericellular fixed charge density, while the superficial collagen fibril stiffness is suggested to contribute secondarily to the cell volume behavior. PMID:23634175

  1. Guiding the orientation of smooth muscle cells on random and aligned polyurethane/collagen nanofibers.

    PubMed

    Jia, Lin; Prabhakaran, Molamma P; Qin, Xiaohong; Ramakrishna, Seeram

    2014-09-01

    Fabricating scaffolds that can simulate the architecture and functionality of native extracellular matrix is a huge challenge in vascular tissue engineering. Various kinds of materials are engineered via nano-technological approaches to meet the current challenges in vascular tissue regeneration. During this study, nanofibers from pure polyurethane and hybrid polyurethane/collagen in two different morphologies (random and aligned) and in three different ratios of polyurethane:collagen (75:25; 50:50; 25:75) are fabricated by electrospinning. The fiber diameters of the nanofibrous scaffolds are in the range of 174-453 nm and 145-419 for random and aligned fibers, respectively, where they closely mimic the nanoscale dimensions of native extracellular matrix. The aligned polyurethane/collagen nanofibers expressed anisotropic wettability with mechanical properties which is suitable for regeneration of the artery. After 12 days of human aortic smooth muscle cells culture on different scaffolds, the proliferation of smooth muscle cells on hybrid polyurethane/collagen (3:1) nanofibers was 173% and 212% higher than on pure polyurethane scaffolds for random and aligned scaffolds, respectively. The results of cell morphology and protein staining showed that the aligned polyurethane/collagen (3:1) scaffold promote smooth muscle cells alignment through contact guidance, while the random polyurethane/collagen (3:1) also guided cell orientation most probably due to the inherent biochemical composition. Our studies demonstrate the potential of aligned and random polyurethane/collagen (3:1) as promising substrates for vascular tissue regeneration.

  2. Influence of saline and pH on collagen type I fibrillogenesis in vitro: fibril polymorphism and colloidal gold labelling.

    PubMed

    Harris, J Robin; Reiber, Andreas

    2007-01-01

    We have produced different collagen type I fibrils by in vitro fibrillogenesis of acetic acid-soluble collagen within the pH range 2.5-9.0, in the presence and absence of 150 mM NaCl. The varying relatively stable molecular assemblies and polymorphic fibrillar end-products produced after 24 h incubation have been assessed and compared by the TEM study of specimens negatively stained with uranyl acetate. In the presence of 150 mM NaCl, the assembly of collagen at low pH (2.5) leads to the formation of initial molecular aggregates that progressively link together at slightly higher pH (5.0) to form sub-fibrils and spindle-shaped D-banded bundles of sub-fibrils. At pH 6.0 these D-banded bundles aggregate into larger spindle-shaped fibrils with lateral misalignment of the D-banding across the bundle. However, at pH 7.0 and 8.0, in the presence of 150 mM NaCl, the characteristic parallel-sided mature D-banded collagen type I fibres are formed. At pH 9.0 more loosely formed parallel-sided D-banded collagen fibrils are present, within which the spindle-shaped sub-fibrils can be defined by negative staining more convincingly than at pH 7-8. In the presence of 50 mM buffer at pH 2.5, but absence of 150 mM NaCl, collagen type I forms disorganized periodic initial molecular aggregates, which have a tendency to link together to form sub-fibrils. Flexuous collagen type I sub-fibrils predominate at pH 5.0, alongside large spindle-shaped fibrils that possess a regular transverse approximately 10 nm periodicity, with an oblique approximately 67 nm periodicity, significantly different to the D-banding periodicity. At pH 7.0 and pH 8 in the absence of saline loosely-formed flexuous and spindle-shaped fibres co-exist, with underlying sub-fibrils visible, but at pH 9.0 only disorganized flexuous fibrillar aggregates are present. Colloidal gold labelling of the characteristic D-banded collagen type I fibrils with 5 nm and 2 nm chemically reactive gold particles reveals a periodic

  3. Modelling the mechanics of partially mineralized collagen fibrils, fibres and tissue

    PubMed Central

    Liu, Yanxin; Thomopoulos, Stavros; Chen, Changqing; Birman, Victor; Buehler, Markus J.; Genin, Guy M.

    2014-01-01

    Progressive stiffening of collagen tissue by bioapatite mineral is important physiologically, but the details of this stiffening are uncertain. Unresolved questions about the details of the accommodation of bioapatite within and upon collagen's hierarchical structure have posed a central hurdle, but recent microscopy data resolve several major questions. These data suggest how collagen accommodates bioapatite at the lowest relevant hierarchical level (collagen fibrils), and suggest several possibilities for the progressive accommodation of bioapatite at higher hierarchical length scales (fibres and tissue). We developed approximations for the stiffening of collagen across spatial hierarchies based upon these data, and connected models across hierarchies levels to estimate mineralization-dependent tissue-level mechanics. In the five possible sequences of mineralization studied, percolation of the bioapatite phase proved to be an important determinant of the degree of stiffening by bioapatite. The models were applied to study one important instance of partially mineralized tissue, which occurs at the attachment of tendon to bone. All sequences of mineralization considered reproduced experimental observations of a region of tissue between tendon and bone that is more compliant than either tendon or bone, but the size and nature of this region depended strongly upon the sequence of mineralization. These models and observations have implications for engineered tissue scaffolds at the attachment of tendon to bone, bone development and graded biomimetic attachment of dissimilar hierarchical materials in general. PMID:24352669

  4. Modelling the mechanics of partially mineralized collagen fibrils, fibres and tissue.

    PubMed

    Liu, Yanxin; Thomopoulos, Stavros; Chen, Changqing; Birman, Victor; Buehler, Markus J; Genin, Guy M

    2014-03-01

    Progressive stiffening of collagen tissue by bioapatite mineral is important physiologically, but the details of this stiffening are uncertain. Unresolved questions about the details of the accommodation of bioapatite within and upon collagen's hierarchical structure have posed a central hurdle, but recent microscopy data resolve several major questions. These data suggest how collagen accommodates bioapatite at the lowest relevant hierarchical level (collagen fibrils), and suggest several possibilities for the progressive accommodation of bioapatite at higher hierarchical length scales (fibres and tissue). We developed approximations for the stiffening of collagen across spatial hierarchies based upon these data, and connected models across hierarchies levels to estimate mineralization-dependent tissue-level mechanics. In the five possible sequences of mineralization studied, percolation of the bioapatite phase proved to be an important determinant of the degree of stiffening by bioapatite. The models were applied to study one important instance of partially mineralized tissue, which occurs at the attachment of tendon to bone. All sequences of mineralization considered reproduced experimental observations of a region of tissue between tendon and bone that is more compliant than either tendon or bone, but the size and nature of this region depended strongly upon the sequence of mineralization. These models and observations have implications for engineered tissue scaffolds at the attachment of tendon to bone, bone development and graded biomimetic attachment of dissimilar hierarchical materials in general.

  5. Molecular and intermolecular effects in collagen fibril mechanics: a multiscale analytical model compared with atomistic and experimental studies.

    PubMed

    Marino, Michele

    2016-02-01

    Both atomistic and experimental studies reveal the dependence of collagen fibril mechanics on biochemical and biophysical features such as, for instance, cross-link density, water content and protein sequence. In order to move toward a multiscale structural description of biological tissues, a novel analytical model for collagen fibril mechanics is herein presented. The model is based on a multiscale approach that incorporates and couples: thermal fluctuations in collagen molecules; the uncoiling of collagen triple helix; the stretching of molecular backbone; the straightening of the telopeptide in which covalent cross-links form; slip-pulse mechanisms due to the rupture of intermolecular weak bonds; molecular interstrand delamination due to the rupture of intramolecular weak bonds; the rupture of covalent bonds within molecular strands. The effectiveness of the proposed approach is verified by comparison with available atomistic results and experimental data, highlighting the importance of cross-link density in tuning collagen fibril mechanics. The typical three-region shape and hysteresis behavior of fibril constitutive response, as well as the transition from a yielding-like to a brittle-like behavior, are recovered with a special insight on the underlying nanoscale mechanisms. The model is based on parameters with a clear biophysical and biochemical meaning, resulting in a promising tool for analyzing the effect of pathological or pharmacological-induced histochemical alterations on the functional mechanical response of collagenous tissues.

  6. Characterization of the viscoelastic behavior of a simplified collagen micro-fibril based on molecular dynamics simulations.

    PubMed

    Ghodsi, Hossein; Darvish, Kurosh

    2016-10-01

    Collagen fibril is a major component of connective tissues such as bone, tendon, blood vessels, and skin. The mechanical properties of this highly hierarchical structure are greatly influenced by the presence of covalent cross-links between individual collagen molecules. This study investigates the viscoelastic behavior of a collagen lysine-lysine cross-link based on creep simulations with applied forces in the range or 10 to 2000pN using steered molecular dynamics (SMD). The viscoelastic model of the cross-link was combined with a system composed by two segments of adjacent collagen molecules hence representing a reduced viscoelastic model for a simplified micro-fibril. It was found that the collagen micro-fibril assembly had a steady-state Young׳s modulus ranging from 2.24 to 3.27GPa, which is in agreement with reported experimental measurements. The propagation of longitudinal force wave along the molecule was implemented by adding a delay element to the model. The force wave speed was found to be correlated with the speed of one-dimensional elastic waves in rods. The presented reduced model with three degrees of freedom can serve as a building block for developing models of the next level of hierarchy, i.e., a collagen fibril. PMID:27341288

  7. Stiparin: a glycoprotein from sea cucumber dermis that aggregates collagen fibrils.

    PubMed

    Trotter, J A; Lyons-Levy, G; Luna, D; Koob, T J; Keene, D R; Atkinson, M A

    1996-07-01

    The interactions between collagen fibrils in many echinoderm connective tissues are rapidly altered by the secretions of resident neurosecretory cells. Recent evidence has suggested that a secreted protein is responsible for the interactions that lead to an increase in tissue stiffness (Trotter and Koob, 1995). Structurally intact collagen fibrils have been isolated from such a connective tissue- the dermis of the sea cucumber Cucumaria frondosa- and used in an assay in vitro to identify a protein that binds to them and causes them to aggregate. This protein has been purified by anion-exchange and molecular sieve chromatography. It is eluted from a MonoQ column at approximately 0.55 M NaCl. Its isoelectric point is 5.2. It elutes from a Superose-6 column in a position corresponding to a molecule with a Stokes radius of 11.5 nm. Its native molecular weight estimated from sedimentation equilibrium analysis under non-denaturing conditions is 375,000, and its monomer molecular weight, estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, is approximately 350,000. Sedimentation velocity measurements indicated for the native molecule a sedimentation coefficient of 11 x 10(-13)s, a diffusion coefficient of 3.274 x 10(-7) cm2s-1, and a frictional ratio of 1.95, which corresponds to a prolate ellipsoid of revolution with an axial ratio of 19. The highly asymmetric structure suggested by the above correlated well with the images obtained by transmission electron microscopy following rotary shadowing, which revealed a flexible structure approximately 125 nm long. Based on its ability to aggregate collagen fibrils, this protein has been named "stiparin," from the Latin stipare, "to pack together."

  8. Molecular properties and fibril ultrastructure of types II and XI collagens in cartilage of mice expressing exclusively the α1(IIA) collagen isoform.

    PubMed

    McAlinden, Audrey; Traeger, Geoffrey; Hansen, Uwe; Weis, Mary Ann; Ravindran, Soumya; Wirthlin, Louisa; Eyre, David R; Fernandes, Russell J

    2014-02-01

    Until now, no biological tools have been available to determine if a cross-linked collagen fibrillar network derived entirely from type IIA procollagen isoforms, can form in the extracellular matrix (ECM) of cartilage. Recently, homozygous knock-in transgenic mice (Col2a1(+ex2), ki/ki) were generated that exclusively express the IIA procollagen isoform during post-natal development while type IIB procollagen, normally present in the ECM of wild type mice, is absent. The difference between these Col2a1 isoforms is the inclusion (IIA) or exclusion (IIB) of exon 2 that is alternatively spliced in a developmentally regulated manner. Specifically, chondroprogenitor cells synthesize predominantly IIA mRNA isoforms while differentiated chondrocytes produce mainly IIB mRNA isoforms. Recent characterization of the Col2a1(+ex2) mice has surprisingly shown that disruption of alternative splicing does not affect overt cartilage formation. In the present study, biochemical analyses showed that type IIA collagen extracted from ki/ki mouse rib cartilage can form homopolymers that are stabilized predominantly by hydroxylysyl pyridinoline (HP) cross-links at levels that differed from wild type rib cartilage. The findings indicate that mature type II collagen derived exclusively from type IIA procollagen molecules can form hetero-fibrils with type XI collagen and contribute to cartilage structure and function. Heteropolymers with type XI collagen also formed. Electron microscopy revealed mainly thin type IIA collagen fibrils in ki/ki mouse rib cartilage. Immunoprecipitation and mass spectrometry of purified type XI collagen revealed a heterotrimeric molecular composition of α1(XI)α2(XI)α1(IIA) chains where the α1(IIA) chain is the IIA form of the α3(XI) chain. Since the N-propeptide of type XI collagen regulates type II collagen fibril diameter in cartilage, the retention of the exon 2-encoded IIA globular domain would structurally alter the N-propeptide of type XI collagen

  9. Pseudo-hyperelastic model of tendon hysteresis from adaptive recruitment of collagen type I fibrils.

    PubMed

    Ciarletta, Pasquale; Dario, Paolo; Micera, Silvestro

    2008-02-01

    Understanding the functional relationship between the viscoelasticity and the morphology of soft collagenous tissues is fundamental for many applications in bioengineering science. This work presents a pseudo-hyperelastic constitutive theory aiming at describing the time-dependant hysteretic response of tendons subjected to uniaxial tensile loads. A macroscopic tendon is modeled as a composite homogeneous tissue with the anisotropic reinforcement of collagen type I fibrils. The tissue microstructure is considered as an adaptive network of fibrillar units connected in temporary junctions. The processes of breakage and reformation of active fibrils are thermally activated, and are occurring at random times. An internal softening variable and a dissipation energy function account for the adaptive arrangement of the fibrillar network in the pseudo-hyperelastic model. Cyclic uniaxial tensile tests have been performed in vitro on porcine flexor digital tendons. The theoretical predictions fit accurately the experimental stress-strain data both for the loading and the unloading processes. The hysteresis behavior reflects the improvement in the efficiency and performance of the motion of the muscle-tendon unit at high strain rates. The results of the model demonstrate the microstructural importance of proteoglycans in determining the functional viscoelastic adaptability of the macroscopic tendon.

  10. A possible role of collagen fibrils in the process of calcification observed in the capsule of the pineal gland in aging rats.

    PubMed

    Humbert, W; Cuisinier, F; Voegel, J C; Pévet, P

    1997-06-01

    The relationship between collagen fibrils and calcified concretions exclusively appearing in the pineal gland of adult/aging rats has been investigated. Deposits of lanthanum, which replace calcium ions are distributed along collagen fibrils with a repeating period of about 70 nm. Calcium has been detected histochemically between collagen bundles surrounding extracellular concretions by means of the pyroantimonate method and by X-ray microanalysis. It is associated with phosphorus. The data presented here suggest that collagen fibrils are involved in the genesis and growth of extracellular concretions located in the connective tissue surrounding the pineal gland of aging rats. PMID:9134857

  11. Development of human corneal epithelium on organized fibrillated transparent collagen matrices synthesized at high concentration.

    PubMed

    Tidu, Aurélien; Ghoubay-Benallaoua, Djida; Lynch, Barbara; Haye, Bernard; Illoul, Corinne; Allain, Jean-Marc; Borderie, Vincent M; Mosser, Gervaise

    2015-08-01

    Several diseases can lead to opacification of cornea requiring transplantation of donor tissue to restore vision. In this context, transparent collagen I fibrillated matrices have been synthesized at 15, 30, 60 and 90 mg/mL. The matrices were evaluated for fibril organizations, transparency, mechanical properties and ability to support corneal epithelial cell culture. The best results were obtained with 90 mg/mL scaffolds. At this concentration, the fibril organization presented some similarities to that found in corneal stroma. Matrices had a mean Young's modulus of 570 kPa and acellular scaffolds had a transparency of 87% in the 380-780 nm wavelength range. Human corneal epithelial cells successfully colonized the surface of the scaffolds and generated an epithelium with characteristics of corneal epithelial cells (i.e. expression of cytokeratin 3 and presence of desmosomes) and maintenance of stemness during culture (i.e. expression of ΔNp63α and formation of holoclones in colony formation assay). Presence of cultured epithelium on the matrices was associated with increased transparency (89%).

  12. Investigation of ethanol infiltration into demineralized dentin collagen fibrils using molecular dynamics simulations.

    PubMed

    Jee, Sang Eun; Zhou, Jienfeng; Tan, Jianquo; Breschi, Lorenzo; Tay, Franklin R; Grégoire, Geneviève; Pashley, David H; Jang, Seung Soon

    2016-05-01

    The purpose of this study is to investigate the interaction of neat ethanol with bound and non-bound water in completely demineralized dentin that is fully hydrated, using molecular dynamics (MD) simulation method. The key to creating ideal resin-dentin bonds is the removal of residual free water layers and its replacement by ethanol solvent in which resin monomers are soluble, using the ethanol wet-bonding technique. The test null hypotheses were that ethanol cannot remove any collagen-bound water, and that ethanol cannot infiltrate into the spacing between collagen triple helix due to narrow interlayer spacing. Collagen fibrillar structures of overlap and gap regions were constructed by aligning the collagen triple helix of infinite length in hexagonal packing. Three layers of the water molecules were specified as the layers of 0.15-0.22nm, 0.22-0.43nm and 0.43-0.63nm from collagen atoms by investigating the water distribution surrounding collagen molecules. Our simulation results show that ethanol molecules infiltrated into the intermolecular spacing in the gap region, which increased due to the lateral shrinkage of the collagen structures in contact with ethanol solution, while there was no ethanol infiltration observed in the overlap region. Infiltrated ethanol molecules in the gap region removed residual water molecules via modifying mostly the third water layer (50% decrease), which would be considered as a loosely-bound water layer. The first and second hydration layers, which would be considered as tightly bound water layers, were not removed by the ethanol molecules, thus maintaining the helical structures of the collagen molecules. PMID:26969524

  13. Rapid biomimetic mineralization of collagen fibrils and combining with human umbilical cord mesenchymal stem cells for bone defects healing.

    PubMed

    Ye, Bihua; Luo, Xueshi; Li, Zhiwen; Zhuang, Caiping; Li, Lihua; Lu, Lu; Ding, Shan; Tian, Jinhuan; Zhou, Changren

    2016-11-01

    Collagen biomineralization is regulated by complicated interactions between the collagen matrix and non-collagenous extracellular proteins. Here, the use of sodium tripolyphosphate to simulate the templating functional motif of the C-terminal fragment of non-collagenous proteins is reported, and a low molecular weight polyacrylic acid served as a sequestration agent to stabilize amorphous calcium phosphate into nanoprecursors. Self-assembled collagen fibrils served as a fixed template for achieving rapid biomimetic mineralization in vitro. Results demonstrated that, during the mineralization process, intrafibrillar and extrafibrillar hydroxyapatite mineral with collagen fibrils formed and did so via bottom-up nanoparticle assembly based on the non-classical crystallization approach in the presence of these dual biomimetic functional analogues. In vitro human umbilical cord mesenchymal stem cell (hUCMSC) culture found that the mineralized scaffolds have a better cytocompatibility in terms of cell viability, adhesion, proliferation, and differentiation into osteoblasts. A rabbit femoral condyle defect model was established to confirm the ability of the n-HA/collagen scaffolds to facilitate bone regeneration and repair. The images of gross anatomy, MRI, CT and histomorphology taken 6 and 12weeks after surgery showed that the biomimetic mineralized collagen scaffolds with hUCMSCs can promote the healing speed of bone defects in vivo, and both of the scaffolds groups performing better than the bone defect control group. As new bone tissue formed, the scaffolds degraded and were gradually absorbed. All these results demonstrated that both of the scaffolds and cells have better histocompatibility. PMID:27523994

  14. Layered chitosan-collagen hydrogel/aligned PLLA nanofiber construct for flexor tendon regeneration.

    PubMed

    Deepthi, S; Nivedhitha Sundaram, M; Deepti Kadavan, J; Jayakumar, R

    2016-11-20

    The aim of our study was to develop a tendon construct of electrospun aligned poly (l-lactic acid) (PLLA) nanofibers, to mimic the aligned collagen fiber bundles and layering PLLA fibers with chitosan-collagen hydrogel, to mimic the glycosaminoglycans of sheath ECM for tendon regeneration. The hydrogel coated electrospun membrane was rolled and an outer coating of alginate gel was given to prevent peritendinous adhesion. The developed constructs were characterized by SEM, FT-IR and tensile testing. Protein adsorption studies showed lower protein adsorption on coated scaffolds compared to uncoated scaffolds. The samples were proven to be non-toxic to tenocytes. The chitosan-collagen/PLLA uncoated scaffolds and alginate gel coated chitosan-collagen/PLLA scaffolds showed good cell proliferation. The tenocytes showed good attachment and spreading on the scaffolds. This study indicated that the developed chitosan-collagen/PLLA/alginate scaffold would be suitable for flexor tendon regeneration. PMID:27561521

  15. Fabrication of fibrillized collagen microspheres with the microstructure resembling an extracellular matrix.

    PubMed

    Matsuhashi, Aki; Nam, Kwangwoo; Kimura, Tsuyoshi; Kishida, Akio

    2015-04-14

    Microspheres using artificial or natural materials have been widely applied in the field of tissue engineering and drug delivery systems. Collagen is being widely used for microspheres because of its abundancy in the extracellular matrix (ECM), and its good biocompatibility. The purpose of this study is to establish the appropriate condition for preparing collagen microspheres (CMS) and fibrillized collagen microspheres (fCMS) using water-in-oil (W/O) emulsion. Collagen can be tailored to mimic the native cell environment possessing a similar microstructure to that of the ECM by conditioning the aqueous solution. We focused on the preparation of stable and injectable CMS and fCMS which is stable and would promote the healing response. Controlling the interfacial properties of hydrophilic-lipophilic balance (HLB), we obtained CMS and fCMS with various sizes and various morphologies. The microsphere prepared with wetting agents showed good microsphere formation, but too low or too high HLB value caused low yield and uncontrollable size distribution. The change in the surfactant amount and the rotor speed also affected the formation of the CMS and fCMS, where the low surfactant amount and fast rotor speed produced smaller CMS and fCMS. In the case of fCMS, the presence of NaCl made it possible to prepare stable fCMS without using any cross-linker due to fibrillogenesis and gelling of collagen molecules. The microstructure of fCMS was similar to that of the native tissue indicating that the fCMS would replicate its function in vivo.

  16. Monitoring the effect of magnetically aligned collagen scaffolds on tendon tissue engineering by PSOCT

    NASA Astrophysics Data System (ADS)

    Yang, Ying; Ahearne, Mark; Wimpenny, Ian; Torbet, Jim

    2009-02-01

    As the repair of injured or degenerated tendon is often compromised by the shortage of suitable donor tissue, other procedures need to be developed. The application of a functional tissue engineered tendon could prove to be a promising alternative therapy. Due to their good biocompatibility, collagen hydrogel based scaffolds have been considered to be potentially suitable for engineering tendon tissue in vitro. One of the major limitations of collagen hydrogels for engineering tissues is the difficulty in controlling their architecture and collagen concentration which results in poor mechanical strength. This study aims to overcome these limitations by creating a highly biocompatible scaffold that is both mechanically robust and aligned. Collagen fibers were pre-aligned under a high magnetic field then concentrated using plastic compression. Primary tenocytes cultured from rats were seeded on the aligned scaffolds. Following a protocol in public domain, thick cultured collagen constructs were rolled up into a spiral after undergoing plastic compressed. Both a light microscopy and a polarization sensitive optical coherence tomography (PSOCT) with central beam at 1300 nm were used to monitor the birefringence in the constructs. Conventional light microscopy showed that the tenocytes aligned along the pre-organized collagen bundles in contrast to the random distributed observed on unaligned scaffolds. PSOCT only revealed weak birefringence from aligned but uncompressed constructs. However, PSOCT images showed contrast band structures in the spiral constructs which suggests that the birefringence signal depends on the density of aligned collagen fibers. The effect of aligned cells, neo-formed matrix and the plastic compression on the birefringence signals are discussed in this paper briefly.

  17. Guiding the orientation of smooth muscle cells on random and aligned polyurethane/collagen nanofibers.

    PubMed

    Jia, Lin; Prabhakaran, Molamma P; Qin, Xiaohong; Ramakrishna, Seeram

    2014-09-01

    Fabricating scaffolds that can simulate the architecture and functionality of native extracellular matrix is a huge challenge in vascular tissue engineering. Various kinds of materials are engineered via nano-technological approaches to meet the current challenges in vascular tissue regeneration. During this study, nanofibers from pure polyurethane and hybrid polyurethane/collagen in two different morphologies (random and aligned) and in three different ratios of polyurethane:collagen (75:25; 50:50; 25:75) are fabricated by electrospinning. The fiber diameters of the nanofibrous scaffolds are in the range of 174-453 nm and 145-419 for random and aligned fibers, respectively, where they closely mimic the nanoscale dimensions of native extracellular matrix. The aligned polyurethane/collagen nanofibers expressed anisotropic wettability with mechanical properties which is suitable for regeneration of the artery. After 12 days of human aortic smooth muscle cells culture on different scaffolds, the proliferation of smooth muscle cells on hybrid polyurethane/collagen (3:1) nanofibers was 173% and 212% higher than on pure polyurethane scaffolds for random and aligned scaffolds, respectively. The results of cell morphology and protein staining showed that the aligned polyurethane/collagen (3:1) scaffold promote smooth muscle cells alignment through contact guidance, while the random polyurethane/collagen (3:1) also guided cell orientation most probably due to the inherent biochemical composition. Our studies demonstrate the potential of aligned and random polyurethane/collagen (3:1) as promising substrates for vascular tissue regeneration. PMID:24682037

  18. Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins.

    PubMed

    Hsiao, Tony W; Tresco, Patrick A; Hlady, Vladimir

    2015-01-01

    To modulate the surface properties of collagen and subsequent cell-surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels.

  19. Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins.

    PubMed

    Hsiao, Tony W; Tresco, Patrick A; Hlady, Vladimir

    2015-01-01

    To modulate the surface properties of collagen and subsequent cell-surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels. PMID:25477179

  20. Purification, characterization and cloning of tensilin, the collagen-fibril binding and tissue-stiffening factor from Cucumaria frondosa dermis.

    PubMed

    Tipper, Jennifer P; Lyons-Levy, Gillian; Atkinson, Mark A L; Trotter, John A

    2002-12-01

    The inner dermis of the sea cucumber, Cucumaria frondosa, is a mutable collagenous tissue characterized by rapid and reversible changes in its mechanical properties regulated by one or more protein effectors that are released from neurosecretory cells. One such effector, tensilin, is a collagen-fibril binding protein, named for its ability to induce dermis stiffening. Tensilin was purified using an affinity column constructed from C. frondosa collagen-fibrils. The protein migrates as a single band on SDS-PAGE (Mr approximately 33 kDa) and has an isoelectric point of 5.8. Equilibrium sedimentation experiments suggest a molecular mass of approximately 28.5-29.4 kDa. Carbohydrate analysis of tensilin revealed no measurable sugar content. The molar amount of tensilin was determined to be 0.38% that of collagen and 47% that of stiparin, a constitutive matrix glycoprotein. A full-length cDNA clone for tensilin was obtained from a C. frondosa inner dermis cDNA expression library. Predicted properties derived from the deduced peptide sequence were in agreement with those of the native protein. A noted feature of tensilin's deduced peptide sequence, particularly in its N-terminal domain, is its homology to tissue inhibitor of metalloproteinases. Tensilin's C-terminal tail has no known homology to other proteins but contains a putative collagen-fibril binding site.

  1. Development of a reinforced electrochemically aligned collagen bioscaffold for tendon tissue engineering applications

    NASA Astrophysics Data System (ADS)

    Uquillas Paredes, Jorge Alfredo

    Type-I collagen is a promising biomaterial that can be used to synthesize bioscaffolds as a strategy to regenerate and repair damaged tendons. The existing in vitro prepared collagen bioscaffolds are in the form of gels, foams, or extruded fibers. These bioscaffolds readily present sites for attachment of biological factors and cells; however, they have extremely poor biomechanical properties in comparison to the properties of native tendons. The biomechanical function of type-I collagen bioscaffolds needs to be elevated to the level of natural tissues for this biomaterial to replace mechanically challenged tendons in a functionally meaningful way. The overall goal of this dissertation is to develop a reinforced electrochemically aligned collagenous bioscaffold for applications in tendon tissue engineering. The bioscaffold is synthesized by a unique electrochemical process via isoelectric focusing (IEF) to attain a very high degree of molecular alignment and packing density. This dissertation presents progress made on four aims: A) development of simple and descriptive electrochemical theory via the mathematical model of IEF and the forces acting on collagen alignment under an electric field; B) optimization of the post-alignment PBS treatment step to achieve d- banding pattern in uncrosslinked electrochemically aligned collagen (ELAC) bioscaffolds; C) optimization of the best crosslinking protocol to produce the strongest possible ELAC biomaterial with excellent cellular compatibility; and D) in vivo evaluation of the biocompatibility and biodegradability properties of electronically aligned collagen bioscaffolds. The results of this dissertation provide strong evidence showing that reinforced ELAC bioscaffolds could be used clinically in the future to repair damaged tendons.

  2. The organisation of collagen fibrils in the superficial zones of articular cartilage.

    PubMed Central

    Clark, J M

    1990-01-01

    The origin and structure of collagen fibres in the surface of articular cartilage were studied using SEM. Cryofracture was used to create orthogonal fracture surfaces in three planes. Fibres which originated in the radial zone could be traced into the surface where they flattened and overlapped in a common direction. Thick fibres from the periosteum ran into the surface as well, but apparently ended there and did not enter the radial zone. The tangential fibres were covered by a dense, separate layer of small fibrils. The fundamental aspects of the model proposed by Benninghoff are supported by these findings. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:2081698

  3. The process of EDC-NHS Cross-linking of reconstituted collagen fibres increases collagen fibrillar order and alignment

    PubMed Central

    Shepherd, D.V.; Shepherd, J.H.; Ghose, S.; Kew, S.J.; Cameron, R.E.; Best, S.M.

    2014-01-01

    We describe the production of collagen fibre bundles through a multi-strand, semi-continuous extrusion process. Cross-linking using an EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), NHS (N-hydroxysuccinimide) combination was considered. Atomic Force Microscopy (AFM) and Raman spectroscopy focused on how cross-linking affected the collagen fibrillar structure. In the cross-linked fibres, a clear fibrillar structure comparable to native collagen was observed which was not observed in the non-cross-linked fibre. The amide III doublet in the Raman spectra provided additional evidence of alignment in the cross-linked fibres. Raman spectroscopy also indicated no residual polyethylene glycol (from the fibre forming buffer) or water in any of the fibres. PMID:25506518

  4. The process of EDC-NHS cross-linking of reconstituted collagen fibres increases collagen fibrillar order and alignment

    SciTech Connect

    Shepherd, D. V. Shepherd, J. H.; Cameron, R. E.; Best, S. M.; Ghose, S.; Kew, S. J.

    2015-01-01

    We describe the production of collagen fibre bundles through a multi-strand, semi-continuous extrusion process. Cross-linking using an EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), NHS (N-hydroxysuccinimide) combination was considered. Atomic Force Microscopy and Raman spectroscopy focused on how cross-linking affected the collagen fibrillar structure. In the cross-linked fibres, a clear fibrillar structure comparable to native collagen was observed which was not observed in the non-cross-linked fibre. The amide III doublet in the Raman spectra provided additional evidence of alignment in the cross-linked fibres. Raman spectroscopy also indicated no residual polyethylene glycol (from the fibre forming buffer) or water in any of the fibres.

  5. Quantification of Interfibrillar Shear Stress in Aligned Soft Collagenous Tissues via Notch Tension Testing

    NASA Astrophysics Data System (ADS)

    Szczesny, Spencer E.; Caplan, Jeffrey L.; Pedersen, Pal; Elliott, Dawn M.

    2015-10-01

    The mechanical function of soft collagenous tissues is largely determined by their hierarchical organization of collagen molecules. While collagen fibrils are believed to be discontinuous and transfer load through shearing of the interfibrillar matrix, interfibrillar shear stresses have never been quantified. Scaling traditional shear testing procedures down to the fibrillar length scale is impractical and would introduce substantial artifacts. Here, through the use of a novel microscopic variation of notch tension testing, we explicitly demonstrate the existence of interfibrillar shear stresses within tendon fascicles and provide the first measurement of their magnitude. Axial stress gradients along the sample length generated by notch tension testing were measured and used to calculate a value of 32 kPa for the interfibrillar shear stress. This estimate is comparable to the interfibrillar shear stress predicted by previous multiscale modeling of tendon fascicles, which supports the hypothesis that fibrils are discontinuous and transmit load through interfibrillar shear. This information regarding the structure-function relationships of tendon and other soft collagenous tissues is necessary to identify potential causes for tissue impairment with degeneration and provide the foundation for developing regenerative repair strategies or engineering biomaterials for tissue replacement.

  6. The nano-morphological relationships between apatite crystals and collagen fibrils in ivory dentine.

    PubMed

    Jantou-Morris, V; Horton, Michael A; McComb, David W

    2010-07-01

    In this work, analytical transmission electron microscopy (TEM) was used to study the nanostructure of mineralised ivory dentine, in order to gain a clearer understanding of the relationship between the organic (collagen fibrils) and inorganic (calcium phosphate apatite crystals) components. Thin sections prepared by both focused ion beam (FIB) milling and ultramicrotomy, in the longitudinal and transverse planes, were investigated using electron energy-loss spectroscopy (EELS) in a monochromated field-emission gun scanning TEM (FEI Titan 80-300 FEGSTEM). Both low- and core-loss spectroscopy were used in the investigation, and the signals from phosphorous, carbon, calcium, nitrogen and oxygen were studied in detail. A combination of HAADF (high-angle annular dark-field)-STEM imaging and EELS analysis was used for simultaneous acquisition of both spatial and spectral information pixel by pixel (spectrum imaging). Across the collagen D banding in longitudinal sections, the relative thickness of the bright bands was significantly higher than that of the dark bands. Core-loss spectroscopy showed that the bright bands were richer in apatite than the dark bands. However, no ELNES variation was observed across the D banding. In transverse sections, significant changes in the carbon edge fine structure were observed at the interface between the extra- and intra-fibrillar regions.

  7. Interleukin-4 Receptor α Signaling in Myeloid Cells Controls Collagen Fibril Assembly in Skin Repair.

    PubMed

    Knipper, Johanna A; Willenborg, Sebastian; Brinckmann, Jürgen; Bloch, Wilhelm; Maaß, Tobias; Wagener, Raimund; Krieg, Thomas; Sutherland, Tara; Munitz, Ariel; Rothenberg, Marc E; Niehoff, Anja; Richardson, Rebecca; Hammerschmidt, Matthias; Allen, Judith E; Eming, Sabine A

    2015-10-20

    Activation of the immune response during injury is a critical early event that determines whether the outcome of tissue restoration is regeneration or replacement of the damaged tissue with a scar. The mechanisms by which immune signals control these fundamentally different regenerative pathways are largely unknown. We have demonstrated that, during skin repair in mice, interleukin-4 receptor α (IL-4Rα)-dependent macrophage activation controlled collagen fibril assembly and that this process was important for effective repair while having adverse pro-fibrotic effects. We identified Relm-α as one important player in the pathway from IL-4Rα signaling in macrophages to the induction of lysyl hydroxylase 2 (LH2), an enzyme that directs persistent pro-fibrotic collagen cross-links, in fibroblasts. Notably, Relm-β induced LH2 in human fibroblasts, and expression of both factors was increased in lipodermatosclerosis, a condition of excessive human skin fibrosis. Collectively, our findings provide mechanistic insights into the link between type 2 immunity and initiation of pro-fibrotic pathways. PMID:26474656

  8. Collagen fibril orientation in ovine and bovine leather affects strength: a small angle X-ray scattering (SAXS) study.

    PubMed

    Basil-Jones, Melissa M; Edmonds, Richard L; Cooper, Sue M; Haverkamp, Richard G

    2011-09-28

    There is a large difference in strength between ovine and bovine leather. The structure and arrangement of fibrous collagen in leather and the relationship between collagen structure and leather strength has until now been poorly understood. Synchrotron based SAXS is used to characterize the fibrous collagen structure in a series of ovine and bovine leathers and to relate it to tear strength. SAXS gives quantitative information on the amount of fibrous collagen, the orientation (direction and spread) of the collagen microfibrils, and the d-spacing of the collagen. The amount of collagen varies through the thickness of the leather from the grain to the corium, with a greater concentration of crystalline collagen measured toward the corium side. The orientation index (OI) is correlated strongly with strength in ovine leather and between ovine and bovine leathers. Stronger leather has the fibrils arranged mostly parallel to the plane of the leather surface (high OI), while weaker leather has more out-of-plane fibrils (low OI). With the measurement taken parallel to the animal's backbone, weak (19.9 N/mm) ovine leather has an OI of 0.422 (0.033), stronger (39.5 N/mm) ovine leather has an OI of 0.452 (0.033), and bovine leather with a strength of (61.5 N/mm) has an OI of 0.493 (0.016). The d-spacing profile through leather thickness also varies according to leather strength, with little variation being detected in weak ovine leather (average=64.3 (0.5) nm), but with strong ovine leather and bovine leather (which is even stronger) exhibiting a dip in d-spacing (from 64.5 nm at the edges dropping to 62 nm in the center). This work provides a clear understanding of a nanostructural characteristic of ovine and bovine leather that leads to differences in strength.

  9. Recombinant human-like collagen directed growth of hydroxyapatite nanocrystals

    NASA Astrophysics Data System (ADS)

    Zhai, Y.; Cui, F. Z.

    2006-05-01

    Bones are biocomposites with hierarchical structure that require controlled mineral deposition during their self-assembly to form tissues with unique mechanical properties. Type I collagen proteins, acidic extracellular matrix proteins, play a critical role in mineral formation and many researches on artificial bones have been made inspired by nature using type I collagen derived from animal tissues. Here we report that recombinant human-like type I collagen, an acidic protein, can direct growth of hydroxyapatite (HA) nanocrystals in vitro in the form of self-assembly of nano-fibrils of mineralized collagen resembling extracellular matrix. The mineralized collagen fibrils aligned parallel to each other to form mineralized collagen fibers. HA nanocrystals grew on the surface of these collagen fibrils with the c-axis of nanocrystals of HA orienting along the longitudinal axis of the fibrils. These artificial analogs of bone have a potential clinical application in bone repair.

  10. Optimising contraction and alignment of cellular collagen hydrogels to achieve reliable and consistent engineered anisotropic tissue.

    PubMed

    O'Rourke, Caitriona; Drake, Rosemary A L; Cameron, Grant W W; Loughlin, A Jane; Phillips, James B

    2015-11-01

    Engineered anisotropic tissue constructs containing aligned cell and extracellular matrix structures are useful as in vitro models and for regenerative medicine. They are of particular interest for nervous system modelling and regeneration, where tracts of aligned neurons and glia are required. The self-alignment of cells and matrix due to tension within tethered collagen gels is a useful tool for generating anisotropic tissues, but requires an optimal balance between cell density, matrix concentration and time to be achieved for each specific cell type. The aim of this study was to develop an assay system based on contraction of free-floating cellular gels in 96-well plates that could be used to investigate cell-matrix interactions and to establish optimal parameters for subsequent self-alignment of cells in tethered gels. Using C6 glioma cells, the relationship between contraction and alignment was established, with 60-80% contraction in the 96-well plate assay corresponding to alignment throughout tethered gels made using the same parameters. The assay system was used to investigate the effect of C6 cell density, collagen concentration and time. It was also used to show that blocking α1 integrin reduced the contraction and self-alignment of these cells, whereas blocking α2 integrin had little effect. The approach was validated by using primary astrocytes in the assay system under culture conditions that modified their ability to contract collagen gels. This detailed investigation describes a robust assay for optimising cellular self-alignment and provides a useful reference framework for future development of self-aligned artificial tissue.

  11. Real-time high-resolution measurement of collagen alignment in dynamically loaded soft tissue.

    PubMed

    York, Timothy; Kahan, Lindsey; Lake, Spencer P; Gruev, Viktor

    2014-06-01

    A technique for creating maps of the direction and strength of fiber alignment in collagenous soft tissues is presented. The method uses a division of focal plane polarimeter to measure circularly polarized light transmitted through the tissue. The architecture of the sensor allows measurement of the retardance and fiber alignment at the full frame rate of the sensor without any moving optics. The technique compares favorably to the standard method of using a rotating polarizer. How the new technique enables real-time capture of the full angular spread of fiber alignment and retardance under various cyclic loading conditions is illustrated. PMID:24972359

  12. Nano measurements with micro-devices: mechanical properties of hydrated collagen fibrils

    PubMed Central

    Eppell, S.J; Smith, B.N; Kahn, H; Ballarini, R

    2005-01-01

    The mechanical response of a biological material to applied forces reflects deformation mechanisms occurring within a hierarchical architecture extending over several distinct length scales. Characterizing and in turn predicting the behaviour of such a material requires an understanding of the mechanical properties of the substructures within the hierarchy, the interaction between the substructures, and the relative influence of each substructure on the overall behaviour. While significant progress has been made in mechanical testing of micrometre to millimetre sized biological specimens, quantitative reproducible experimental techniques for making mechanical measurements on specimens with characteristic dimensions in the smaller range of 10–1000 nm are lacking. Filling this void in experimentation is a necessary step towards the development of realistic multiscale computational models useful to predict and mitigate the risk of bone fracture, design improved synthetic replacements for bones, tendons and ligaments, and engineer bioinspired efficient and environmentally friendly structures. Here, we describe a microelectromechanical systems device for directly measuring the tensile strength, stiffness and fatigue behaviour of nanoscale fibres. We used the device to obtain the first stress–strain curve of an isolated collagen fibril producing the modulus and some fatigue properties of this soft nanofibril. PMID:16849223

  13. Ultrastructure Organization of Collagen Fibrils and Proteoglycans of Stingray and Shark Corneal Stroma

    PubMed Central

    Alanazi, Saud A.; Almubrad, Turki; AlIbrahim, Ahmad I. A.; Khan, Adnan A.; Akhtar, Saeed

    2015-01-01

    We report here the ultrastructural organization of collagen fibrils (CF) and proteoglycans (PGs) of the corneal stroma of both the stingray and the shark. Three corneas from three stingrays and three corneas from three sharks were processed for electron microscopy. Tissues were embedded in TAAB 031 resin. The corneal stroma of both the stingray and shark consisted of parallel running lamellae of CFs which were decorated with PGs. In the stingray, the mean area of PGs in the posterior stroma was significantly larger than the PGs of the anterior and middle stroma, whereas, in the shark, the mean area of PGs was similar throughout the stroma. The mean area of PGs of the stingray was significantly larger compared to the PGs, mean area of the shark corneal stroma. The CF diameter of the stingray was significantly smaller compared to the CF diameter in the shark. The ultrastructural features of the corneal stroma of both the stingray and the shark were similar to each other except for the CFs and PGs. The PGs in the stingray and shark might be composed of chondroitin sulfate (CS)/dermatan sulfate (DS) PGs and these PGs with sutures might contribute to the nonswelling properties of the cornea of the stingray and shark. PMID:26167294

  14. Ultrastructure Organization of Collagen Fibrils and Proteoglycans of Stingray and Shark Corneal Stroma.

    PubMed

    Alanazi, Saud A; Almubrad, Turki; AlIbrahim, Ahmad I A; Khan, Adnan A; Akhtar, Saeed

    2015-01-01

    We report here the ultrastructural organization of collagen fibrils (CF) and proteoglycans (PGs) of the corneal stroma of both the stingray and the shark. Three corneas from three stingrays and three corneas from three sharks were processed for electron microscopy. Tissues were embedded in TAAB 031 resin. The corneal stroma of both the stingray and shark consisted of parallel running lamellae of CFs which were decorated with PGs. In the stingray, the mean area of PGs in the posterior stroma was significantly larger than the PGs of the anterior and middle stroma, whereas, in the shark, the mean area of PGs was similar throughout the stroma. The mean area of PGs of the stingray was significantly larger compared to the PGs, mean area of the shark corneal stroma. The CF diameter of the stingray was significantly smaller compared to the CF diameter in the shark. The ultrastructural features of the corneal stroma of both the stingray and the shark were similar to each other except for the CFs and PGs. The PGs in the stingray and shark might be composed of chondroitin sulfate (CS)/dermatan sulfate (DS) PGs and these PGs with sutures might contribute to the nonswelling properties of the cornea of the stingray and shark. PMID:26167294

  15. Langevin dynamics modeling of the water diffusion tensor in partially aligned collagen networks

    NASA Astrophysics Data System (ADS)

    Powell, Sean K.; Momot, Konstantin I.

    2012-09-01

    In this work, a Langevin dynamics model of the diffusion of water in articular cartilage was developed. Numerical simulations of the translational dynamics of water molecules and their interaction with collagen fibers were used to study the quantitative relationship between the organization of the collagen fiber network and the diffusion tensor of water in model cartilage. Langevin dynamics was used to simulate water diffusion in both ordered and partially disordered cartilage models. In addition, an analytical approach was developed to estimate the diffusion tensor for a network comprising a given distribution of fiber orientations. The key findings are that (1) an approximately linear relationship was observed between collagen volume fraction and the fractional anisotropy of the diffusion tensor in fiber networks of a given degree of alignment, (2) for any given fiber volume fraction, fractional anisotropy follows a fiber alignment dependency similar to the square of the second Legendre polynomial of cos(θ), with the minimum anisotropy occurring at approximately the magic angle (θMA), and (3) a decrease in the principal eigenvalue and an increase in the transverse eigenvalues is observed as the fiber orientation angle θ progresses from 0∘ to 90∘. The corresponding diffusion ellipsoids are prolate for θ<θMA, spherical for θ≈θMA, and oblate for θ>θMA. Expansion of the model to include discrimination between the combined effects of alignment disorder and collagen fiber volume fraction on the diffusion tensor is discussed.

  16. Changes in collagen fibril network organization and proteoglycan distribution in equine articular cartilage during maturation and growth.

    PubMed

    Hyttinen, Mika M; Holopainen, Jaakko; van Weeren, P René; Firth, Elwyn C; Helminen, Heikki J; Brama, Pieter A J

    2009-11-01

    The aim of this study was to record growth-related changes in collagen network organization and proteoglycan distribution in intermittently peak-loaded and continuously lower-level-loaded articular cartilage. Cartilage from the proximal phalangeal bone of the equine metacarpophalangeal joint at birth, at 5, 11 and 18 months, and at 6-10 years of age was collected from two sites. Site 1, at the joint margin, is unloaded at slow gaits but is subjected to high-intensity loading during athletic activity; site 2 is a continuously but less intensively loaded site in the centre of the joint. The degree of collagen parallelism was determined with quantitative polarized light microscopy and the parallelism index for collagen fibrils was computed from the cartilage surface to the osteochondral junction. Concurrent changes in the proteoglycan distribution were quantified with digital densitometry. We found that the parallelism index increased significantly with age (up to 90%). At birth, site 2 exhibited a more organized collagen network than site 1. In adult horses this situation was reversed. The superficial and intermediate zones exhibited the greatest reorganization of collagen. Site 1 had a higher proteoglycan content than site 2 at birth but here too the situation was reversed in adult horses. We conclude that large changes in joint loading during growth and maturation in the period from birth to adulthood profoundly affect the architecture of the collagen network in equine cartilage. In addition, the distribution and content of proteoglycans are modified significantly by altered joint use. Intermittent peak-loading with shear seems to induce higher collagen parallelism and a lower proteoglycan content in cartilage than more constant weight-bearing. Therefore, we hypothesize that the formation of mature articular cartilage with a highly parallel collagen network and relatively low proteoglycan content in the peak-loaded area of a joint is needed to withstand

  17. Articular cartilage superficial zone collagen birefringence reduced and cartilage thickness increased before surface fibrillation in experimental osteoarthritis

    PubMed Central

    Panula, H.; Hyttinen, M.; Arokoski, J.; Langsjo, T.; Pelttari, A.; Kiviranta, I.; Helminen, H.

    1998-01-01

    OBJECTIVES—To investigate articular cartilage collagen network, thickness of birefringent cartilage zones, and glycosaminoglycan concentration in macroscopically normal looking knee joint cartilage of young beagles subjected to experimental slowly progressive osteoarthritis (OA).
METHODS—OA was induced by a tibial 30° valgus osteotomy in 15 female beagles at the age of 3 months. Fifteen sisters were controls. Cartilage specimens were collected seven (Group 1) and 18 months (Group 2) postoperatively. Collagen induced optical path difference and cartilage zone thickness measurements were determined from histological sections of articular cartilage with smooth and intact surface by computer assisted quantitative polarised light microscopy. Volume density of cartilage collagen fibrils was determined by image analysis from transmission electron micrographs and content of glycosaminoglycans by quantitative digital densitometry from histological sections.
Results—In the superficial zone of the lateral tibial and femoral cartilage, the collagen induced optical path difference (birefringence) decreased by 19 to 71% (p < 0.05) seven months postoperatively. This suggests that severe superficial collagen fibril network deterioration took place, as 18 months postoperatively, macroscopic and microscopic OA was present in many cartilage areas. Thickness of the uncalcified cartilage increased while the superficial zone became thinner in the same sites. In operated dogs, glycosaminoglycan content first increased (Group 1) in the lateral tibial condyle and then decreased (Group 2) (p < 0.05).
Conclusion—In this OA model, derangement of the superficial zone collagen network was the probable reason for birefringence reduction. This change occurred well before macroscopic OA.

 Keywords: cartilage; birefringence PMID:9709181

  18. Quantitative mapping of collagen fiber alignment in thick tissue samples using transmission polarized-light microscopy.

    PubMed

    Yakovlev, Dmitry D; Shvachkina, Marina E; Sherman, Maria M; Spivak, Andrey V; Pravdin, Alexander B; Yakovlev, Dmitry A

    2016-07-01

    Immersion optical clearing makes it possible to use transmission polarized-light microscopy for characterization of thick (200 to 2000  μm) layers of biological tissues. We discuss polarization properties of thick samples in the context of the problem of characterization of collagen fiber alignment in connective tissues such as sclera and dermis. Optical chirality caused by azimuthal variations of the macroscopic (effective) optic axis of the medium across the sample thickness should be considered in polarization mapping of thick samples of these tissues. We experimentally evaluate to what extent the optical chirality affects the measurement results in typical situations and show under what conditions it can be easily taken into account and does not hinder, but rather helps, in characterization of collagen fiber alignment.

  19. Quantitative mapping of collagen fiber alignment in thick tissue samples using transmission polarized-light microscopy

    NASA Astrophysics Data System (ADS)

    Yakovlev, Dmitry D.; Shvachkina, Marina E.; Sherman, Maria M.; Spivak, Andrey V.; Pravdin, Alexander B.; Yakovlev, Dmitry A.

    2016-07-01

    Immersion optical clearing makes it possible to use transmission polarized-light microscopy for characterization of thick (200 to 2000 μm) layers of biological tissues. We discuss polarization properties of thick samples in the context of the problem of characterization of collagen fiber alignment in connective tissues such as sclera and dermis. Optical chirality caused by azimuthal variations of the macroscopic (effective) optic axis of the medium across the sample thickness should be considered in polarization mapping of thick samples of these tissues. We experimentally evaluate to what extent the optical chirality affects the measurement results in typical situations and show under what conditions it can be easily taken into account and does not hinder, but rather helps, in characterization of collagen fiber alignment.

  20. Tendon and ligament fibrillar crimps give rise to left-handed helices of collagen fibrils in both planar and helical crimps.

    PubMed

    Franchi, Marco; Ottani, Vittoria; Stagni, Rita; Ruggeri, Alessandro

    2010-03-01

    Collagen fibres in tendons and ligaments run straight but in some regions they show crimps which disappear or appear more flattened during the initial elongation of tissues. Each crimp is formed of collagen fibrils showing knots or fibrillar crimps at the crimp top angle. The present study analyzes by polarized light microscopy, scanning electron microscopy, transmission electron microscopy the 3D morphology of fibrillar crimp in tendons and ligaments of rat demonstrating that each fibril in the fibrillar region always twists leftwards changing the plane of running and sharply bends modifying the course on a new plane. The morphology of fibrillar crimp in stretched tendons fulfills the mechanical role of the fibrillar crimp acting as a particular knot/biological hinge in absorbing tension forces during fibril strengthening and recoiling collagen fibres when stretching is removed. The left-handed path of fibrils in the fibrillar crimp region gives rise to left-handed fibril helices observed both in isolated fibrils and sections of different tendons and ligaments (flexor digitorum profundus muscle tendon, Achilles tendon, tail tendon, patellar ligament and medial collateral ligament of the knee). The left-handed path of fibrils represents a new final suprafibrillar level of the alternating handedness which was previously described only from the molecular to the microfibrillar level. When the width of the twisting angle in the fibrillar crimp is nearly 180 degrees the fibrils appear as left-handed flattened helices forming crimped collagen fibres previously described as planar crimps. When fibrils twist with different subsequent rotational angles (< 180 degrees ) they always assume a left-helical course but, running in many different nonplanar planes, they form wider helical crimped fibres.

  1. SU-E-J-107: Supervised Learning Model of Aligned Collagen for Human Breast Carcinoma Prognosis

    SciTech Connect

    Bredfeldt, J; Liu, Y; Conklin, M; Keely, P; Eliceiri, K; Mackie, T

    2014-06-01

    Purpose: Our goal is to develop and apply a set of optical and computational tools to enable large-scale investigations of the interaction between collagen and tumor cells. Methods: We have built a novel imaging system for automating the capture of whole-slide second harmonic generation (SHG) images of collagen in registry with bright field (BF) images of hematoxylin and eosin stained tissue. To analyze our images, we have integrated a suite of supervised learning tools that semi-automatically model and score collagen interactions with tumor cells via a variety of metrics, a method we call Electronic Tumor Associated Collagen Signatures (eTACS). This group of tools first segments regions of epithelial cells and collagen fibers from BF and SHG images respectively. We then associate fibers with groups of epithelial cells and finally compute features based on the angle of interaction and density of the collagen surrounding the epithelial cell clusters. These features are then processed with a support vector machine to separate cancer patients into high and low risk groups. Results: We validated our model by showing that eTACS produces classifications that have statistically significant correlation with manual classifications. In addition, our system generated classification scores that accurately predicted breast cancer patient survival in a cohort of 196 patients. Feature rank analysis revealed that TACS positive fibers are more well aligned with each other, generally lower density, and terminate within or near groups of epithelial cells. Conclusion: We are working to apply our model to predict survival in larger cohorts of breast cancer patients with a diversity of breast cancer types, predict response to treatments such as COX2 inhibitors, and to study collagen architecture changes in other cancer types. In the future, our system may be used to provide metastatic potential information to cancer patients to augment existing clinical assays.

  2. The effect of ultrasonic irrigation before and after citric acid treatment on collagen fibril exposure: an in vitro SEM study.

    PubMed

    Higashi, T; Okamoto, H

    1995-10-01

    The surface characteristics of periodontally diseased human teeth after two treatments were compared both before and after partial demineralization with citric acid. Thirteen teeth were obtained from patients with advanced periodontal disease. Three teeth were selected for control groups and 10 were used for experimental groups. All diseased root surfaces were identified and outlined. The roots were cut longitudinally into two sections. They were then scaled and root planed and the paired sections were separately classified into two control or two experimental groups. Three sections in control group 1 were rinsed by syringe with saline solution. The three sections in control group 2 were treated with ultrasonic irrigation. The 10 sections in experimental group 1 were rinsed by syringe with saline solution before and after citric acid application; the 10 sections in experimental group 2 were irrigated ultrasonically before and after citric acid application. The concentration of the citric acid was 25% (pH 1.62) and the immersion time was 3 minutes. The root samples were examined by scanning electron microscope. A significant amount of grinding debris covered on all the root surfaces in control group 1, whereas smear was removed in control group 2. The features of root surfaces of the two experimental groups differed considerably. All specimens in experimental group 2 exhibited collagen fibrils exposed as a consequence of citric acid etching. On the other hand, the smear layer was not thoroughly removed from the root surface in experimental group 1, which meant that few collagen fibrils were exposed after partial demineralization. From these results, ultrasonic irrigation before and after citric acid application improves exposure of collagen fibrils, which may be desirable for clinical success in periodontal regenerative therapy.

  3. Anisotropic collagen fibrillogenesis within microfabricated scaffolds: implications for biomimetic tissue engineering.

    PubMed

    Jean, Aurélie; Engelmayr, George C

    2012-01-11

    Anisotropic collagen fibrillogenesis is demonstrated within the pores of an accordion-like honeycomb poly(glycerol sebacate) tissue engineering scaffold. Confocal reflectance microscopy and image analysis demonstrate increased fibril distribution order, fibril density, and alignment in accordion-like honeycomb pores compared with collagen gelled unconstrained. Finite element modeling predicts how collagen gel and scaffold mechanics couple in matching native heart muscle stiffness and anisotropy. PMID:23184695

  4. Dynamics of Structural Parameters and Accumulation of Collagen Fibrils in Rat Lung after Inhalations of Surfactant-BL at Various Terms of Bleomycin-Induced Alveolitis.

    PubMed

    Volchkov, V A; Dubrovskaya, V F; Valkovich, A A; Klestova, O V; Serzhanina, V A; Zhuikov, A G; Seiliev, A A; Rosenberg, O A

    2016-08-01

    Rats were subjected to surfactant-BL inhalations at the early and late phases of bleomycininduced alveolitis. In both regimens, the drug reduced the severity of inflammation. In the acute phase of alveolitis, the therapeutic effect of inhalation was accompanied by activation of the synthesis of fine lose collagen fibrils. In the late phase of alveolitis, inhalation of surfactant-BL thickened the fibrils and diminished their population in alveolar walls. PMID:27591866

  5. Softenin, a novel protein that softens the connective tissue of sea cucumbers through inhibiting interaction between collagen fibrils.

    PubMed

    Takehana, Yasuhiro; Yamada, Akira; Tamori, Masaki; Motokawa, Tatsuo

    2014-01-01

    The dermis in the holothurian body wall is a typical catch connective tissue or mutable collagenous tissue that shows rapid changes in stiffness. Some chemical factors that change the stiffness of the tissue were found in previous studies, but the molecular mechanisms of the changes are not yet fully understood. Detection of factors that change the stiffness by working directly on the extracellular matrix was vital to clarify the mechanisms of the change. We isolated from the body wall of the sea cucumber Stichopus chloronotus a novel protein, softenin, that softened the body-wall dermis. The apparent molecular mass was 20 kDa. The N-terminal sequence of 17 amino acids had low homology to that of known proteins. We performed sequential chemical and physical dissections of the dermis and tested the effects of softenin on each dissection stage by dynamic mechanical tests. Softenin softened Triton-treated dermis whose cells had been disrupted by detergent. The Triton-treated dermis was subjected to repetitive freeze-and-thawing to make Triton-Freeze-Thaw (TFT) dermis that was softer than the Triton-treated dermis, implying that some force-bearing structure had been disrupted by this treatment. TFT dermis was stiffened by tensilin, a stiffening protein of sea cucumbers. Softenin softened the tensilin-stiffened TFT dermis while it had no effect on the TFT dermis without tensilin treatment. We isolated collagen from the dermis. When tensilin was applied to the suspending solution of collagen fibrils, they made a large compact aggregate that was dissolved by the application of softenin or by repetitive freeze-and-thawing. These results strongly suggested that softenin decreased dermal stiffness through inhibiting cross-bridge formation between collagen fibrils; the formation was augmented by tensilin and the bridges were broken by the freeze-thaw treatment. Softenin is thus the first softener of catch connective tissue shown to work on the cross-bridges between

  6. Softenin, a Novel Protein That Softens the Connective Tissue of Sea Cucumbers through Inhibiting Interaction between Collagen Fibrils

    PubMed Central

    Takehana, Yasuhiro; Yamada, Akira; Tamori, Masaki; Motokawa, Tatsuo

    2014-01-01

    The dermis in the holothurian body wall is a typical catch connective tissue or mutable collagenous tissue that shows rapid changes in stiffness. Some chemical factors that change the stiffness of the tissue were found in previous studies, but the molecular mechanisms of the changes are not yet fully understood. Detection of factors that change the stiffness by working directly on the extracellular matrix was vital to clarify the mechanisms of the change. We isolated from the body wall of the sea cucumber Stichopus chloronotus a novel protein, softenin, that softened the body-wall dermis. The apparent molecular mass was 20 kDa. The N-terminal sequence of 17 amino acids had low homology to that of known proteins. We performed sequential chemical and physical dissections of the dermis and tested the effects of softenin on each dissection stage by dynamic mechanical tests. Softenin softened Triton-treated dermis whose cells had been disrupted by detergent. The Triton-treated dermis was subjected to repetitive freeze-and-thawing to make Triton-Freeze-Thaw (TFT) dermis that was softer than the Triton-treated dermis, implying that some force-bearing structure had been disrupted by this treatment. TFT dermis was stiffened by tensilin, a stiffening protein of sea cucumbers. Softenin softened the tensilin-stiffened TFT dermis while it had no effect on the TFT dermis without tensilin treatment. We isolated collagen from the dermis. When tensilin was applied to the suspending solution of collagen fibrils, they made a large compact aggregate that was dissolved by the application of softenin or by repetitive freeze-and-thawing. These results strongly suggested that softenin decreased dermal stiffness through inhibiting cross-bridge formation between collagen fibrils; the formation was augmented by tensilin and the bridges were broken by the freeze-thaw treatment. Softenin is thus the first softener of catch connective tissue shown to work on the cross-bridges between

  7. Three-dimensional imaging of collagen fibril organization in rat circumferential lamellar bone using a dual beam electron microscope reveals ordered and disordered sub-lamellar structures.

    PubMed

    Reznikov, Natalie; Almany-Magal, Rotem; Shahar, Ron; Weiner, Steve

    2013-02-01

    Lamellar bone is a major component of most mammalian skeletons. A prominent component of individual lamellae are parallel arrays of mineralized type I collagen fibrils, organized in a plywood like motif. Here we use a dual beam microscope and the serial surface view (SSV) method to investigate the three dimensional collagen organization of circumferential lamellar bone from rat tibiae after demineralization and osmium staining. Fast Fourier transform analysis is used to quantitatively identify the mean collagen array orientations and local collagen fibril dispersion. Based on collagen fibril array orientations and variations in fibril dispersion, we identify 3 distinct sub-lamellar structural motifs: a plywood-like fanning sub-lamella, a unidirectional sub-lamella and a disordered sub-lamella. We also show that the disordered sub-lamella is less mineralized than the other sub-lamellae. The hubs and junctions of the canalicular network, which connect radially oriented canaliculi, are intimately associated with the disordered sub-lamella. We also note considerable variations in the proportions of these 3 sub-lamellar structural elements among different lamellae. This new application of Serial Surface View opens the way to quantitatively compare lamellar bone from different sources, and to clarify the 3-dimensional structures of other bone types, as well as other biological structural materials. PMID:23153959

  8. Manipulation of in vitro collagen matrix architecture for scaffolds of improved physiological relevance

    NASA Astrophysics Data System (ADS)

    Hapach, Lauren A.; VanderBurgh, Jacob A.; Miller, Joseph P.; Reinhart-King, Cynthia A.

    2015-12-01

    Type I collagen is a versatile biomaterial that is widely used in medical applications due to its weak antigenicity, robust biocompatibility, and its ability to be modified for a wide array of applications. As such, collagen has become a major component of many tissue engineering scaffolds, drug delivery platforms, and substrates for in vitro cell culture. In these applications, collagen constructs are fabricated to recapitulate a diverse set of conditions. Collagen fibrils can be aligned during or post-fabrication, cross-linked via numerous techniques, polymerized to create various fibril sizes and densities, and copolymerized into a wide array of composite scaffolds. Here, we review approaches that have been used to tune collagen to better recapitulate physiological environments for use in tissue engineering applications and studies of basic cell behavior. We discuss techniques to control fibril alignment, methods for cross-linking collagen constructs to modulate stiffness, and composite collagen constructs to better mimic physiological extracellular matrix.

  9. Manipulation of in vitro collagen matrix architecture for scaffolds of improved physiological relevance.

    PubMed

    Hapach, Lauren A; VanderBurgh, Jacob A; Miller, Joseph P; Reinhart-King, Cynthia A

    2015-01-01

    Type I collagen is a versatile biomaterial that is widely used in medical applications due to its weak antigenicity, robust biocompatibility, and its ability to be modified for a wide array of applications. As such, collagen has become a major component of many tissue engineering scaffolds, drug delivery platforms, and substrates for in vitro cell culture. In these applications, collagen constructs are fabricated to recapitulate a diverse set of conditions. Collagen fibrils can be aligned during or post-fabrication, cross-linked via numerous techniques, polymerized to create various fibril sizes and densities, and copolymerized into a wide array of composite scaffolds. Here, we review approaches that have been used to tune collagen to better recapitulate physiological environments for use in tissue engineering applications and studies of basic cell behavior. We discuss techniques to control fibril alignment, methods for cross-linking collagen constructs to modulate stiffness, and composite collagen constructs to better mimic physiological extracellular matrix. PMID:26689380

  10. Structural constraints on the evolution of the collagen fibril: convergence on a 1014-residue COL domain.

    PubMed

    Slatter, David Anthony; Farndale, Richard William

    2015-05-01

    Type I collagen is the fundamental component of the extracellular matrix. Its α1 gene is the direct descendant of ancestral fibrillar collagen and contains 57 exons encoding the rod-like triple-helical COL domain. We trace the evolution of the COL domain from a primordial collagen 18 residues in length to its present 1014 residues, the limit of its possible length. In order to maintain and improve the essential structural features of collagen during evolution, exons can be added or extended only in permitted, non-random increments that preserve the position of spatially sensitive cross-linkage sites. Such sites cannot be maintained unless the twist of the triple helix is close to 30 amino acids per turn. Inspection of the gene structure of other long structural proteins, fibronectin and titin, suggests that their evolution might have been subject to similar constraints.

  11. Age-associated reduction of cellular spreading/mechanical force up-regulates matrix metalloproteinase-1 expression and collagen fibril fragmentation via c-Jun/AP-1 in human dermal fibroblasts.

    PubMed

    Qin, Zhaoping; Voorhees, John J; Fisher, Gary J; Quan, Taihao

    2014-12-01

    The dermal compartment of human skin is largely composed of dense collagen-rich fibrils, which provide structural and mechanical support. Skin dermal fibroblasts, the major collagen-producing cells, are interact with collagen fibrils to maintain cell spreading and mechanical force for function. A characteristic feature of aged human skin is fragmentation of collagen fibrils, which is initiated by matrix metalloproteinase 1 (MMP-1). Fragmentation impairs fibroblast attachment and thereby reduces spreading. Here, we investigated the relationship among fibroblast spreading, mechanical force, MMP-1 expression, and collagen fibril fragmentation. Reduced fibroblast spreading due to cytoskeletal disruption was associated with reduced cellular mechanical force, as determined by atomic force microscopy. These reductions substantially induced MMP-1 expression, which led to collagen fibril fragmentation and disorganization in three-dimensional collagen lattices. Constraining fibroblast size by culturing on slides coated with collagen micropatterns also significantly induced MMP-1 expression. Reduced spreading/mechanical force induced transcription factor c-Jun and its binding to a canonical AP-1 binding site in the MMP-1 proximal promoter. Blocking c-Jun function with dominant negative mutant c-Jun significantly reduced induction of MMP-1 expression in response to reduced spreading/mechanical force. Furthermore, restoration of fibroblast spreading/mechanical force led to decline of c-Jun and MMP-1 levels and eliminated collagen fibril fragmentation and disorganization. These data reveal a novel mechanism by which alteration of fibroblast shape/mechanical force regulates c-Jun/AP-1-dependent expression of MMP-1 and consequent collagen fibril fragmentation. This mechanism provides a foundation for understanding the cellular and molecular basis of age-related collagen fragmentation in human skin.

  12. Effects of Phosphate Buffered Saline Concentration and Incubation Time on the Mechanical and Structural Properties of Electrochemically Aligned Collagen Threads

    PubMed Central

    Uquillas, Jorge Alfredo; Kishore, Vipuil; Akkus, Ozan

    2011-01-01

    A key step during the synthesis of collagen constructs is the incubation of monomeric collagen in phosphate buffer saline (PBS) to promote fibrillogenesis in the collagen network. Optimal PBS treatment conditions for monomeric collagen solutions to induce gelation are well established in the literature. Recently, a report in the literature[1] showed a novel method to fabricate highly oriented electrochemically aligned collagen (ELAC) threads which have orders of magnitude greater packing density than collagen gels. The optimal PBS treatment conditions for induction of D-banding pattern in such dense and anisotropic collagen network are unknown. This study aimed to optimize PBS treatment of ELAC threads by investigating the effect of phosphate ion concentration (0.5×, 1×, 5× or 10×) and incubation time (3, 12 or 96 hours) on the mechanical strength and ultrastructural organization by monotonic mechanical testing, small angle X-ray scattering and transmission electron microscopy. ELAC threads incubated in water (No PBS) served as the control. ELAC threads incubated in 1× PBS showed significantly higher extensibility compared to 0.5× or 10× PBS along with the presence of D-banded patterns with a periodicity of 63.83 nm. Incubation of ELAC threads in 1× PBS for 96 hours resulted in significantly higher ultimate stress compared to 3 or 12 hours. However, these threads lacked D-banding pattern. TEM showed no significant differences in the microfibril diameter distribution of ELAC threads treated with or without PBS. This indicates that microfibrils lacked D-banding following electrochemical alignment and the subsequent PBS treatment induced D-banding by reorganization within microfibrils. It was concluded that incubation of aligned collagen in 1× PBS for 12 hours results in mechanically competent, D-banded ELAC threads which can be used for the regeneration of load bearing tissues such as tendons and ligaments. PMID:21540522

  13. A Novel 3D Fibril Force Assay Implicates Src in Tumor Cell Force Generation in Collagen Networks

    PubMed Central

    Polackwich, Robert J.; Koch, Daniel; Arevalo, Richard; Miermont, Anne M.; Jee, Kathleen J.; Lazar, John; Urbach, Jeffrey; Mueller, Susette C.; McAllister, Ryan G.

    2013-01-01

    New insight into the biomechanics of cancer cell motility in 3D extracellular matrix (ECM) environments would significantly enhance our understanding of aggressive cancers and help identify new targets for intervention. While several methods for measuring the forces involved in cell-matrix interactions have been developed, previous to this study none have been able to measure forces in a fibrillar environment. We have developed a novel assay for simultaneously measuring cell mechanotransduction and motility in 3D fibrillar environments. The assay consists of a controlled-density fibrillar collagen gel atop a controlled-stiffness polyacrylamide (PAA) surface. Forces generated by living cells and their migration in the 3D collagen gel were measured with the 3D motion of tracer beads within the PAA layer. Here, this 3D fibril force assay is used to study the role of the invasion-associated protein kinase Src in mechanotransduction and motility. Src expression and activation are linked with proliferation, invasion, and metastasis, and have been shown to be required in 2D for invadopodia membranes to direct and mediate invasion. Breast cancer cell line MDA-MD-231 was stably transfected with GFP-tagged constitutively active Src or wild-type Src. In 3D fibrillar collagen matrices we found that, relative to wild-type Src, constitutively active Src: 1) increased the strength of cell-induced forces on the ECM, 2) did not significantly change migration speed, and 3) increased both the duration and the length, but not the number, of long membrane protrusions. Taken together, these results support the hypothesis that Src controls invasion by controlling the ability of the cell to form long lasting cellular protrusions to enable penetration through tissue barriers, in addition to its role in promoting invadopodia matrix-degrading activity. PMID:23536784

  14. DYNAMIC SHEAR-INFLUENCED COLLAGEN SELF-ASSEMBLY

    PubMed Central

    Saeidi, Nima; Sander, Edward A.

    2011-01-01

    The ability to influence the direction of polymerization of a self-assembling biomolecular system has the potential to generate materials with extremely high anisotropy. In biological systems where highly-oriented cellular populations give rise to aligned and often load-bearing tissue such organized molecular scaffolds could aid in the contact guidance of cells for engineered tissue constructs (e.g cornea and tendon). In this investigation we examine the detailed dynamics of pepsin-extracted type I bovine collagen assembly on a glass surface under the influence of flow between two plates. Differential Interference Contrast (DIC) imaging (60x-1.4NA) with focal plane stabilization was used to resolve and track the growth of collagen aggregates on borosilicate glass for 4 different shear rates (500, 80, 20, and 9 s-1). The detailed morphology of the collagen fibrils/aggregates was examined using Quick Freeze Deep Etch electron microscopy. Nucleation of fibrils on the glass was observed to occur rapidly (~2 min) followed by continued growth of the fibrils. The growth rates were dependent on flow in a complex manner with the highest rate of axial growth (0.1 microns/sec) occurring at a shear rate of 9 s-1. The lowest growth rate occurred at the highest shear. Fibrils were observed to both branch and join during the experiments. The best alignment of fibrils was observed at intermediate shear rates of 20 and 80s-1. However, the investigation revealed that fibril directional growth was not stable. At high shear rates, fibrils would often turn downstream forming what we term “hooks” which are likely the combined result of monomer interaction with the initial collagen layer or “mat” and the high shear rate. Further, QFDE examination of fibril morphology demonstrated that the assembled fibrillar structure did not possess native D-periodicity. Instead, fibrils comprised a collection of generally aligned, monomers which were self-assembled to form a fibril

  15. Mineralization of collagen may occur on fibril surfaces: evidence from conventional and high-voltage electron microscopy and three-dimensional imaging

    NASA Technical Reports Server (NTRS)

    Landis, W. J.; Hodgens, K. J.; Song, M. J.; Arena, J.; Kiyonaga, S.; Marko, M.; Owen, C.; McEwen, B. F.

    1996-01-01

    The interaction between collagen and mineral crystals in the normally calcifying leg tendons from the domestic turkey, Meleagris gallopavo, has been investigated at an ultrastructural level with conventional and high-voltage electron microscopy, computed tomography, and three-dimensional image reconstruction methods. Specimens treated by either aqueous or anhydrous techniques and resin-embedded were appropriately sectioned and regions of early tendon mineralization were photographed. On the basis of individual photomicrographs, stereoscopic pairs of images, and tomographic three-dimensional image reconstructions, platelet-shaped crystals may be demonstrated for the first time in association with the surface of collagen fibrils. Mineral is also observed in closely parallel arrays within collagen hole and overlap zones. The mineral deposition at these spatially distinct locations in the tendon provides insight into possible means by which calcification is mediated by collagen as a fundamental event in skeletal and dental formation among vertebrates.

  16. Quantification of Collagen Organization in the Peripheral Human Cornea at Micron-Scale Resolution

    PubMed Central

    Boote, Craig; Kamma-Lorger, Christina S.; Hayes, Sally; Harris, Jonathan; Burghammer, Manfred; Hiller, Jennifer; Terrill, Nicholas J.; Meek, Keith M.

    2011-01-01

    The collagen microstructure of the peripheral cornea is important in stabilizing corneal curvature and refractive status. However, the manner in which the predominantly orthogonal collagen fibrils of the central cornea integrate with the circumferential limbal collagen is unknown. We used microfocus wide-angle x-ray scattering to quantify the relative proportion and orientation of collagen fibrils over the human corneolimbal interface at intervals of 50 μm. Orthogonal fibrils changed direction 1–1.5 mm before the limbus to integrate with the circumferential limbal fibrils. Outside the central 6 mm, additional preferentially aligned collagen was found to reinforce the cornea and limbus. The manner of integration and degree of reinforcement varied significantly depending on the direction along which the limbus was approached. We also employed small-angle x-ray scattering to measure the average collagen fibril diameter from central cornea to limbus at 0.5 mm intervals. Fibril diameter was constant across the central 6 mm. More peripherally, fibril diameter increased, indicative of a merging of corneal and scleral collagen. The point of increase varied with direction, consistent with a scheme in which the oblique corneal periphery is reinforced by chords of scleral collagen. The results have implications for the cornea's biomechanical response to ocular surgeries involving peripheral incision. PMID:21723812

  17. Collagen biogenesis and assembly into fibrils as shown by ultrastructural and 3H-proline radioautographic studies on the fibroblasts of the rat food pad

    SciTech Connect

    Marchi, F.; Leblond, C.P.

    1983-10-01

    To examine whether collagen is assembled into fibrils within or outside fibroblasts, the connective tissue of the rat foot pad was investigated by electron microscopy and by radioautography at times varying from 4 min to 3 days after an intravenous injection of 3H-proline. The fibroblasts of the rat food pad are long polarized cells with the nucleus at one end, the Golgi apparatus in the center, and a region with long processes at the other end. This region contains secretory granules and is considered to be the secretory pole of the cell. In the Golgi apparatus the stacks of saccules are separated from rough endoplasmic reticulum (rER) by groups of ''intermediate vesicles'' including similarly structured tubules which may be over 300 nm long and are referred to as ''intermediate tubules.'' The Golgi saccules exhibit distended portions which differ at the various levels of the stack. Finally, the fibroblasts are associated with two types of collagen fibrils: extracellular ones arranged into large groups between the cells and intracellular ones located within long intracytoplasmic channels. Quantitative radioautography after 3H-proline injection reveals that the number of silver grains per unit area reaches a peak over the rER at 4-10 min, Golgi apparatus at 40 min, secretory granules at 60 min, and extracellular collagen fibrils at 3 h. At no time are intracellular collagen fibrils labeled. Qualitative observations further indicate that spherical Golgi distentions are mainly labeled at 40 min, and cylindrical distentions, at 60 min. In addition, from 20 min to 3 hr, some lysosomal elements are labeled.

  18. Genetic linkage of type VII collagen (COL7A1) to dominant dystrophic epidermolysis bullosa in families with abnormal anchoring fibrils.

    PubMed Central

    Ryynänen, M; Ryynänen, J; Sollberg, S; Iozzo, R V; Knowlton, R G; Uitto, J

    1992-01-01

    Epidermolysis bullosa (EB) in a group of genodermatoses characterized by the fragility of skin. Previous studies on the dystrophic (scarring) forms of EB have suggested abnormalities in anchoring fibrils, morphologically recognizable attachment structures that provide stability to the association of the cutaneous basement membrane to the underlying dermis. Since type VII collagen is the major component of the anchoring fibrils, we examined the genetic linkage of dominant dystrophic EB (EBDD) and the type VII collagen gene (COL7A1) locus, which we have recently mapped to chromosome 3p, in three large kindreds with abnormal anchoring fibrils. Strong genetic linkage of EBDD and COL7A1 loci was demonstrated with the maximum logarithm of odds (LOD) score of 8.77 at theta = 0. This linkage was further confirmed with two additional markers in this region of the short arm of chromosome 3, and these analyses allowed further refinement of the map locus of COL7A1. Since there were no recombinants between the COL7A1 and EBDD loci, our findings suggest that type VII collagen is the candidate gene that may harbor the mutations responsible for the EB phenotype in these three families. Images PMID:1347297

  19. Aligned nanofibrillar collagen scaffolds - Guiding lymphangiogenesis for treatment of acquired lymphedema.

    PubMed

    Hadamitzky, Catarina; Zaitseva, Tatiana S; Bazalova-Carter, Magdalena; Paukshto, Michael V; Hou, Luqia; Strassberg, Zachary; Ferguson, James; Matsuura, Yuka; Dash, Rajesh; Yang, Phillip C; Kretchetov, Shura; Vogt, Peter M; Rockson, Stanley G; Cooke, John P; Huang, Ngan F

    2016-09-01

    Secondary lymphedema is a common disorder associated with acquired functional impairment of the lymphatic system. The goal of this study was to evaluate the therapeutic efficacy of aligned nanofibrillar collagen scaffolds (BioBridge) positioned across the area of lymphatic obstruction in guiding lymphatic regeneration. In a porcine model of acquired lymphedema, animals were treated with BioBridge scaffolds, alone or in conjunction with autologous lymph node transfer as a source of endogenous lymphatic growth factor. They were compared with a surgical control group and a second control group in which the implanted BioBridge was supplemented with exogenous vascular endothelial growth factor-C (VEGF-C). Three months after implantation, immunofluorescence staining of lymphatic vessels demonstrated a significant increase in lymphatic collectors within close proximity to the scaffolds. To quantify the functional impact of scaffold implantation, bioimpedance was used as an early indicator of extracellular fluid accumulation. In comparison to the levels prior to implantation, the bioimpedance ratio was significantly improved only in the experimental BioBridge recipients with or without lymph node transfer, suggesting restoration of functional lymphatic drainage. These results further correlated with quantifiable lymphatic collectors, as visualized by contrast-enhanced computed tomography. They demonstrate the therapeutic potential of BioBridge scaffolds in secondary lymphedema. PMID:27348849

  20. Influence of collagen source on fibrillar architecture and properties of vitrified collagen membranes.

    PubMed

    Majumdar, Shoumyo; Guo, Qiongyu; Garza-Madrid, Marcos; Calderon-Colon, Xiomara; Duan, Derek; Carbajal, Priscilla; Schein, Oliver; Trexler, Morgana; Elisseeff, Jennifer

    2016-02-01

    Collagen vitrigel membranes are transparent biomaterials characterized by a densely organized, fibrillar nanostructure that show promise in the treatment of corneal injury and disease. In this study, the influence of different type I collagen sources and processing techniques, including acid-solubilized collagen from bovine dermis (Bov), pepsin-solubilized collagen from human fibroblast cell culture (HuCC), and ficin-solubilized collagen from recombinant human collagen expressed in tobacco leaves (rH), on the properties of the vitrigel membranes was evaluated. Postvitrification carbodiimide crosslinking (CX) was also carried out on the vitrigels from each collagen source, forming crosslinked counterparts BovXL, HuCCXL, and rHXL, respectively. Collagen membrane ultrastructure and biomaterial properties were found to rely heavily on both collagen source and crosslinking. Bov and HuCC samples showed a random fibrillar organization of collagen, whereas rH vitrigels showed remarkable regional fibril alignment. After CX, light transmission was enhanced in all groups. Denaturation temperatures after CX increased in all membranes, of which the highest increase was seen in rH (14.71°C), suggesting improved thermal stability of the collagen fibrils in the membranes. Noncrosslinked rH vitrigels may be reinforced through CX to reach levels of mechanical strength and thermal stability comparable to Bov.

  1. Influence of Cyclic Mechanical Stretch and Tissue Constraints on Cellular and Collagen Alignment in Fibroblast-Derived Cell Sheets

    PubMed Central

    Weidenhamer, Nathan K.

    2013-01-01

    Mechanical forces play an important role in shaping the organization of the extracellular matrix (ECM) in developing and mature tissues. The resulting organization gives the tissue its unique functional properties. Understanding how mechanical forces influence the alignment of the ECM is important in tissue engineering, where recapitulating the alignment of the native tissue is essential for appropriate mechanical anisotropy. In this work, a novel method was developed to create and stretch tubular cell sheets by seeding neonatal dermal fibroblasts onto a rotating silicone tube. We show the fibroblasts proliferated to create a confluent monolayer around the tube and a collagenous, isotropic tubular tissue over 4 weeks of static culture. These silicone tubes with overlying tubular tissue constructs were mounted into a cyclic distension bioreactor and subjected to cyclic circumferential stretch at 5% strain, 0.5 Hz for 3 weeks. We found that the tissue subjected to cyclic stretch compacted axially over the silicone tube in comparison to static controls, leading to a circumferentially aligned tissue with higher membrane stiffness and maximum tension. In a subsequent study, the tissue constructs were constrained against axial compaction during cyclic stretching. The resulting alignment of fibroblasts and collagen was perpendicular (axial) to the stretch direction (circumferential). When the cells were devitalized with sodium azide before stretching, similarly constrained tissue did not develop strong axial alignment. This work suggests that both mechanical stretching and mechanical constraints are important in determining tissue organization, and that this organization is dependent on an intact cytoskeleton. PMID:23126441

  2. Accurate Determination of Interstrand Distances and Alignment in Amyloid Fibrils by Magic Angle Spinning NMR

    PubMed Central

    Caporini, Marc A.; Bajaj, Vikram S.; Veshtort, Mikhail; Fitzpatrick, Anthony; MacPhee, Cait E; Vendruscolo, Michele; Dobson, Christopher M.; Griffin, Robert G.

    2010-01-01

    Amyloid fibrils are structurally ordered aggregates of proteins whose formation is associated with many neurodegenerative and other diseases. For that reason, their high resolution structures are of considerable interest and have been studied using a wide range of techniques, notably electron microscopy, x-ray diffraction, and magic angle spinning (MAS) NMR. Because of the excellent resolution in the spectra, MAS NMR is uniquely capable of delivering site-specific, atomic resolution information about all levels of amyloid structure: (1) the monomer, which packs into several (2) protofilaments that in turn associate to form a (3) fibril. Building upon our high resolution structure of the monomer of an amyloid-forming peptide from transthyretin (TTR105-115), we introduce single 1-13C labeled amino acids at seven different sites in the peptide and measure intermolecular carbonyl-carbonyl distances with an accuracy of ~0.11 A. Our results conclusively establish a parallel, in register, topology for the packing of this peptide into a β-sheet and provide constraints essential for the determination of an atomic resolution structure of the fibril. Furthermore, the approach we employ, based on a combination of a double-quantum filtered variant of the DRAWS recoupling sequence and multispin numerical simulations in SPINEVOLUTION, is general and should be applicable to a wide range of systems. PMID:20925357

  3. Epitaxially guided assembly of collagen layers on mica surfaces.

    PubMed

    Leow, Wee Wen; Hwang, Wonmuk

    2011-09-01

    Ordered assembly of collagen molecules on flat substrates has potential for various applications and serves as a model system for studying the assembly process. While previous studies demonstrated self-assembly of collagen on muscovite mica into highly ordered layers, the mechanism by which different conditions affect the resulting morphology remains to be elucidated. Using atomic force microscopy, we follow the assembly of collagen on muscovite mica at a concentration lower than the critical fibrillogenesis concentration in bulk. Initially, individual collagen molecules adsorb to mica and subsequently nucleate into fibrils possessing the 67 nm D-periodic bands. Emergence of fibrils aligned in parallel despite large interfibril distances agrees with an alignment mechanism guided by the underlying mica. The epitaxial growth was further confirmed by the formation of novel triangular networks of collagen fibrils on phlogopite mica, whose surface lattice is known to have a hexagonal symmetry, whereas the more widely used muscovite does not. Comparing collagen assembly on the two types of mica at different potassium concentrations revealed that potassium binds to the negatively charged mica surface and neutralizes it, thereby reducing the binding affinity of collagen and enhancing surface diffusion. These results suggest that collagen assembly on mica follows the surface adsorption, diffusion, nucleation, and growth pathway, where the growth direction is determined at the nucleation step. Comparison with other molecules that assemble similarly on mica supports generality of the proposed assembly mechanism, the knowledge of which will be useful for controlling the resulting surface morphologies.

  4. Location of 3-hydroxyproline residues in collagen types I, II, III, and V/XI implies a role in fibril supramolecular assembly.

    PubMed

    Weis, Mary Ann; Hudson, David M; Kim, Lammy; Scott, Melissa; Wu, Jiann-Jiu; Eyre, David R

    2010-01-22

    Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro(986) in A-clade chains alpha1(I), alpha1(II), and alpha2(V). Two partially modified sites (A2 and A3) were found at Pro(944) in alpha1(II) and alpha2(V) and Pro(707) in alpha2(I) and alpha2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro(470) in alpha2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian alpha1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures.

  5. Quantitative measurement of the distribution and alignment of collagen fibers in unfixed aortic tissues.

    PubMed

    Sugita, Shukei; Matsumoto, Takeo

    2013-04-26

    Determination of the local amount and direction of collagen fibers during deformation is crucial for an understanding of the mechanical behavior of aortic tissues. Since most conventional methods cannot be used for this purpose, we propose a method to quantify the local amount and direction of fibers by simply measuring the optical properties of the specimen. After confirming the linear correlation between the retardance and thickness of sections of porcine thoracic aortas (PTAs) ranging from 15 to 300 μm, we investigated the effects of their structural components, i.e., smooth muscle cells (SMCs), elastin and collagen, on the retardance of whole tissues. Decellularization of SMCs did not change the retardance of PTA sections significantly. Patterns in autofluorescent and immunofluorescent images of elastin purified from bovine nuchal ligaments did not match those in retardance images. Images of collagen in PTA sections stained with picrosirius red were similar to corresponding retardance images. The slow axis azimuth corresponded to the circumferential direction of the aorta. Results indicate that collagen in aortas can be quantified by measuring the retardance and slow axis azimuth of whole aortic tissues. Application of this technique to PTAs showed that retardance was higher in dorsal and distal regions than ventral and proximal regions, respectively, indicating that the aortas contain more collagen in distal and dorsal regions than proximal and ventral regions, respectively. Both results were in accordance with previous findings. Measurement of retardance is useful to quantify the amount of collagen in unfixed aortas.

  6. Molecular Crowding of Collagen: A Pathway to Produce Highly-Organized Collagenous Structures

    PubMed Central

    Saeidi, Nima; Karmelek, Kathryn N.; Paten, Jeffrey. A; Zareian, Ramin; DiMasi, Elaine

    2013-01-01

    Collagen in vertebrate animals is often arranged in alternating lamellae or in bundles of aligned fibrils which are designed to withstand in vivo mechanical loads. The formation of these organized structures is thought to result from a complex, large-area integration of individual cell motion and locally-controlled synthesis of fibrillar arrays via cell-surface fibripositors (direct matrix printing). The difficulty of reproducing such a process in vitro has prevented tissue engineers from constructing clinically useful load-bearing connective tissue directly from collagen. However, we and others have taken the view that long-range organizational information is potentially encoded into the structure of the collagen molecule itself, allowing the control of fibril organization to extend far from cell (or bounding) surfaces. We here demonstrate a simple, fast, cell-free method capable of producing highly-organized, anistropic collagen fibrillar lamellae de novo which persist over relatively long-distances (tens to hundreds of microns). Our approach to nanoscale organizational control takes advantage of the intrinsic physiochemical properties of collagen molecules by inducing collagen association through molecular crowding and geometric confinement. To mimic biological tissues which comprise planar, aligned collagen lamellae (e.g. cornea, lamellar bone or annulus fibrosus), type I collagen was confined to a thin, planar geometry, concentrated through molecular crowding and polymerized. The resulting fibrillar lamellae show a striking resemblance to native load-bearing lamellae in that the fibrils are small, generally aligned in the plane of the confining space and change direction en masse throughout the thickness of the construct. The process of organizational control is consistent with embryonic development where the bounded planar cell sheets produced by fibroblasts suggest a similar confinement/concentration strategy. Such a simple approach to nanoscale

  7. Molecular crowding of collagen: a pathway to produce highly-organized collagenous structures.

    PubMed

    Saeidi, Nima; Karmelek, Kathryn P; Paten, Jeffrey A; Zareian, Ramin; DiMasi, Elaine; Ruberti, Jeffrey W

    2012-10-01

    Collagen in vertebrate animals is often arranged in alternating lamellae or in bundles of aligned fibrils which are designed to withstand in vivo mechanical loads. The formation of these organized structures is thought to result from a complex, large-area integration of individual cell motion and locally-controlled synthesis of fibrillar arrays via cell-surface fibripositors (direct matrix printing). The difficulty of reproducing such a process in vitro has prevented tissue engineers from constructing clinically useful load-bearing connective tissue directly from collagen. However, we and others have taken the view that long-range organizational information is potentially encoded into the structure of the collagen molecule itself, allowing the control of fibril organization to extend far from cell (or bounding) surfaces. We here demonstrate a simple, fast, cell-free method capable of producing highly-organized, anistropic collagen fibrillar lamellae de novo which persist over relatively long-distances (tens to hundreds of microns). Our approach to nanoscale organizational control takes advantage of the intrinsic physiochemical properties of collagen molecules by inducing collagen association through molecular crowding and geometric confinement. To mimic biological tissues which comprise planar, aligned collagen lamellae (e.g. cornea, lamellar bone or annulus fibrosus), type I collagen was confined to a thin, planar geometry, concentrated through molecular crowding and polymerized. The resulting fibrillar lamellae show a striking resemblance to native load-bearing lamellae in that the fibrils are small, generally aligned in the plane of the confining space and change direction en masse throughout the thickness of the construct. The process of organizational control is consistent with embryonic development where the bounded planar cell sheets produced by fibroblasts suggest a similar confinement/concentration strategy. Such a simple approach to nanoscale

  8. Mineralisation of reconstituted collagen using polyvinylphosphonic acid/polyacrylic acid templating matrix protein analogues in the presence of calcium, phosphate and hydroxyl ions

    PubMed Central

    Kim, Young Kyung; Gu, Li-sha; Bryan, Thomas E.; Kim, Jong Ryul; Chen, Liang; Liu, Yan; Yoon, James C.; Breschi, Lorenzo; Pashley, David H.; Tay, Franklin R.

    2010-01-01

    The complex morphologies of mineralised collagen fibrils are regulated through interactions between the collagen matrix and non-collagenous extracellular proteins. In the present study, polyvinylphosphonic acid, a biomimetic analogue of matrix phosphoproteins, was synthesised and confirmed with FTIR and NMR. Biomimetic mineralisation of reconstituted collagen fibrils devoid of natural non-collagenous proteins was demonstrated with TEM using a Portland cement-containing resin composite and a phosphate-containing fluid in the presence of polyacrylic acid as sequestration, and polyvinylphosphonic acid as templating matrix protein analogues. In the presence of these dual biomimetic analogues in the mineralisation medium, intrafibrillar and extrafibrillar mineralisation via bottom-up nanoparticle assembly based on the nonclassical crystallisation pathway could be identified. Conversely, only large mineral spheres with no preferred association with collagen fibrils were observed in the absence of biomimetic analogues in the medium. Mineral phases were evident within the collagen fibrils as early as 4 hours after the initially-formed amorphous calcium phosphate nanoprecursors were transformed into apatite nanocrystals. Selected area electron diffraction patterns of highly mineralised collagen fibrils were nearly identical to those of natural bone, with apatite crystallites preferentially aligned along the collagen fibril axes. PMID:20621767

  9. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase*

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

  10. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.

    PubMed

    Kalamajski, Sebastian; Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W

    2016-04-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

  11. The effect of anisotropic collagen-GAG scaffolds and growth factor supplementation on tendon cell recruitment, alignment, and metabolic activity

    PubMed Central

    Caliari, Steven R.; Harley, Brendan A.C.

    2014-01-01

    Current surgical and tissue engineering approaches for treating tendon injuries have shown limited success, suggesting the need for new biomaterial strategies. Here we describe the development of an anisotropic collagen-glycosaminoglycan (CG) scaffold and use of growth factor supplementation strategies to create a 3D platform for tendon tissue engineering. We fabricated cylindrical CG scaffolds with aligned tracks of ellipsoidal pores that mimic the native physiology of tendon by incorporating a directional solidification step into a conventional lyophilization strategy. By modifying the freezing temperature, we created a homologous series of aligned CG scaffolds with constant relative density and degree of anisotropy but a range of pore sizes (55–243 μm). Equine tendon cells showed greater levels of attachment, metabolic activity, and alignment as well as less cell-mediated scaffold contraction, when cultured in anisotropic scaffolds compared to an isotropic CG scaffold control. The anisotropic CG scaffolds also provided critical contact guidance cues for cell alignment. While tendon cells were randomly oriented in the isotropic control scaffold and the transverse (unaligned) plane of the anisotropic scaffolds, significant cell alignment was observed in the direction of the contact guidance cues in the longitudinal plane of the anisotropic scaffolds. Scaffold pore size was found to significantly influence tendon cell viability, proliferation, penetration into the scaffold, and metabolic activity in a manner predicted by cellular solids arguments. Finally, the addition of the growth factors PDGF-BB and IGF-1 to aligned CG scaffolds was found to enhance tendon cell motility, viability, and metabolic activity in dose-dependent manners. This work suggests a composite strategy for developing bioactive, 3D material systems for tendon tissue engineering. PMID:21550653

  12. The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension.

    PubMed

    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E; Qvortrup, Klaus; Baar, Keith; Svensson, René B; Magnusson, S Peter; Krogsgaard, Michael; Koch, Manuel; Kjaer, Michael

    2010-06-01

    Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts to initiate collagen fibrillogenesis when cultured in fixed-length fibrin gels. Fibroblasts were dissected from semitendinosus and gracilis tendons from healthy humans and cultured in 3D linear fibrin gels. The fibroblasts synthesized an extracellular matrix of parallel collagen fibrils that were aligned along the axis of tension. The fibrils had a homogeneous narrow diameter that was similar to collagen fibrils occurring in embryonic tendon. Immunostaining showed colocalization of collagen type I with collagen III, XII and XIV. A fibronectin network was formed in parallel with the collagen, and fibroblasts stained positive for integrin alpha(5). Finally, the presence of cell extensions into the extracellular space with membrane-enclosed fibrils in fibripositors indicated characteristics of embryonic tendon. We conclude that mature human tendon fibroblasts retain an intrinsic capability to perform collagen fibrillogenesis similar to that of developing tendon, which implies that the hormonal/mechanical milieu, rather than intrinsic cellular function, inhibits regenerative potential in mature tendon.

  13. A collagen and elastic network in the wing of the bat.

    PubMed Central

    Holbrook, K A; Odland, G F

    1978-01-01

    Bundles of collagen fibrils, elastic fibres and fibroblasts are organized into a network that lies in the plane of a large portion of the bat wing. By ultrastructural (TEM and SEM) and biochemical analyses it was found that individual bundles of the net are similar to elastic ligaments. Although elastic fibres predominate, they are integrated and aligned in parallel with small bundles of collagen. A reticulum of fibroblasts, joined by focal junctions, forms a cellular framework throughout each bundle. Because of the unique features of the fibre bundles of the bat's wing, in particular their accessibility, and the parallel alignment of the collagen fibrils and elastic fibres in each easily isolatable fibre bundle, they should prove a most valuable model for connective tissue studies, particularly for the study of collagen-elastin interactions. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 PMID:649500

  14. Fiber/collagen composites for ligament tissue engineering: influence of elastic moduli of sparse aligned fibers on mesenchymal stem cells.

    PubMed

    Thayer, Patrick S; Verbridge, Scott S; Dahlgren, Linda A; Kakar, Sanjeev; Guelcher, Scott A; Goldstein, Aaron S

    2016-08-01

    Electrospun microfibers are attractive for the engineering of oriented tissues because they present instructive topographic and mechanical cues to cells. However, high-density microfiber networks are too cell-impermeable for most tissue applications. Alternatively, the distribution of sparse microfibers within a three-dimensional hydrogel could present instructive cues to guide cell organization while not inhibiting cell behavior. In this study, thin (∼5 fibers thick) layers of aligned microfibers (0.7 μm) were embedded within collagen hydrogels containing mesenchymal stem cells (MSCs), cultured for up to 14 days, and assayed for expression of ligament markers and imaged for cell organization. These microfibers were generated through the electrospinning of polycaprolactone (PCL), poly(ester-urethane) (PEUR), or a 75/25 PEUR/PCL blend to produce microfiber networks with elastic moduli of 31, 15, and 5.6 MPa, respectively. MSCs in composites containing 5.6 MPa fibers exhibited increased expression of the ligament marker scleraxis and the contractile phenotype marker α-smooth muscle actin versus the stiffer fiber composites. Additionally, cells within the 5.6 MPa microfiber composites were more oriented compared to cells within the 15 and 31 MPa microfiber composites. Together, these data indicate that the mechanical properties of microfiber/collagen composites can be tuned for the engineering of ligament and other target tissues. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1894-1901, 2016.

  15. Nanomechanics of Type I Collagen.

    PubMed

    Varma, Sameer; Orgel, Joseph P R O; Schieber, Jay D

    2016-07-12

    Type I collagen is the predominant collagen in mature tendons and ligaments, where it gives them their load-bearing mechanical properties. Fibrils of type I collagen are formed by the packing of polypeptide triple helices. Higher-order structures like fibril bundles and fibers are assembled from fibrils in the presence of other collagenous molecules and noncollagenous molecules. Curiously, however, experiments show that fibrils/fibril bundles are less resistant to axial stress compared to their constituent triple helices-the Young's moduli of fibrils/fibril bundles are an order-of-magnitude smaller than the Young's moduli of triple helices. Given the sensitivity of the Young's moduli of triple helices to solvation environment, a plausible explanation is that the packing of triple helices into fibrils perhaps reduces the Young's modulus of an individual triple helix, which results in fibrils having smaller Young's moduli. We find, however, from molecular dynamics and accelerated conformational sampling simulations that the Young's modulus of the buried core of the fibril is of the same order as that of a triple helix in aqueous phase. These simulations, therefore, suggest that the lower Young's moduli of fibrils/fibril bundles cannot be attributed to the specific packing of triple helices in the fibril core. It is not the fibril core that yields initially to axial stress. Rather, it must be the portion of the fibril exposed to the solvent and/or the fibril-fibril interface that bears the initial strain. Overall, this work provides estimates of Young's moduli and persistence lengths at two levels of collagen's structural assembly, which are necessary to quantitatively investigate the response of various biological factors on collagen mechanics, including congenital mutations, posttranslational modifications and ligand binding, and also engineer new collagen-based materials. PMID:27410733

  16. Electron microscopic stereological study of collagen fibrils in bovine articular cartilage: volume and surface densities are best obtained indirectly (from length densities and diameters) using isotropic uniform random sampling

    PubMed Central

    LÅNGSJÖ, TEEMU K.; HYTTINEN, MIKA; PELTTARI, ALPO; KIRALY, KARI; AROKOSKI, JARI; HELMINEN, HEIKKI J.

    1999-01-01

    Results obtained by the indirect zonal isotropic uniform random (IUR) estimation were compared with those obtained by the direct point and interception counting methods on vertical (VS) or IUR sections in a stereological study of bovine articular cartilage collagen fibrils at the ultrastructural level. Besides comparisons between the direct and indirect estimations (direct IUR vs indirect IUR estimations) and between different sampling methods (VS vs IUR sampling), simultaneous comparison of the 2 issues took place (direct VS vs indirect IUR estimation). Using the direct VS method, articular cartilage superficial zone collagen volume fraction (Vv 41%) was 67% and fibril surface density (Sv 0.030 nm2/nm3) 15% higher (P<0.05) than values obtained by the indirect IUR method (Vv 25% and Sv 0.026 nm2/nm3). The same was observed when the direct IUR method was used: collagen volume fraction (Vv 40%) was 63% and fibril surface density (Sv 0.032 nm2/nm3) 21% higher (P<0.05) than those obtained by the indirect IUR technique. Similarly, in the deep zone of articular cartilage direct VS and direct IUR methods gave 50 and 55% higher (P<0.05) collagen fibril volume fractions (Vv 43 and 44% vs 29%) and the direct IUR method 25% higher (P<0.05) fibril surface density values (Sv 0.025 vs 0.020 nm2/nm3) than the indirect IUR estimation. On theoretical grounds, scrutiny calculations, as well as earlier reports, it is concluded that the direct VS and direct IUR methods systematically overestimated the Vv and Sv of collagen fibrils. This bias was due to the overprojection which derives from the high section thickness in relation to collagen fibril diameter. On the other hand, factors that during estimation tend to underestimate Vv and Sv, such as profile overlapping and truncation (‘fuzzy’ profiles), seemed to cause less bias. As length density (Lv) and collagen fibril diameter are minimally biased by the high relative section thickness, the indirect IUR method, based on

  17. Alignment hierarchies: engineering architecture from the nanometre to the micrometre scale

    PubMed Central

    Kureshi, Alvena; Cheema, Umber; Alekseeva, Tijna; Cambrey, Alison; Brown, Robert

    2010-01-01

    Natural tissues are built of metabolites, soluble proteins and solid extracellular matrix components (largely fibrils) together with cells. These are configured in highly organized hierarchies of structure across length scales from nanometre to millimetre, with alignments that are dominated by anisotropies in their fibrillar matrix. If we are to successfully engineer tissues, these hierarchies need to be mimicked with an understanding of the interaction between them. In particular, the movement of different elements of the tissue (e.g. molecules, cells and bulk fluids) is controlled by matrix structures at distinct scales. We present three novel systems to introduce alignment of collagen fibrils, cells and growth factor gradients within a three-dimensional collagen scaffold using fluid flow, embossing and layering of construct. Importantly, these can be seen as different parts of the same hierarchy of three-dimensional structure, as they are all formed into dense collagen gels. Fluid flow aligns collagen fibrils at the nanoscale, embossed topographical features provide alignment cues at the microscale and introducing layered configuration to three-dimensional collagen scaffolds provides microscale- and mesoscale-aligned pathways for protein factor delivery as well as barriers to confine protein diffusion to specific spatial directions. These seemingly separate methods can be employed to increase complexity of simple extracellular matrix scaffolds, providing insight into new approaches to directly fabricate complex physical and chemical cues at different hierarchical scales, similar to those in natural tissues. PMID:20926429

  18. Alignment hierarchies: engineering architecture from the nanometre to the micrometre scale.

    PubMed

    Kureshi, Alvena; Cheema, Umber; Alekseeva, Tijna; Cambrey, Alison; Brown, Robert

    2010-12-01

    Natural tissues are built of metabolites, soluble proteins and solid extracellular matrix components (largely fibrils) together with cells. These are configured in highly organized hierarchies of structure across length scales from nanometre to millimetre, with alignments that are dominated by anisotropies in their fibrillar matrix. If we are to successfully engineer tissues, these hierarchies need to be mimicked with an understanding of the interaction between them. In particular, the movement of different elements of the tissue (e.g. molecules, cells and bulk fluids) is controlled by matrix structures at distinct scales. We present three novel systems to introduce alignment of collagen fibrils, cells and growth factor gradients within a three-dimensional collagen scaffold using fluid flow, embossing and layering of construct. Importantly, these can be seen as different parts of the same hierarchy of three-dimensional structure, as they are all formed into dense collagen gels. Fluid flow aligns collagen fibrils at the nanoscale, embossed topographical features provide alignment cues at the microscale and introducing layered configuration to three-dimensional collagen scaffolds provides microscale- and mesoscale-aligned pathways for protein factor delivery as well as barriers to confine protein diffusion to specific spatial directions. These seemingly separate methods can be employed to increase complexity of simple extracellular matrix scaffolds, providing insight into new approaches to directly fabricate complex physical and chemical cues at different hierarchical scales, similar to those in natural tissues.

  19. Collagen IX is required for the integrity of collagen II fibrils and the regulation of vascular plexus formation in zebrafish caudal fins.

    PubMed

    Huang, Cheng-chen; Wang, Tai-Chuan; Lin, Bo-Hung; Wang, Yi-Wen; Johnson, Stephen L; Yu, John

    2009-08-15

    Capillary plexuses form during both vasculogenesis and angiogenesis and are remodeled into mature vessel types and patterns which are delicately orchestrated with the sizes and shapes of other tissues and organs. We isolated a zebrafish mutation named prp (for persistent plexus) that causes persistent formation of vascular plexuses in the caudal fins and consequent mispatterning of bony fin rays and the fin shape. Detailed analyses revealed that the prp mutation causes a significant reduction in the size and dramatic structural defects in collagen II-rich extracellular matrices called actinotrichia of both embryonic finfolds and adult fins. prp was mapped to chromosome 19 and found to encode the zebrafish collagen9alpha1 (col9alpha1) gene which is abundantly expressed in developing finfolds. A point mutation resulting in a leucine-to-histidine change was detected in the thrombospondin domain of the col9alpha1 gene in prp. Morpholino-mediated knockdown of col9alpha1 phenocopied the prp small-finfold phenotype in wild-type embryos, and an injection of plasmids containing the col9alpha1 cDNA into prp embryos locally restored the finfold size. Furthermore, we found that osteoblasts in prp mutants were mispatterned apparently following the abnormal vascular plexus pattern, demonstrating that blood vessels play an important role in the patterning of bony rays in zebrafish caudal fins. PMID:19501583

  20. Ovine tendon collagen: Extraction, characterisation and fabrication of thin films for tissue engineering applications.

    PubMed

    Fauzi, M B; Lokanathan, Y; Aminuddin, B S; Ruszymah, B H I; Chowdhury, S R

    2016-11-01

    Collagen is the most abundant extracellular matrix (ECM) protein in the human body, thus widely used in tissue engineering and subsequent clinical applications. This study aimed to extract collagen from ovine (Ovis aries) Achilles tendon (OTC), and to evaluate its physicochemical properties and its potential to fabricate thin film with collagen fibrils in a random or aligned orientation. Acid-solubilized protein was extracted from ovine Achilles tendon using 0.35M acetic acid, and 80% of extracted protein was measured as collagen. SDS-PAGE and mass spectrometry analysis revealed the presence of alpha 1 and alpha 2 chain of collagen type I (col I). Further analysis with Fourier transform infrared spectrometry (FTIR), X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDS) confirms the presence of triple helix structure of col I, similar to commercially available rat tail col I. Drying the OTC solution at 37°C resulted in formation of a thin film with randomly orientated collagen fibrils (random collagen film; RCF). Introduction of unidirectional mechanical intervention using a platform rocker prior to drying facilitated the fabrication of a film with aligned orientation of collagen fibril (aligned collagen film; ACF). It was shown that both RCF and ACF significantly enhanced human dermal fibroblast (HDF) attachment and proliferation than that on plastic surface. Moreover, cells were distributed randomly on RCF, but aligned with the direction of mechanical intervention on ACF. In conclusion, ovine tendon could be an alternative source of col I to fabricate scaffold for tissue engineering applications. PMID:27524008

  1. Ovine tendon collagen: Extraction, characterisation and fabrication of thin films for tissue engineering applications.

    PubMed

    Fauzi, M B; Lokanathan, Y; Aminuddin, B S; Ruszymah, B H I; Chowdhury, S R

    2016-11-01

    Collagen is the most abundant extracellular matrix (ECM) protein in the human body, thus widely used in tissue engineering and subsequent clinical applications. This study aimed to extract collagen from ovine (Ovis aries) Achilles tendon (OTC), and to evaluate its physicochemical properties and its potential to fabricate thin film with collagen fibrils in a random or aligned orientation. Acid-solubilized protein was extracted from ovine Achilles tendon using 0.35M acetic acid, and 80% of extracted protein was measured as collagen. SDS-PAGE and mass spectrometry analysis revealed the presence of alpha 1 and alpha 2 chain of collagen type I (col I). Further analysis with Fourier transform infrared spectrometry (FTIR), X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDS) confirms the presence of triple helix structure of col I, similar to commercially available rat tail col I. Drying the OTC solution at 37°C resulted in formation of a thin film with randomly orientated collagen fibrils (random collagen film; RCF). Introduction of unidirectional mechanical intervention using a platform rocker prior to drying facilitated the fabrication of a film with aligned orientation of collagen fibril (aligned collagen film; ACF). It was shown that both RCF and ACF significantly enhanced human dermal fibroblast (HDF) attachment and proliferation than that on plastic surface. Moreover, cells were distributed randomly on RCF, but aligned with the direction of mechanical intervention on ACF. In conclusion, ovine tendon could be an alternative source of col I to fabricate scaffold for tissue engineering applications.

  2. Imaging Analysis of Collagen Fiber Networks in Cusps of Porcine Aortic Valves: Effect of their Local Distribution and Alignment on Valve Functionality

    PubMed Central

    Mega, Mor; Marom, Gil; Halevi, Rotem; Hamdan, Ashraf; Bluestein, Danny; Haj-Ali, Rami

    2015-01-01

    The cusps of native Aortic Valve (AV) are composed of collagen bundles embedded in soft tissue, creating a heterogenic tissue with asymmetric alignment in each cusp. This study compares native collagen fiber networks (CFNs) with a goal to better understand their influence on stress distribution and valve kinematics. Images of CFNs from five porcine tricuspid AVs are analyzed and fluid-structure interaction models are generated based on them. Although the valves had similar overall kinematics, the CFNs had distinctive influence on local mechanics. The regions with dilute CFN are more prone to damage since they are subjected to higher stress magnitudes. PMID:26406926

  3. Use of magnetically oriented orthogonal collagen scaffolds for hemi-corneal reconstruction and regeneration.

    PubMed

    Builles, Nicolas; Janin-Manificat, Hélène; Malbouyres, Marilyne; Justin, Virginie; Rovère, Marie-Rose; Pellegrini, Graziella; Torbet, Jim; Hulmes, David J S; Burillon, Carole; Damour, Odile; Ruggiero, Florence

    2010-11-01

    We recently showed that the highly organized architecture of the corneal stroma could be reproduced using scaffolds consisting of orthogonally aligned multilayers of collagen fibrils prepared using a high magnetic field. Here we show that such scaffolds permit the reconstruction in vitro of human hemi-corneas (stroma + epithelium), using primary human keratocytes and limbal stem cell derived human keratinocytes. On the surface of these hemi-corneas, a well-differentiated epithelium was formed, as determined both histologically and ultrastructurally and by the expression of characteristic markers. Within the stroma, the keratocytes aligned with the directions of the fibrils in the scaffold and synthesized a new extracellular matrix with typical collagen markers and small, uniform diameter fibrils. Finally, in vivo experiments using a rabbit model showed that these orthogonally oriented multi-layer scaffolds could be used to repair the anterior region of the stroma, leading to re-epithelialization and recovery of both transparency and ultrastructural organization. PMID:20708260

  4. Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities.

    PubMed

    Igoucheva, Olga; Alexeev, Vitali; Halabi, Carmen M; Adams, Sheila M; Stoilov, Ivan; Sasaki, Takako; Arita, Machiko; Donahue, Adele; Mecham, Robert P; Birk, David E; Chu, Mon-Li

    2015-08-28

    Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.

  5. Abnormal arrangement of a collagen/apatite extracellular matrix orthogonal to osteoblast alignment is constructed by a nanoscale periodic surface structure.

    PubMed

    Matsugaki, Aira; Aramoto, Gento; Ninomiya, Takafumi; Sawada, Hiroshi; Hata, Satoshi; Nakano, Takayoshi

    2015-01-01

    Morphological and directional alteration of cells is essential for structurally appropriate construction of tissues and organs. In particular, osteoblast alignment is crucial for the realization of anisotropic bone tissue microstructure. In this article, the orientation of a collagen/apatite extracellular matrix (ECM) was established by controlling osteoblast alignment using a surface geometry with nanometer-sized periodicity induced by laser ablation. Laser irradiation induced self-organized periodic structures (laser-induced periodic surface structures; LIPSS) with a spatial period equal to the wavelength of the incident laser on the surface of biomedical alloys of Ti-6Al-4V and Co-Cr-Mo. Osteoblast orientation was successfully induced parallel to the grating structure. Notably, both the fibrous orientation of the secreted collagen matrix and the c-axis of the produced apatite crystals were orientated orthogonal to the cell direction. To the best of our knowledge, this is the first report demonstrating that bone tissue anisotropy is controllable, including the characteristic organization of a collagen/apatite composite orthogonal to the osteoblast orientation, by controlling the cell alignment using periodic surface geometry.

  6. Fabrication of electrospun poly(L-lactide-co-ε-caprolactone)/collagen nanoyarn network as a novel, three-dimensional, macroporous, aligned scaffold for tendon tissue engineering.

    PubMed

    Xu, Yuan; Wu, Jinglei; Wang, Haoming; Li, Hanqin; Di, Ning; Song, Lei; Li, Sontao; Li, Dianwei; Xiang, Yang; Liu, Wei; Mo, Xiumei; Zhou, Qiang

    2013-12-01

    Tissue engineering techniques using novel scaffolding materials offer potential alternatives for managing tendon disorders. An ideal tendon tissue engineered scaffold should mimic the three-dimensional (3D) structure of the natural extracellular matrix (ECM) of the native tendon. Here, we propose a novel electrospun nanoyarn network that is morphologically and structurally similar to the ECM of native tendon tissues. The nanoyarn, random nanofiber, and aligned nanofiber scaffolds of a synthetic biodegradable polymer, poly(L-lactide-co-ε-caprolactone) [P(LLA-CL)], and natural collagen I complex were fabricated using electrospinning. These scaffolds were characterized in terms of fiber morphology, pore size, porosity, and chemical and mechanical properties for the purpose of culturing tendon cells (TCs) for tendon tissue engineering. The results indicated a fiber diameter of 632 ± 81 nm for the random nanofiber scaffold, 643 ± 97 nm for the aligned nanofiber scaffold, and 641 ± 68 nm for the nanoyarn scaffold. The yarn in the nanoyarn scaffold was twisted by many nanofibers similar to the structure and inherent nanoscale organization of tendons, indicating an increase in the diameter of 9.51 ± 3.62 μm. The nanoyarn scaffold also contained 3D aligned microstructures with large interconnected pores and high porosity. Fourier transform infrared analyses revealed the presence of collagen in the three scaffolds. The mechanical properties of the sample scaffolds indicated that the scaffolds had desirable mechanical properties for tissue regeneration. Further, the results revealed that TC proliferation and infiltration, and the expression of tendon-related ECM genes, were significantly enhanced on the nanoyarn scaffold compared with that on the random nanofiber and aligned nanofiber scaffolds. This study demonstrates that electrospun P(LLA-CL)/collagen nanoyarn is a novel, 3D, macroporous, aligned scaffold that has potential application in tendon tissue engineering.

  7. Fabrication of Electrospun Poly(L-Lactide-co-ɛ-Caprolactone)/Collagen Nanoyarn Network as a Novel, Three-Dimensional, Macroporous, Aligned Scaffold for Tendon Tissue Engineering

    PubMed Central

    Xu, Yuan; Wu, Jinglei; Wang, Haoming; Li, Hanqin; Di, Ning; Song, Lei; Li, Sontao; Li, Dianwei; Xiang, Yang; Liu, Wei

    2013-01-01

    Tissue engineering techniques using novel scaffolding materials offer potential alternatives for managing tendon disorders. An ideal tendon tissue engineered scaffold should mimic the three-dimensional (3D) structure of the natural extracellular matrix (ECM) of the native tendon. Here, we propose a novel electrospun nanoyarn network that is morphologically and structurally similar to the ECM of native tendon tissues. The nanoyarn, random nanofiber, and aligned nanofiber scaffolds of a synthetic biodegradable polymer, poly(l-lactide-co-ɛ-caprolactone) [P(LLA-CL)], and natural collagen I complex were fabricated using electrospinning. These scaffolds were characterized in terms of fiber morphology, pore size, porosity, and chemical and mechanical properties for the purpose of culturing tendon cells (TCs) for tendon tissue engineering. The results indicated a fiber diameter of 632±81 nm for the random nanofiber scaffold, 643±97 nm for the aligned nanofiber scaffold, and 641±68 nm for the nanoyarn scaffold. The yarn in the nanoyarn scaffold was twisted by many nanofibers similar to the structure and inherent nanoscale organization of tendons, indicating an increase in the diameter of 9.51±3.62 μm. The nanoyarn scaffold also contained 3D aligned microstructures with large interconnected pores and high porosity. Fourier transform infrared analyses revealed the presence of collagen in the three scaffolds. The mechanical properties of the sample scaffolds indicated that the scaffolds had desirable mechanical properties for tissue regeneration. Further, the results revealed that TC proliferation and infiltration, and the expression of tendon-related ECM genes, were significantly enhanced on the nanoyarn scaffold compared with that on the random nanofiber and aligned nanofiber scaffolds. This study demonstrates that electrospun P(LLA-CL)/collagen nanoyarn is a novel, 3D, macroporous, aligned scaffold that has potential application in tendon tissue engineering

  8. Fibrillogenesis in Continuously Spun Synthetic Collagen Fiber

    PubMed Central

    Caves, Jeffrey M.; Kumar, Vivek A.; Wen, Jing; Cui, Wanxing; Martinez, Adam; Apkarian, Robert; Coats, Julie E.; Berland, Keith; Chaikof, Elliot L.

    2013-01-01

    The universal structural role of collagen fiber networks has motivated the development of collagen gels, films, coatings, injectables, and other formulations. However, reported synthetic collagen fiber fabrication schemes have either culminated in short, discontinuous fiber segments at unsuitably low production rates, or have incompletely replicated the internal fibrillar structure that dictates fiber mechanical and biological properties. We report a continuous extrusion system with an off-line phosphate buffer incubation step for the manufacture of synthetic collagen fiber. Fiber with a cross-section of 53±14 by 21±3 µm and an ultimate tensile strength of 94±19 MPa was continuously produced at 60 m/hr from an ultrafiltered monomeric collagen solution. The effect of collagen solution concentration, flow rate, and spinneret size on fiber size was investigated. The fiber was further characterized by microdifferential scanning calorimetry, transmission electron microscopy (TEM), second harmonic generation (SHG) analysis, and in a subcutaneous murine implant model. Calorimetry demonstrated stabilization of the collagen triple helical structure, while TEM and SHG revealed a dense, axially aligned D-periodic fibril structure throughout the fiber cross-section. Implantation of glutaraldehyde crosslinked and non-crosslinked fiber in the subcutaneous tissue of mice demonstrated limited inflammatory response and biodegradation after a 6-week implant period. PMID:20024969

  9. Collagen V-heterozygous and -null supraspinatus tendons exhibit altered dynamic mechanical behaviour at multiple hierarchical scales.

    PubMed

    Connizzo, Brianne K; Han, Lin; Birk, David E; Soslowsky, Louis J

    2016-02-01

    Tendons function using a unique set of mechanical properties governed by the extracellular matrix and its ability to respond to varied multi-axial loads. Reduction of collagen V expression, such as in classic Ehlers-Danlos syndrome, results in altered fibril morphology and altered macroscale mechanical function in both clinical and animal studies, yet the mechanism by which changes at the fibril level lead to macroscale functional changes has not yet been investigated. This study addresses this by defining the multiscale mechanical response of wild-type, collagen V-heterozygous and -null supraspinatus tendons. Tendons were subjected to mechanical testing and analysed for macroscale properties, as well as microscale (fibre re-alignment) and nanoscale (fibril deformation and sliding) responses. In many macroscale parameters, results showed a dose-dependent response with severely decreased properties in the null group. In addition, both heterozygous and null groups responded to load faster than in wild-type tendons via earlier fibre re-alignment and fibril stretch. However, the heterozygous group exhibited increased fibril sliding, while the null group exhibited no fibril sliding. These studies demonstrate that dynamic responses play an important role in determining overall function and that collagen V is a critical regulator in the development of these relationships. PMID:26855746

  10. Microfluidics-Produced Collagen Fibers Show Extraordinary Mechanical Properties.

    PubMed

    Haynl, Christian; Hofmann, Eddie; Pawar, Kiran; Förster, Stephan; Scheibel, Thomas

    2016-09-14

    Collagens are widely used as biomaterials in drug-delivery and tissue engineering applications due to their biodegradability, biocompatibility and hypoallergenicity. Besides gelatin-based materials, collagen microfibers are in the focus of biomedical research. Commonly, man-made fibers are produced by wet-spinning yielding fiber diameters higher than 8 μm. Here, assembly and continuous production of single collagen type I microfibers were established using a microfluidic chip. Microfluidics-produced microfibers exhibited tensile strength and Young's modulus exceeding that of fibers produced in classical wet-spinning devices and even that of natural tendon and they showed lower diameters. Their structural orientation was examined by polarized Fourier transform infrared spectroscopy (FTIR) showing fibril alignment within the microfiber. Cell culture tests using the neuronal cell line NG108-15 showed cell alignment and axon growth along the microfiber axes inaugurating potential applications in, for example, peripheral nerve repair. PMID:27513098

  11. The Effect of Gradations in Mineral Content, Matrix Alignment, and Applied Strain on Human Mesenchymal Stem Cell Morphology within Collagen Biomaterials.

    PubMed

    Mozdzen, Laura C; Thorpe, Stephen D; Screen, Hazel R C; Harley, Brendan A C

    2016-07-01

    The tendon-bone junction is a unique, mechanically dynamic, structurally graded anatomical zone, which transmits tensile loads between tendon and bone. Current surgical repair techniques rely on mechanical fixation and can result in high re-failure rates. A new class of collagen biomaterial that contains discrete mineralized and structurally aligned regions linked by a continuous interface to mimic the graded osteotendinous insertion has been recently described. Here the combined influence of graded biomaterial environment and increasing levels of applied strain (0%-20%) on mesenchymal stem cell (MSC) orientation and alignment have been reported. In osteotendinous scaffolds, which contain opposing gradients of mineral content and structural alignment characteristic of the native osteotendinous interface, MSC nuclear, and actin alignment is initially dictated by the local pore architecture, while applied tensile strain enhances cell alignment in the direction of strain. Comparatively, in layered scaffolds that do not contain any structural alignment cues, MSCs are randomly oriented in the unstrained condition, then become oriented in a direction perpendicular to applied strain. These findings provide an initial understanding of how scaffold architecture can provide significant, potentially competitive, feedback influencing MSC orientation under applied strain, and form the basis for future tissue engineering efforts to regenerate the osteotendinous enthesis.

  12. Development of collagen-hydroxyapatite nanostructured composites via a calcium phosphate precursor mechanism

    NASA Astrophysics Data System (ADS)

    Jee, Sang Soo

    of poly-aspartic acid affects the degree of intrafibrillar mineralization of collagen scaffolds. High molecular weight poly-aspartic acid could produce a stable and dispersed amorphous precursor, leading to a high degree of intrafibrillar mineralization. The mineral content of the collagen sponge mineralized using high molecular weight poly-aspartic acid was equivalent to the mineral content of bone. According to X-ray diffraction analysis of the mineralized collagen, the size and composition of the intrafibrillar hydroxyapatite produced by the PILP process were almost identical to carbonated hydroxyapatite in bone. The selective area electron diffraction patterns indicated that the [001] direction of hydroxyapatite is roughly aligned along the c-axis of collagen fibril, leading to the formation 002 arcs. Using dark field imaging, it was possible to visualize the preferentially oriented hydroxyapatite in TEM. Thermal analysis of mineralized collagen also showed a reduction in the thermal stability of collagen, which is similar to that observed in the collagen in bone, due to the presence of intrafibrillar hydroxyapatite. Now, we confidently suggest that the PILP process can provide a new way to develop synthetic bone-like composites whose nano-structure is very close to the nano-structure of natural bone. Moreover, we hope that our successful intrafibrillar mineralization of collagen via the precursor mechanism revives discussion of hypothesis of bone mineralization via the amorphous calcium phosphate phase.

  13. Collagen orientation and leather strength for selected mammals.

    PubMed

    Sizeland, Katie H; Basil-Jones, Melissa M; Edmonds, Richard L; Cooper, Sue M; Kirby, Nigel; Hawley, Adrian; Haverkamp, Richard G

    2013-01-30

    Collagen is the main structural component of leather, skin, and some other applications such as medical scaffolds. All of these materials have a mechanical function, so the manner in which collagen provides them with their strength is of fundamental importance and was investigated here. This study shows that the tear strength of leather across seven species of mammals depends on the degree to which collagen fibrils are aligned in the plane of the tissue. Tear-resistant material has the fibrils contained within parallel planes with little crossover between the top and bottom surfaces. The fibril orientation is observed using small-angle X-ray scattering in leather, produced from skin, with tear strengths (normalized for thickness) of 20-110 N/mm. The orientation index, 0.420-0.633, is linearly related to tear strength such that greater alignment within the plane of the tissue results in stronger material. The statistical confidence and diversity of animals suggest that this is a fundamental determinant of strength in tissue. This insight is valuable in understanding the performance of leather and skin in biological and industrial applications.

  14. Type XIV Collagen Regulates Fibrillogenesis

    PubMed Central

    Ansorge, Heather L.; Meng, Xianmin; Zhang, Guiyun; Veit, Guido; Sun, Mei; Klement, John F.; Beason, David P.; Soslowsky, Louis J.; Koch, Manuel; Birk, David E.

    2009-01-01

    Type XIV collagen is a fibril-associated collagen with an interrupted triple helix. This collagen interacts with the fibril surface and has been implicated as a regulator of fibrillogenesis; however, a specific role has not been elucidated. Functional roles for type XIV collagen were defined utilizing a new type XIV collagen-deficient mouse line. This line was produced using a conventional targeted knock-out approach. Col14a1(–/–) mice were devoid of type XIV collagen, whereas heterozygous mice had reduced synthesis. Both mutant Col14a1 genotypes were viable with a grossly normal phenotype; however, mature skin exhibited altered mechanical properties. Prior to evaluating tendon fibrillogenesis in type XIV collagen-deficient mice, the developmental expression patterns were analyzed in wild-type flexor digitorum longus (FDL) tendons. Analyses of mRNA and protein expression indicated tissue-specific temporal expression that was associated with the early stages in fibrillogenesis. Ultrastructural analyses of wild-type and null tendons demonstrated premature fibril growth and larger fibril diameters in tendons from null mice at postnatal day 4 (P4). However, fibril structure in mature tendons was normal. Biomechanical studies established a direct structure/function relationship with reduced strength in P7-null tendons. However, the biomechanical properties in P60 tendons were comparable in null and wild-type mice. Our results indicate a regulatory function for type XIV collagen in early stages of collagen fibrillogenesis with tissue differences. PMID:19136672

  15. MMP16 Mediates a Proteolytic Switch to Promote Cell-Cell Adhesion, Collagen Alignment, and Lymphatic Invasion in Melanoma.

    PubMed

    Tatti, Olga; Gucciardo, Erika; Pekkonen, Pirita; Holopainen, Tanja; Louhimo, Riku; Repo, Pauliina; Maliniemi, Pilvi; Lohi, Jouko; Rantanen, Ville; Hautaniemi, Sampsa; Alitalo, Kari; Ranki, Annamari; Ojala, Päivi M; Keski-Oja, Jorma; Lehti, Kaisa

    2015-05-15

    Lymphatic invasion and accumulation of continuous collagen bundles around tumor cells are associated with poor melanoma prognosis, but the underlying mechanisms and molecular determinants have remained unclear. We show here that a copy-number gain or overexpression of the membrane-type matrix metalloproteinase MMP16 (MT3-MMP) is associated with poor clinical outcome, collagen bundle assembly around tumor cell nests, and lymphatic invasion. In cultured WM852 melanoma cells derived from human melanoma metastasis, silencing of MMP16 resulted in cell-surface accumulation of the MMP16 substrate MMP14 (MT1-MMP) as well as L1CAM cell adhesion molecule, identified here as a novel MMP16 substrate. When limiting the activities of these trans-membrane protein substrates toward pericellular collagen degradation, cell junction disassembly, and blood endothelial transmigration, MMP16 supported nodular-type growth of adhesive collagen-surrounded melanoma cell nests, coincidentally steering cell collectives into lymphatic vessels. These results uncover a novel mechanism in melanoma pathogenesis, whereby restricted collagen infiltration and limited mesenchymal invasion are unexpectedly associated with the properties of the most aggressive tumors, revealing MMP16 as a putative indicator of adverse melanoma prognosis. PMID:25808867

  16. Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography

    NASA Technical Reports Server (NTRS)

    Landis, W. J.; Hodgens, K. J.; Arena, J.; Song, M. J.; McEwen, B. F.

    1996-01-01

    bone collagen. The results suggest that platelet-shaped crystals are arranged in channels or grooves which are formed by collagen hole zones in register and that crystal sizes may exceed the dimensions of hole zones. Such data agree with those from mineral-matrix interaction in normally calcifying avian tendon obtained by similar high voltage tomographic means, but in addition they indicate a possible gradual and continuous deposition of crystals in collagen of bone unlike tendon and imply that individual collagen fibrils in local regions of osteoid are organized such that they all may be aligned in a coherent manner.

  17. Studies of chain substitution caused sub-fibril level differences in stiffness and ultrastructure of wildtype and oim/oim collagen fibers using multifrequency-AFM and molecular modeling.

    PubMed

    Li, Tao; Chang, Shu-Wei; Rodriguez-Florez, Naiara; Buehler, Markus J; Shefelbine, Sandra; Dao, Ming; Zeng, Kaiyang

    2016-11-01

    Molecular alteration in type I collagen, i.e., substituting the α2 chain with α1 chain in tropocollagen molecule, can cause osteogenesis imperfecta (OI), a brittle bone disease, which can be represented by a mouse model (oim/oim). In this work, we use dual-frequency Atomic Force Microscopy (AFM) and incorporated with molecular modeling to quantify the ultrastructure and stiffness of the individual native collagen fibers from wildtype (+/+) and oim/oim diseased mice humeri. Our work presents direct experimental evidences that the +/+ fibers have highly organized and compact ultrastructure and corresponding ordered stiffness distribution. In contrast, oim/oim fibers have ordered but loosely packed ultrastructure with uncorrelated stiffness distribution, as well as local defects. The molecular model also demonstrates the structural and molecular packing differences between +/+ and oim/oim collagens. The molecular mutation significantly altered sub-fibril structure and mechanical property of collagen fibers. This study can give the new insight for the mechanisms and treatment of the brittle bone disease.

  18. Studies of chain substitution caused sub-fibril level differences in stiffness and ultrastructure of wildtype and oim/oim collagen fibers using multifrequency-AFM and molecular modeling.

    PubMed

    Li, Tao; Chang, Shu-Wei; Rodriguez-Florez, Naiara; Buehler, Markus J; Shefelbine, Sandra; Dao, Ming; Zeng, Kaiyang

    2016-11-01

    Molecular alteration in type I collagen, i.e., substituting the α2 chain with α1 chain in tropocollagen molecule, can cause osteogenesis imperfecta (OI), a brittle bone disease, which can be represented by a mouse model (oim/oim). In this work, we use dual-frequency Atomic Force Microscopy (AFM) and incorporated with molecular modeling to quantify the ultrastructure and stiffness of the individual native collagen fibers from wildtype (+/+) and oim/oim diseased mice humeri. Our work presents direct experimental evidences that the +/+ fibers have highly organized and compact ultrastructure and corresponding ordered stiffness distribution. In contrast, oim/oim fibers have ordered but loosely packed ultrastructure with uncorrelated stiffness distribution, as well as local defects. The molecular model also demonstrates the structural and molecular packing differences between +/+ and oim/oim collagens. The molecular mutation significantly altered sub-fibril structure and mechanical property of collagen fibers. This study can give the new insight for the mechanisms and treatment of the brittle bone disease. PMID:27589372

  19. Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct in vitro

    PubMed Central

    Martens, Wendy; Sanen, Kathleen; Georgiou, Melanie; Struys, Tom; Bronckaers, Annelies; Ameloot, Marcel; Phillips, James; Lambrichts, Ivo

    2014-01-01

    In the present study, we evaluated the differentiation potential of human dental pulp stem cells (hDPSCs) toward Schwann cells, together with their functional capacity with regard to myelination and support of neurite outgrowth in vitro. Successful Schwann cell differentiation was confirmed at the morphological and ultrastructural level by transmission electron microscopy. Furthermore, compared to undifferentiated hDPSCs, immunocytochemistry and ELISA tests revealed increased glial marker expression and neurotrophic factor secretion of differentiated hDPSCs (d-hDPSCs), which promoted survival and neurite outgrowth in 2-dimensional dorsal root ganglia cultures. In addition, neurites were myelinated by d-hDPSCs in a 3-dimensional collagen type I hydrogel neural tissue construct. This engineered construct contained aligned columns of d-hDPSCs that supported and guided neurite outgrowth. Taken together, these findings provide the first evidence that hDPSCs are able to undergo Schwann cell differentiation and support neural outgrowth in vitro, proposing them to be good candidates for cell-based therapies as treatment for peripheral nerve injury.—Martens, W., Sanen, K., Georgiou, M., Struys, T., Bronckaers, A., Ameloot, M., Phillips, J., Lambrichts, I. Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct in vitro. PMID:24352035

  20. Aligning health care policy with evidence-based medicine: the case for funding direct oral anticoagulants in atrial fibrillation.

    PubMed

    Stone, James A; Earl, Karen M; O'Neill, Blair J; Sharma, Mukul; Huynh, Thao; Leblanc, Kori; Ward, Richard; Teal, Philip A; Cox, Jafna L

    2014-10-01

    Misalignment between evidence-informed clinical care guideline recommendations and reimbursement policy has created care gaps that lead to suboptimal outcomes for patients denied access to guideline-based therapies. The purpose of this article is to make the case for addressing this growing access barrier to optimal care. Stroke prevention in atrial fibrillation (AF) is discussed as an example. Stroke is an extremely costly disease, imposing a significant human, societal, and economic burden. Stroke in the setting of AF carries an 80% probability of death or disability. Although two-thirds of these strokes are preventable with appropriate anticoagulation, this has historically been underprescribed and poorly managed. National and international guidelines endorse the direct oral anticoagulants as first-line therapy for this indication. However, no Canadian province has provided these agents with an unrestricted listing. These decisions appear to be founded on silo-based cost assessment-the drug costs rather than the total system costs-and thus overlook several important cost-drivers in stroke. The discordance between best scientific evidence and public policy requires health care providers to use a potentially suboptimal therapy in contravention of guideline recommendations. It represents a significant obstacle for knowledge translation efforts that aim to increase the appropriate anticoagulation of Canadians with AF. As health care professionals, we have a responsibility to our patients to engage with policy-makers in addressing and resolving this barrier to optimal patient care.

  1. Inhibition of collagen-induced platelet aggregation by antibodies to distinct types of collagens.

    PubMed Central

    Balleisen, L; Nowack, H; Gay, S; Timpl, R

    1979-01-01

    Aggregation of platelets by fibrils formed from collagens type I, II and III could be inhibited by coating the fibrils with anti-collagen antibodies or Fab fragments. Similar results were obtained in a clot-retraction assay. Inhibition was achieved with stoichiometric amounts of antibodies and was specific for each type of collagen. Aggregation caused by a mixture of type-I and -III collagens could only be inhibited by a mixture of antibodies against both collagens. The data show that each interstitial collagen is capable of interacting with platelets and do not support the concept of an outstanding activity of type-III collagen. Images PLATE 1 PMID:395952

  2. Liquid crystallinity in condensed type I collagen solutions. A clue to the packing of collagen in extracellular matrices.

    PubMed

    Giraud-Guille, M M

    1992-04-01

    We recently described a new type of assembly of collagen molecules, forming typical liquid crystalline phases in highly concentrated solutions after sonication. The present work shows that intact 300 nm long collagen molecules also form cholesteric liquid crystalline domains, but the time required is much longer, several weeks instead of several days. Differential calorimetry and X-ray diffraction show that sonication does not alter the triple-helical structure of the collagen fragments. In the viscous solutions, observed between crossed polars in optical microscopy, the textures vary as a function of the concentration. Molecules first align near the air interface at the coverslip edge, then as the concentration increases by slow evaporation of the solvent, the birefringence extends inwards and liquid crystalline domains progressively appear. For concentrations estimated to be above 100 mg/ml, typical textures and defects of cholesteric phases are obtained, at lower concentrations zig-zag extinction patterns and banded patterns are observed; all these textures are described and interpreted. The cholesteric packing of collagen fibrils in various extracellular matrices is known, and the relationship that can be made between the ordered phases obtained with collagen molecules in vitro and the related geometrical structures observed between fibrils in vivo is thoroughly discussed.

  3. Hydrodynamic alignment and assembly of nano-fibrillated cellulose in the laminar extensional flow: Effects of solidifying agents

    NASA Astrophysics Data System (ADS)

    Mittal, Nitesh; Lundell, Fredrik; Soderberg, Daniel

    2015-11-01

    There are several fiber production technologies that are based on wet-spinning processes. Many such processes rely on the transformation of a liquid solution into a solid filament. The kinetics of solidification depends largely on the diffusion of the solvents, additives and polymer molecules, which make such systems quite complex and differ from a system to another as a function of the specific chemical, physical and structural features of the used material components. Moreover, tuning the orientation of the polymers in the liquid suspensions makes it further possible to control their structure, which in turn can lead to materials having improved properties. By keeping in mind the facts mentioned above, the aim of the current study is to utilize benefits of a flow focusing approach to align carboxymethylated cellulose nanofibrils (CNF), as a colloidal dispersion, with the help of a laminar elongational flow-field followed by the solidification using different solidifying agents or molecules (with dissimilar diffusion behavior based on their size and charges) to synthesize fibers with enhanced mechanical properties. CNF are charged elongated particles obtained from woods with diameter of 4-10 nm and length of 1-1.5 μm, and they are completely biodegradable.

  4. A microscopic and macroscopic study of aging collagen on its molecular structure, mechanical properties, and cellular response.

    PubMed

    Wilson, Samantha L; Guilbert, Marie; Sulé-Suso, Josep; Torbet, Jim; Jeannesson, Pierre; Sockalingum, Ganesh D; Yang, Ying

    2014-01-01

    During aging, collagen structure changes, detrimentally affecting tissues' biophysical and biomechanical properties due to an accumulation of advanced glycation end-products (AGEs). In this investigation, we conducted a parallel study of microscopic and macroscopic properties of different-aged collagens from newborn to 2-yr-old rats, to examine the effect of aging on fibrillogenesis, mechanical and contractile properties of reconstituted hydrogels from these collagens seeded with or without fibroblasts. In addition to fibrillogenesis of collagen under the conventional conditions, some fibrillogenesis was conducted alongside a 12-T magnetic field, and gelation rate and AGE content were measured. A nondestructive indentation technique and optical coherence tomography were used to determine the elastic modulus and dimensional changes, respectively. It was revealed that in comparison to younger specimens, older collagens exhibited higher viscosity, faster gelation rates, and a higher AGE-specific fluorescence. Exceptionally, only young collagens formed highly aligned fibrils under magnetic fields. The youngest collagen demonstrated a higher elastic modulus and contraction in comparison to the older collagen. We conclude that aging changes collagen monomer structure, which considerably affects the fibrillogenesis process, the architecture of the resulting collagen fibers and the global network, and the macroscopic properties of the formed constructs.

  5. In the beginning there were soft collagen-cell gels: towards better 3D connective tissue models?

    PubMed

    Brown, Robert A

    2013-10-01

    In the 40 years since Elsdale and Bard's analysis of fibroblast culture in collagen gels we have moved far beyond the concept that such 3D fibril network systems are better models than monolayer cultures. This review analyses key aspects of that progression of models, against a background of what exactly each model system tries to mimic. This story tracks our increasing understanding of fibroblast responses to soft collagen gels, in particularly their cytoskeletal contraction, migration and integrin attachment. The focus on fibroblast mechano-function has generated models designed to directly measure the overall force generated by fibroblast populations, their reaction to external loads and the role of the matrix structure. Key steps along this evolution of 3D collagen models have been designed to mimic normal skin, wound repair, tissue morphogenesis and remodelling, growth and contracture during scarring/fibrosis. As new models are developed to understand cell-mechanical function in connective tissues the collagen material has become progressively more important, now being engineered to mimic more complex aspects of native extracellular matrix structure. These have included collagen fibril density, alignment and hierarchical structure, controlling material stiffness and anisotropy. But of these, tissue-like collagen density is key in that it contributes to control of the others. It is concluded that across this 40 year window major progress has been made towards establishing a family of 3D experimental collagen tissue-models, suitable to investigate normal and pathological fibroblast mechano-functions.

  6. The fibrillar collagen family.

    PubMed

    Exposito, Jean-Yves; Valcourt, Ulrich; Cluzel, Caroline; Lethias, Claire

    2010-01-01

    Collagens, or more precisely collagen-based extracellular matrices, are often considered as a metazoan hallmark. Among the collagens, fibrillar collagens are present from sponges to humans, and are involved in the formation of the well-known striated fibrils. In this review we discuss the different steps in the evolution of this protein family, from the formation of an ancestral fibrillar collagen gene to the formation of different clades. Genomic data from the choanoflagellate (sister group of Metazoa) Monosiga brevicollis, and from diploblast animals, have suggested that the formation of an ancestral alpha chain occurred before the metazoan radiation. Phylogenetic studies have suggested an early emergence of the three clades that were first described in mammals. Hence the duplication events leading to the formation of the A, B and C clades occurred before the eumetazoan radiation. Another important event has been the two rounds of "whole genome duplication" leading to the amplification of fibrillar collagen gene numbers, and the importance of this diversification in developmental processes. We will also discuss some other aspects of fibrillar collagen evolution such as the development of the molecular mechanisms involved in the formation of procollagen molecules and of striated fibrils. PMID:20386646

  7. Collagen: Biochemistry, biomechanics, biotechnology

    SciTech Connect

    Nimni, M.E.

    1988-01-01

    This book is an up-to-date reference for new ideas, information, and concepts in collagen research. The first volume emphasizes the relationship between the molecular structure and function of collagen, including descriptions of collagen types which exist in tissues as well as how these molecules organize into fibrils and the nature of the chemical crosslinks which stabilize them. In Volume II the biomechanical behavior of various specialized tissues, abnormal accumulation of collagen in the form of scars of fibrous infiltration are examined/and wound healing, tissue regulation and repair are covered in detail. Volume III explores the increasing application of collagen technology to the field of bioprosthesis, including the production of heart valve bioprosthesis, blood vessels, ligament substitutes, and bone substitutes.

  8. Chick tendon fibroblast transcriptome and shape depend on whether the cell has made its own collagen matrix

    PubMed Central

    Yeung, Ching-Yan Chloé; Zeef, Leo A. H.; Lallyett, Chloe; Lu, Yinhui; Canty-Laird, Elizabeth G.; Kadler, Karl E.

    2015-01-01

    Collagen- and fibrin-based gels are extensively used to study cell behaviour. However, 2D–3D and collagen-fibrin comparisons of gene expression, cell shape and mechanotransduction, with an in vivo reference, have not been reported. Here we compared chick tendon fibroblasts (CTFs) at three stages of embryonic development with CTFs cultured in collagen- or fibrin-based tissue engineered constructs (TECs). CTFs synthesised their own collagen matrix in fibrin-based TECs and better recapitulated the gene expression, collagen fibril alignment and cell shape seen in vivo. In contrast, cells in 3D collagen gels exhibited a 2D-like morphology and expressed fewer of the genes expressed in vivo. Analysis of YAP/TAZ target genes showed that collagen gels desensitise mechanotransduction pathways. In conclusion, gene expression and cell shape are similar on plastic and 3D collagen whereas cells in 3D fibrin have a shape and transcriptome better resembling the in vivo situation. Implications for wound healing are discussed. PMID:26337655

  9. The structural and optical properties of type III human collagen biosynthetic corneal substitutes

    PubMed Central

    Hayes, Sally; Lewis, Phillip; Islam, M. Mirazul; Doutch, James; Sorensen, Thomas; White, Tomas; Griffith, May; Meek, Keith M.

    2015-01-01

    The structural and optical properties of clinically biocompatible, cell-free hydrogels comprised of synthetically cross-linked and moulded recombinant human collagen type III (RHCIII) with and without the incorporation of 2-methacryloyloxyethyl phosphorylcholine (MPC) were assessed using transmission electron microscopy (TEM), X-ray scattering, spectroscopy and refractometry. These findings were examined alongside similarly obtained data from 21 human donor corneas. TEM demonstrated the presence of loosely bundled aggregates of fine collagen filaments within both RHCIII and RHCIII-MPC implants, which X-ray scattering showed to lack D-banding and be preferentially aligned in a uniaxial orientation throughout. This arrangement differs from the predominantly biaxial alignment of collagen fibrils that exists in the human cornea. By virtue of their high water content (90%), very fine collagen filaments (2–9 nm) and lack of cells, the collagen hydrogels were found to transmit almost all incident light in the visible spectrum. They also transmitted a large proportion of UV light compared to the cornea which acts as an effective UV filter. Patients implanted with these hydrogels should be cautious about UV exposure prior to regrowth of the epithelium and in-growth of corneal cells into the implants. PMID:26159106

  10. The structural and optical properties of type III human collagen biosynthetic corneal substitutes.

    PubMed

    Hayes, Sally; Lewis, Phillip; Islam, M Mirazul; Doutch, James; Sorensen, Thomas; White, Tomas; Griffith, May; Meek, Keith M

    2015-10-01

    The structural and optical properties of clinically biocompatible, cell-free hydrogels comprised of synthetically cross-linked and moulded recombinant human collagen type III (RHCIII) with and without the incorporation of 2-methacryloyloxyethyl phosphorylcholine (MPC) were assessed using transmission electron microscopy (TEM), X-ray scattering, spectroscopy and refractometry. These findings were examined alongside similarly obtained data from 21 human donor corneas. TEM demonstrated the presence of loosely bundled aggregates of fine collagen filaments within both RHCIII and RHCIII-MPC implants, which X-ray scattering showed to lack D-banding and be preferentially aligned in a uniaxial orientation throughout. This arrangement differs from the predominantly biaxial alignment of collagen fibrils that exists in the human cornea. By virtue of their high water content (90%), very fine collagen filaments (2-9 nm) and lack of cells, the collagen hydrogels were found to transmit almost all incident light in the visible spectrum. They also transmitted a large proportion of UV light compared to the cornea which acts as an effective UV filter. Patients implanted with these hydrogels should be cautious about UV exposure prior to regrowth of the epithelium and in-growth of corneal cells into the implants. PMID:26159106

  11. Enhanced stabilization of collagen by furfural.

    PubMed

    Lakra, Rachita; Kiran, Manikantan Syamala; Usha, Ramamoorthy; Mohan, Ranganathan; Sundaresan, Raja; Korrapati, Purna Sai

    2014-04-01

    Furfural (2-furancarboxaldehyde), a product derived from plant pentosans, has been investigated for its interaction with collagen. Introduction of furfural during fibril formation enhanced the thermal and mechanical stability of collagen. Collagen films treated with furfural exhibited higher denaturation temperature (Td) (p<0.04) and showed a 3-fold increase in Young's modulus (p<0.04) at higher concentration. Furfural and furfural treated collagen films did not have any cytotoxic effect. Rheological characterization showed an increase in shear stress and shear viscosity with increasing shear rate for treated collagen. Circular dichroism (CD) studies indicated that the furfural did not have any impact on triple helical structure of collagen. Scanning electron microscopy (SEM) of furfural treated collagen exhibited small sized porous structure in comparison with untreated collagen. Thus this study provides an alternate ecologically safe crosslinking agent for improving the stability of collagen for biomedical and industrial applications.

  12. The evolution of fibrillar collagens: a sea-pen collagen shares common features with vertebrate type V collagen.

    PubMed

    Tillet, E; Franc, J M; Franc, S; Garrone, R

    1996-02-01

    The extracellular matrix of marine primitive invertebrates (sponges, polyps and jellyfishes) contains collagen fibrils with narrow diameters. From various data, it has been hypothesized that these primitive collagens could represent ancestral forms of the vertebrate minor collagens, i.e., types V or XI. Recently we have isolated a primitive collagen from the soft tissues of the sea-pen Veretillum cynomorium. This report examines whether the sea-pen collagen shares some features with vertebrate type V collagen. Rotary shadowed images of acid-soluble collagen molecules extracted from beta-APN treated animals, positive staining of segment-long-spacing crystallites precipitated from pepsinized collagen, Western blots of the pepsinized alpha1 and alpha2 chains with antibodies to vertebrate types I, III and V collagens, and in situ gold immunolabeling of ECM collagen fibrils were examined. Our results showed that the tissue form of the sea-pen collagen is a 340-nm threadlike molecule, which is close to the vertebrate type V collagen with its voluminous terminal globular domain, the distribution of most of its polar amino-acid residues, and its antigenic properties.

  13. The evolution of fibrillar collagens: a sea-pen collagen shares common features with vertebrate type V collagen.

    PubMed

    Tillet, E; Franc, J M; Franc, S; Garrone, R

    1996-02-01

    The extracellular matrix of marine primitive invertebrates (sponges, polyps and jellyfishes) contains collagen fibrils with narrow diameters. From various data, it has been hypothesized that these primitive collagens could represent ancestral forms of the vertebrate minor collagens, i.e., types V or XI. Recently we have isolated a primitive collagen from the soft tissues of the sea-pen Veretillum cynomorium. This report examines whether the sea-pen collagen shares some features with vertebrate type V collagen. Rotary shadowed images of acid-soluble collagen molecules extracted from beta-APN treated animals, positive staining of segment-long-spacing crystallites precipitated from pepsinized collagen, Western blots of the pepsinized alpha1 and alpha2 chains with antibodies to vertebrate types I, III and V collagens, and in situ gold immunolabeling of ECM collagen fibrils were examined. Our results showed that the tissue form of the sea-pen collagen is a 340-nm threadlike molecule, which is close to the vertebrate type V collagen with its voluminous terminal globular domain, the distribution of most of its polar amino-acid residues, and its antigenic properties. PMID:8653581

  14. Fluorescent nanonetworks: A novel bioalley for collagen scaffolds and Tissue Engineering

    PubMed Central

    Nidhin, Marimuthu; Vedhanayagam, Mohan; Sangeetha, Selvam; Kiran, Manikantan Syamala; Nazeer, Shaiju S.; Jayasree, Ramapurath S.; Sreeram, Kalarical Janardhanan; Nair, Balachandran Unni

    2014-01-01

    Native collagen is arranged in bundles of aligned fibrils to withstand in vivo mechanical loads. Reproducing such a process under in vitro conditions has not met with major success. Our approach has been to induce nanolinks, during the self-assembly process, leading to delayed rather than inhibited fibrillogenesis. For this, a designed synthesis of nanoparticles - using starch as a template and a reflux process, which would provide a highly anisotropic (star shaped) nanoparticle, with large surface area was adopted. Anisotropy associated decrease in Morin temperature and superparamagnetic behavior was observed. Polysaccharide on the nanoparticle surface provided aqueous stability and low cytotoxicity. Starch coated nanoparticles was utilized to build polysaccharide - collagen crosslinks, which supplemented natural crosslinks in collagen, without disturbing the conformation of collagen. The resulting fibrillar lamellae showed a striking resemblance to native lamellae, but had a melting and denaturation temperature higher than native collagen. The biocompatibility and superparamagnetism of the nanoparticles also come handy in the development of stable collagen constructs for various biomedical applications, including that of MRI contrast agents. PMID:25095810

  15. Fluorescent nanonetworks: a novel bioalley for collagen scaffolds and tissue engineering.

    PubMed

    Nidhin, Marimuthu; Vedhanayagam, Mohan; Sangeetha, Selvam; Kiran, Manikantan Syamala; Nazeer, Shaiju S; Jayasree, Ramapurath S; Sreeram, Kalarical Janardhanan; Nair, Balachandran Unni

    2014-08-06

    Native collagen is arranged in bundles of aligned fibrils to withstand in vivo mechanical loads. Reproducing such a process under in vitro conditions has not met with major success. Our approach has been to induce nanolinks, during the self-assembly process, leading to delayed rather than inhibited fibrillogenesis. For this, a designed synthesis of nanoparticles - using starch as a template and a reflux process, which would provide a highly anisotropic (star shaped) nanoparticle, with large surface area was adopted. Anisotropy associated decrease in Morin temperature and superparamagnetic behavior was observed. Polysaccharide on the nanoparticle surface provided aqueous stability and low cytotoxicity. Starch coated nanoparticles was utilized to build polysaccharide - collagen crosslinks, which supplemented natural crosslinks in collagen, without disturbing the conformation of collagen. The resulting fibrillar lamellae showed a striking resemblance to native lamellae, but had a melting and denaturation temperature higher than native collagen. The biocompatibility and superparamagnetism of the nanoparticles also come handy in the development of stable collagen constructs for various biomedical applications, including that of MRI contrast agents.

  16. Fluorescent nanonetworks: A novel bioalley for collagen scaffolds and Tissue Engineering

    NASA Astrophysics Data System (ADS)

    Nidhin, Marimuthu; Vedhanayagam, Mohan; Sangeetha, Selvam; Kiran, Manikantan Syamala; Nazeer, Shaiju S.; Jayasree, Ramapurath S.; Sreeram, Kalarical Janardhanan; Nair, Balachandran Unni

    2014-08-01

    Native collagen is arranged in bundles of aligned fibrils to withstand in vivo mechanical loads. Reproducing such a process under in vitro conditions has not met with major success. Our approach has been to induce nanolinks, during the self-assembly process, leading to delayed rather than inhibited fibrillogenesis. For this, a designed synthesis of nanoparticles - using starch as a template and a reflux process, which would provide a highly anisotropic (star shaped) nanoparticle, with large surface area was adopted. Anisotropy associated decrease in Morin temperature and superparamagnetic behavior was observed. Polysaccharide on the nanoparticle surface provided aqueous stability and low cytotoxicity. Starch coated nanoparticles was utilized to build polysaccharide - collagen crosslinks, which supplemented natural crosslinks in collagen, without disturbing the conformation of collagen. The resulting fibrillar lamellae showed a striking resemblance to native lamellae, but had a melting and denaturation temperature higher than native collagen. The biocompatibility and superparamagnetism of the nanoparticles also come handy in the development of stable collagen constructs for various biomedical applications, including that of MRI contrast agents.

  17. Role of xenogenous bovine platelet gel embedded within collagen implant on tendon healing: an in vitro and in vivo study

    PubMed Central

    Oryan, Ahmad; Meimandi-Parizi, Abdolhamid; Maffulli, Nicola

    2015-01-01

    Surgical reconstruction of large Achilles tendon defects is demanding. Platelet concentrates may be useful to favor healing in such conditions. The characteristics of bovine platelet-gel embedded within a collagen-implant were determined in vitro, and its healing efficacy was examined in a large Achilles tendon defect in rabbits. Two cm of the left Achilles tendon of 60 rabbits were excised, and the animals were randomly assigned to control (no implant), collagen-implant, or bovine-platelet-gel-collagen-implant groups. The tendon edges were maintained aligned using a Kessler suture. No implant was inserted in the control group. In the two other groups, a collagen-implant or bovine-platelet-gel-collagen-implant was inserted in the defect. The bioelectricity and serum platelet-derived growth factor levels were measured weekly and at 60 days post injury, respectively. After euthanasia at 60 days post injury, the tendons were tested at macroscopic, microscopic, and ultrastructural levels, and their dry matter and biomechanical performances were also assessed. Another 60 rabbits were assigned to receive no implant, a collagen-implant, or a bovine-platelet-gel-collagen-implant, euthanized at 10, 20, 30, and 40 days post injury, and their tendons were evaluated grossly and histologically to determine host-graft interactions. Compared to the control and collagen-implant, treatment with bovine-platelet-gel-collagen-implant improved tissue bioelectricity and serum platelet-derived growth factor levels, and increased cell proliferation, differentiation, and maturation. It also increased number, diameter, and density of the collagen fibrils, alignment and maturation of the collagen fibrils and fibers, biomechanical properties and dry matter content of the injured tendons at 60 days post injury. The bovine-platelet-gel-collagen-implant also increased biodegradability, biocompatibility, and tissue incorporation behavior of the implant compared to the collagen-implant alone

  18. Kinetic theory of amyloid fibril templating.

    PubMed

    Schmit, Jeremy D

    2013-05-14

    The growth of amyloid fibrils requires a disordered or partially unfolded protein to bind to the fibril and adapt the same conformation and alignment established by the fibril template. Since the H-bonds stabilizing the fibril are interchangeable, it is inevitable that H-bonds form between incorrect pairs of amino acids which are either incorporated into the fibril as defects or must be broken before the correct alignment can be found. This process is modeled by mapping the formation and breakage of H-bonds to a one-dimensional random walk. The resulting microscopic model of fibril growth is governed by two timescales: the diffusion time of the monomeric proteins, and the time required for incorrectly bound proteins to unbind from the fibril. The theory predicts that the Arrhenius behavior observed in experiments is due to off-pathway states rather than an on-pathway transition state. The predicted growth rates are in qualitative agreement with experiments on insulin fibril growth rates as a function of protein concentration, denaturant concentration, and temperature. These results suggest a templating mechanism where steric clashes due to a single mis-aligned molecule prevent the binding of additional molecules.

  19. Atrial Fibrillation

    MedlinePlus

    ... with the speed or rhythm of the heartbeat. Atrial fibrillation (AF) is the most common type of arrhythmia. The ... the heart's electrical system. Often, people who have AF may not even feel symptoms. But you may ...

  20. Ventricular fibrillation

    MedlinePlus

    ... seconds, it can lead to fainting (syncope) or cardiac arrest. Fibrillation is an uncontrolled twitching or quivering of ... pubmed/23801105 . Myerburg RJ, Castellanos A. Approach to cardiac arrest and life-threatening arrhythmias. In: Goldman L, Schafer ...

  1. Birefringence and second harmonic generation on tendon collagen following red linearly polarized laser irradiation.

    PubMed

    Silva, Daniela Fátima Teixeira; Gomes, Anderson Stevens Leonidas; de Campos Vidal, Benedicto; Ribeiro, Martha Simões

    2013-04-01

    Regarding the importance of type I collagen in understanding the mechanical properties of a range of tissues, there is still a gap in our knowledge of how proteins perform such work. There is consensus in literature that the mechanical characteristics of a tissue are primarily determined by the organization of its molecules. The purpose of this study was to characterize the organization of non-irradiated and irradiated type I collagen. Irradiation was performed with a linearly polarized HeNe laser (λ = 632.8 nm) and characterization was undertaken using polarized light microscopy to investigate the birefringence and second harmonic generation to analyze nonlinear susceptibility. Rats received laser irradiation (P = 6.0 mW, I = 21.2 mW/cm(2), E ≈ 0.3 J, ED = 1.0 J/cm(2)) on their healthy Achilles tendons, which after were extracted to prepare the specimens. Our results show that irradiated samples present higher birefringence and greater non-linear susceptibility than non-irradiated samples. Under studied conditions, we propose that a red laser with polarization direction aligned in parallel to the tendon long axis promotes further alignment on the ordered healthy collagen fibrils towards the electric field incident. Thus, prospects for biomedical applications for laser polarized radiation on type I collagen are encouraging since it supports greater tissue organization. PMID:23247985

  2. Collagen-silica hybrid materials: sodium silicate and sodium chloride effects on type I collagen fibrillogenesis.

    PubMed

    Eglin, David; Coradin, Thibaud; Giraud Guille, Marie M; Helary, Christophe; Livage, Jacques

    2005-01-01

    Collagen-silica hybrid materials have been considered for potential biomedical applications. Understanding of the collagen-silica interactions is the key to control hybrids structure and properties. For this purpose, the effect of sodium silicate and sodium chloride addition at two concentrations, 0.83 and 10 mM, on the kinetic of the type I collagen fibrillogenesis at 20 degrees C, and pH 7.4 were studied. Absorbance profiles of fibrillogenesis experiments were collected together with measures of silicic acid concentration and transmission electron microscopy analysis. The specific effect of silica addition on the collagen fibrils self-assembly mechanisms was demonstrated by comparison with the sodium chloride. Sodium silicate at 10 mM inhibited the collagen fibrillogenesis. At the same concentration, the sodium chloride decreased the rate of the collagen fibril assembly. Collagen fibrillogenesis kinetic was not significantly disturbed by the presence of 0.83 mM of sodium chloride. However, the same concentration of sodium silicate modified the collagen fibrillogenesis kinetic. Transmission electron microscopy indicated for experiment with 0.83 mM of sodium silicate, the formation of longer and wider fibrils than for the equivalent collagen fibrillogenesis experiment with sodium chloride. The effect of sodium chloride is explained in terms of osmotic exclusion and influence on electrostatic interactions between collagen fibrils. The specific involvement of silicic acid in collagen helices hydrogen-bond interactions is suggested. Finally, the results of this study are discussed regarding the preparation of composites by co-gelation of type I collagen and sodium silicate, for potential application as bone repair device.

  3. Type I Collagen and Collagen Mimetics as Angiogenesis Promoting Superpolymers

    SciTech Connect

    Twardowski, T.; Fertala, A.; Orgel, J.P.R.O.; San Antonio, J.D.

    2008-07-18

    Angiogenesis, the development of blood vessels from the pre-existing vasculature, is a key component of embryogenesis and tissue regeneration. Angiogenesis also drives pathologies such as tumor growth and metastasis, and hemangioma development in newborns. On the other hand, promotion of angiogenesis is needed in tissues with vascular insufficiencies, and in bioengineering, to endow tissue substitutes with appropriate microvasculatures. Therefore, much research has focused on defining mechanisms of angiogenesis, and identifying pro- and anti-angiogenic molecules. Type I collagen, the most abundant protein in humans, potently stimulates angiogenesis in vitro and in vivo. Crucial to its angiogenic activity appears to be ligation and possibly clustering of endothelial cell (EC) surface {alpha}1{beta}1/{alpha}2{beta}1 integrin receptors by the GFPGER502-507 sequence of the collagen fibril. However, additional aspects of collagen structure and function that may modulate its angiogenic properties are discussed. Moreover, type I collagen and fibrin, another angiogenic polymer, share several structural features. These observations suggest strategies for creating 'angiogenic superpolymers', including: modifying type I collagen to influence its biological half-life, immunogenicity, and integrin binding capacity; genetically engineering fibrillar collagens to include additional integrin binding sites or angiogenic determinants, and remove unnecessary or deleterious sequences without compromising fibril integrity; and exploring the suitability of poly(ortho ester), PEG-lysine copolymer, tubulin, and cholesteric cuticle as collagen mimetics, and suggesting means of modifying them to display ideal angiogenic properties. The collagenous and collagen mimetic angiogenic superpolymers described here may someday prove useful for many applications in tissue engineering and human medicine.

  4. WOUND HEALING AND COLLAGEN FORMATION

    PubMed Central

    Ross, Russell; Benditt, Earl P.

    1961-01-01

    The regular sequence encountered in healing guinea pig skin wounds has been examined by methods of light and electron microscopy. Observations on cell populations, their fine structure, and fibril formation in the connective tissue have been made. Linear incisions in the skin of normal female guinea pigs weighing 300 to 350 grams were allowed to heal. The wounds were then excised, fixed with buffered 2 per cent osmium tetroxide, and postfixed in neutral buffered formalin, at 16 and 24 hours and at 3, 5, 9, and 14 days after wounding. They were then embedded in epoxy resin. In the inflammatory phase the exudate observed in the early wounds consists largely of polymorphonuclear neutrophilic leukocytes, macrophages, fibrin, and free extracellular organelles from the disrupted inflammatory cells. These organelles later appear in vacuoles in the cytoplasm of the macrophages. Fibroblasts first appear at 24 hours, and show extensive development and dilatation of the endoplasmic reticulum, which sometimes contains moderately dense flocculent material. In addition, these fibroblasts have enlarged mitochondria and condensations of filamentous material within the cytoplasm near the cell surface. Occasional myelin figures and moderately dense, 0.5 to 1.0 micron bodies are found within the cytoplasm of the early fibroblasts. Collagen fibrils are first seen at 3 days extracellularly near the cell surfaces. They appear at the later times in two populations of sizes. With increasing wound age the fibroblasts retain their morphology and the wounds decrease in cellularity concomitantly with the formation of increasing amounts of collagen. Several proposed mechanisms of collagen fibril formation are discussed in relation to the observed phenomena. The problem of correlating fibril diameter with the appearance of the periodic structure of collagen in relation to the minimal size fibril which would be anticipated to display this appearance is discussed. PMID:14494202

  5. Spiroplasma fibrils.

    PubMed

    Williamson, D L; Brink, P R; Zieve, G W

    1984-09-01

    A fundamental question in biology concerns the morphology of spiroplasmas: How does a wall-less microorganism maintain its characteristic morphology as a helical filament? An answer to this question began to form when it was discovered that spiroplasmas treated with any of a number of detergents (sodium deoxycholate, Triton X-100, Nonidet P-40) release their cytoplasmic contents. If this procedure is performed on a formvar-coated electron microscope grid and the resultant preparation negatively stained and observed by transmission electron microscopy, numerous striated microfibrils can be seen where spiroplasmas once were. The fibrils are of varying lengths, 4 nm in width, and show a striation repeat at 9 nm along their length. It is not possible to discern from the pattern of the released fibrils just how they are organized within the intact spiroplasma; nor is it yet possible to identify a fibrillar substructure in thin sections or in freeze-fractured organisms. Townsend and his colleagues at the John Innes Institute in Norwich, UK, purified fibrils by density gradient centrifugation. SDS-PAGE showed the fibrils to consist of a 55,000-dalton protein recognizable in the four serogroups tested by protein blotting with an antiserum made against the PAGE-separated protein. The presence of fibrils is a feature common to all spiroplasma, regardless of whether they are helical or nonhelical, as in the Ixodes tick-derived spiroplasma or Townsend's ASP-1 strain of Spiroplasma citri. We have employed gentle demembranation treatments that preserve filamentous substructure in an effort to elucidate the organization of the fibrils within the spiroplasma cell.

  6. A continuum model for hierarchical fibril assembly

    NASA Astrophysics Data System (ADS)

    van Lith, B. S.; Muntean, A.; Storm, C.

    2014-06-01

    Most of the biological polymers that make up our cells and tissues are hierarchically structured. For biopolymers ranging from collagen, to actin, to fibrin and amyloid fibrils this hierarchy provides vitally important versatility. The structural hierarchy must be encoded in the self-assembly process, from the earliest stages onward, in order to produce the appropriate substructures. In this letter, we explore the kinetics of multistage self-assembly processes in a model system which allows comparison to bulk probes such as light scattering. We apply our model to recent turbidimetry data on the self-assembly of collagen fibrils. Our analysis suggests a connection between diffusion-limited aggregation kinetics and fibril growth, supported by slow, power-law growth at very long time scales.

  7. Collagenous gastroduodenitis.

    PubMed

    Rustagi, Tarun; Rai, Mridula; Scholes, John V

    2011-10-01

    Collagenous gastroduodenitis is a rare histopathologic entity characterized by marked subepithelial collagen deposition with associated mucosal inflammatory infiltrate. Only 4 cases have been reported, of which 3 had associated collagenous colitis. Collagenous gastroduodenitis without colonic involvement is exceptionally rare with only 1 case reported so far in the literature. We present a case of a 68-year-old woman with dyspepsia and mild anemia, who was found to have nodular gastric and duodenal mucosa on endoscopic examination. Histopathology showed collagenous gastroduodenitis. To the best of our knowledge, this is the second (and first in English literature) reported case of isolated collagenous gastroduodenitis.

  8. Collagenous gastroduodenitis.

    PubMed

    Rustagi, Tarun; Rai, Mridula; Scholes, John V

    2011-10-01

    Collagenous gastroduodenitis is a rare histopathologic entity characterized by marked subepithelial collagen deposition with associated mucosal inflammatory infiltrate. Only 4 cases have been reported, of which 3 had associated collagenous colitis. Collagenous gastroduodenitis without colonic involvement is exceptionally rare with only 1 case reported so far in the literature. We present a case of a 68-year-old woman with dyspepsia and mild anemia, who was found to have nodular gastric and duodenal mucosa on endoscopic examination. Histopathology showed collagenous gastroduodenitis. To the best of our knowledge, this is the second (and first in English literature) reported case of isolated collagenous gastroduodenitis. PMID:21346601

  9. Magnetic Resonance Microscopy of Collagen Mineralization

    PubMed Central

    Chesnick, Ingrid E.; Mason, Jeffrey T.; Giuseppetti, Anthony A.; Eidelman, Naomi; Potter, Kimberlee

    2008-01-01

    A model mineralizing system was subjected to magnetic resonance microscopy to investigate how water proton transverse (T2) relaxation times and magnetization transfer ratios can be applied to monitor collagen mineralization. In our model system, a collagen sponge was mineralized with polymer-stabilized amorphous calcium carbonate. The lower hydration and water proton T2 values of collagen sponges during the initial mineralization phase were attributed to the replacement of the water within the collagen fibrils by amorphous calcium carbonate. The significant reduction in T2 values by day 6 (p < 0.001) was attributed to the appearance of mineral crystallites, which were also detected by x-ray diffraction and scanning electron microscopy. In the second phase, between days 6 and 13, magnetic resonance microscopy properties appear to plateau as amorphous calcium carbonate droplets began to coalesce within the intrafibrillar space of collagen. In the third phase, after day 15, the amorphous mineral phase crystallized, resulting in a reduction in the absolute intensity of the collagen diffraction pattern. We speculate that magnetization transfer ratio values for collagen sponges, with similar collagen contents, increased from 0.25 ± 0.02 for control strips to a maximum value of 0.31 ± 0.04 at day 15 (p = 0.03) because mineral crystals greatly reduce the mobility of the collagen fibrils. PMID:18487295

  10. Collagens in the aged human macula.

    PubMed

    Marshall, G E; Konstas, A G; Reid, G G; Edwards, J G; Lee, W R

    1994-03-01

    Immunogold cytochemistry was used to investigate the fine structural distribution of collagen types I-VI in Bruch's membrane and choroid of the aged human macula. Macular tissue was obtained from ten eyes, and processed for cryoultramicrotomy and London Resin white embedding. Striated collagen fibrils within the inner and outer collagenous layers were found to contain collagen types I, III and V. In addition, type V collagen was also present in the basement membrane of the choriocapillaris. Gross thickening of the choriocapillaris basement membrane was attributed to the deposition of type IV collagen. However, type IV collagen appeared to be absent from the basement membrane of the retinal pigment epithelium. The interesting location of type VI collagen on the choroidal side of the choriocapillaris suggested that its function is to anchor the choriocapillaris onto the choroid. The collagens studied were absent from fibrous banded material, long-spacing collagen, the elastic layer and amorphous granular material. It was concluded that, of the collagen types studied, only the deposition of type IV collagen contributes to the age-related thickening of Bruch's membrane.

  11. In-situ Damage Assessment of Collagen within Ancient Manuscripts Written on Parchment: A Polarized Raman Spectroscopy Approach

    NASA Astrophysics Data System (ADS)

    Schütz, R.; Rabin, I.; Hahn, O.; Fratzl, P.; Masic, A.

    2010-08-01

    The collection generally known as Qumran scrolls or Dead Sea Scrolls (DSS) comprises some 900 highly fragmented manuscripts (mainly written on parchment) from the Second Temple period. In the years since their manufacture the writing materials have undergone serious deterioration due to a combination of natural ageing and environmental effects. Therefore, understanding quantitatively state of conservation of such manuscripts is a challenging task and a deep knowledge of damage pathways on all hierarchical levels (from molecular up to macroscopic) results of fundamental importance for a correct protection and conservation strategy. However, the degradation of parchments is very complex and not well understood process. Parchment is a final product of processing of animal skin and consist mainly of type I collagen, which is the most abundant constituent of the dermal matrix. Collagen molecule is built by folding of three polypeptide α-chains into a right-handed triple helix. Every α-chain is made by a repetitive sequence of (Gly-X-Y)n, where X and Y are often proline and hydroxyproline. Parallel and staggered collagen triple helices associate into fibrils, which than assemble into fibers. Deterioration of parchment is caused by chemical changes due to gelatinization, oxidation and hydrolysis of the collagen chains, promoted by several factors, summarized as biological and microbiological (bacteria, fungi etc.), heat, light, humidity and pollutants (1, 2). In this work we have focused on studying the collagen within parchments on two different levels of organization (molecular and fibrilar) by applying polarized Raman spectroscopic technique. Beside spectral information related to chemical bonding, polarization anisotropy of some collagen bands (i.e. amide I) has been used to explore organization of collagen on higher levels (three-dimensional arrangement of the triple-helix molecules and their alignment within a fibril of collagen). To this aim we have compared

  12. Collagen fillers.

    PubMed

    Baumann, Leslie; Kaufman, Joely; Saghari, Sogol

    2006-01-01

    Collagen implants, both animal and human derived, have been used for soft tissue augmentation for many years. Bovine collagen fillers were the most popular injectable implants for nearly two decades in the United States. Since then, human bioengineered collagen products have been available in addition to hyaluronic acid-containing fillers. This article outlines the different types of injectable collagen implants, injection techniques, preferred methods of treatment, and possible adverse reactions to the injectable materials.

  13. Atrial Fibrillation.

    PubMed

    Goralnick, Eric; Bontempo, Laura J

    2015-08-01

    Atrial fibrillation (AF) is a supraventricular tachyarrhythmia that results from the chaotic depolarization of atrial tissue. AF is the most common sustained cardiac dysrhythmia and the most common dysrhythmia diagnosed in US emergency departments. All patients with AF must have their cardioembolic risk assessed, even if sinus rhythm is restored. Novel oral anticoagulants may be considered instead of vitamin K antagonists for anticoagulation in patients with nonvalvular AF. PMID:26226868

  14. Ultrastructural and biochemical characterization of mechanically adaptable collagenous structures in the edible sea urchin Paracentrotus lividus.

    PubMed

    Barbaglio, Alice; Tricarico, Serena; Ribeiro, Ana R; Di Benedetto, Cristiano; Barbato, Marta; Dessì, Desirèe; Fugnanesi, Valeria; Magni, Stefano; Mosca, Fabio; Sugni, Michela; Bonasoro, Francesco; Barbosa, Mario A; Wilkie, Iain C; Candia Carnevali, M Daniela

    2015-06-01

    The viscoelastic properties of vertebrate connective tissues rarely undergo significant changes within physiological timescales, the only major exception being the reversible destiffening of the mammalian uterine cervix at the end of pregnancy. In contrast to this, the connective tissues of echinoderms (sea urchins, starfish, sea cucumbers, etc.) can switch reversibly between stiff and compliant conditions in timescales of around a second to minutes. Elucidation of the molecular mechanism underlying such mutability has implications for the zoological, ecological and evolutionary field. Important information could also arise for veterinary and biomedical sciences, particularly regarding the pathological plasticization or stiffening of connective tissue structures. In the present investigation we analyzed aspects of the ultrastructure and biochemistry in two representative models, the compass depressor ligament and the peristomial membrane of the edible sea urchin Paracentrotus lividus, compared in three different mechanical states. The results provide further evidence that the mechanical adaptability of echinoderm connective tissues does not necessarily imply changes in the collagen fibrils themselves. The higher glycosaminoglycan (GAG) content registered in the peristomial membrane with respect to the compass depressor ligament suggests a diverse role of these molecules in the two mutable collagenous tissues. The possible involvement of GAG in the mutability phenomenon will need further clarification. During the shift from a compliant to a standard condition, significant changes in GAG content were detected only in the compass depressor ligament. Similarities in terms of ultrastructure (collagen fibrillar assembling) and biochemistry (two alpha chains) were found between the two models and mammalian collagen. Nevertheless, differences in collagen immunoreactivity, alpha chain migration on SDS-PAGE and BLAST alignment highlighted the uniqueness of sea urchin

  15. Anisotropy of chemical bonds in collagen molecules studied by X-ray absorption near-edge structure (XANES) spectroscopy.

    PubMed

    Lam, Raymond S K; Metzler, Rebecca A; Gilbert, Pupa U P A; Beniash, Elia

    2012-03-16

    Collagen type I fibrils are the major building blocks of connective tissues. Collagen fibrils are anisotropic supramolecular structures, and their orientation can be revealed by polarized light microscopy and vibrational microspectroscopy. We hypothesized that the anisotropy of chemical bonds in the collagen molecules, and hence their orientation, might also be detected by X-ray photoemission electron spectromicroscopy (X-PEEM) and X-ray absorption near-edge structure (XANES) spectroscopy, which use linearly polarized synchrotron light. To test this hypothesis, we analyzed sections of rat-tail tendon, composed of parallel arrays of collagen fibrils. The results clearly indicate that XANES-PEEM is sensitive to collagen fibril orientation and, more specifically, to the orientations of carbonyl and amide bonds in collagen molecules. These data suggest that XANES-PEEM is a promising technique for characterizing the chemical composition and structural organization at the nanoscale of collagen-based connective tissues, including tendons, cartilage, and bone.

  16. Collagen interactions: Drug design and delivery.

    PubMed

    An, Bo; Lin, Yu-Shan; Brodsky, Barbara

    2016-02-01

    Collagen is a major component in a wide range of drug delivery systems and biomaterial applications. Its basic physical and structural properties, together with its low immunogenicity and natural turnover, are keys to its biocompatibility and effectiveness. In addition to its material properties, the collagen triple-helix interacts with a large number of molecules that trigger biological events. Collagen interactions with cell surface receptors regulate many cellular processes, while interactions with other ECM components are critical for matrix structure and remodeling. Collagen also interacts with enzymes involved in its biosynthesis and degradation, including matrix metalloproteinases. Over the past decade, much information has been gained about the nature and specificity of collagen interactions with its partners. These studies have defined collagen sequences responsible for binding and the high-resolution structures of triple-helical peptides bound to its natural binding partners. Strategies to target collagen interactions are already being developed, including the use of monoclonal antibodies to interfere with collagen fibril formation and the use of triple-helical peptides to direct liposomes to melanoma cells. The molecular information about collagen interactions will further serve as a foundation for computational studies to design small molecules that can interfere with specific interactions or target tumor cells. Intelligent control of collagen biological interactions within a material context will expand the effectiveness of collagen-based drug delivery.

  17. Collagen telopeptides (cross-linking sites) play a role in collagen gel lattice contraction

    NASA Technical Reports Server (NTRS)

    Woodley, D. T.; Yamauchi, M.; Wynn, K. C.; Mechanic, G.; Briggaman, R. A.

    1991-01-01

    Solubilized interstitial collagens will form a fibrillar, gel-like lattice when brought to physiologic conditions. In the presence of human dermal fibroblasts the collagen lattice will contract. The rate of contraction can be determined by computer-assisted planemetry. The mechanisms involved in contraction are as yet unknown. Using this system it was found that the rate of contraction was markedly decreased when collagen lacking telopeptides was substituted for native collagen. Histidinohydroxylysinonorleucine (HHL) is a major stable trifunctional collagen cross-link in mature skin that involves a carboxyl terminal, telopeptide site 16c, the sixteenth amino acid residue from the carboxy terminal of the telopeptide region of alpha 1 (I) in type I collagen. Little, if any, HHL was present in native, purified, reconstituted, soluble collagen fibrils from 1% acetic acid-extracted 2-year-old bovine skin. In contrast, HHL cross-links were present (0.22 moles of cross-link per mole of collagen) in lattices of the same collagen contracted by fibroblasts. However, rat tail tendon does not contain HHL cross-links, and collagen lattices made of rat tail tendon collagen are capable of contraction. This suggests that telopeptide sites, and not mature HHL cross-links per se, are essential for fibroblasts to contract collagen lattices. Beta-aminopropionitrile fumarate (BAPN), a potent lathyrogen that perturbs collagen cross-linking by inhibition of lysyl oxidase, also inhibited the rate of lattice cell contraction in lattices composed of native collagen. However, the concentrations of BAPN that were necessary to inhibit the contraction of collagen lattices also inhibited fibroblast growth suggestive of cellular toxicity. In accordance with other studies, we found no inhibition of the rate of lattice contraction when fibronectin-depleted serum was used. Electron microscopy of contracted gels revealed typical collagen fibers with a characteristic axial periodicity. The data

  18. Micromechanical Model of a Surrogate for Collagenous Soft Tissues: Development, Validation and Analysis of Mesoscale Size Effects

    PubMed Central

    Reese, Shawn P.; Ellis, Benjamin J.; Weiss, Jeffrey A.

    2013-01-01

    Aligned, collagenous tissues such as tendons and ligaments are composed primarily of water and type I collagen, organized hierarchically into nanoscale fibrils, microscale fibers and mesoscale fascicles. Force transfer across scales is complex and poorly understood. Since innervation, the vasculature, damage mechanisms and mechanotransduction occur at the microscale and mesoscale, understanding multiscale interactions is of high importance. This study used a physical model in combination with a computational model to isolate and examine the mechanisms of force transfer between scales. A collagen-based surrogate served as the physical model. The surrogate consisted of extruded collagen fibers embedded within a collagen gel matrix. A micromechanical finite element model of the surrogate was validated using tensile test data that was recorded using a custom tensile testing device mounted on a confocal microscope. Results demonstrated that the experimentally measured macroscale strain was not representative of the microscale strain, which was highly inhomogeneous. The micromechanical model, in combination with a macroscopic continuum model, revealed that the microscale inhomogeneity resulted from size effects in the presence of a constrained boundary. A sensitivity study indicated that significant scale effects would be present over a range of physiologically relevant inter-fiber spacing values and matrix material properties. The results indicate that the traditional continuum assumption is not valid for describing the macroscale behavior of the surrogate, and that boundary-induced size effects are present. PMID:23400805

  19. Micromechanical model of a surrogate for collagenous soft tissues: development, validation and analysis of mesoscale size effects.

    PubMed

    Reese, Shawn P; Ellis, Benjamin J; Weiss, Jeffrey A

    2013-11-01

    Aligned, collagenous tissues such as tendons and ligaments are composed primarily of water and type I collagen, organized hierarchically into nanoscale fibrils, microscale fibers and mesoscale fascicles. Force transfer across scales is complex and poorly understood. Since innervation, the vasculature, damage mechanisms and mechanotransduction occur at the microscale and mesoscale, understanding multiscale interactions is of high importance. This study used a physical model in combination with a computational model to isolate and examine the mechanisms of force transfer between scales. A collagen-based surrogate served as the physical model. The surrogate consisted of extruded collagen fibers embedded within a collagen gel matrix. A micromechanical finite element model of the surrogate was validated using tensile test data that were recorded using a custom tensile testing device mounted on a confocal microscope. Results demonstrated that the experimentally measured macroscale strain was not representative of the microscale strain, which was highly inhomogeneous. The micromechanical model, in combination with a macroscopic continuum model, revealed that the microscale inhomogeneity resulted from size effects in the presence of a constrained boundary. A sensitivity study indicated that significant scale effects would be present over a range of physiologically relevant inter-fiber spacing values and matrix material properties. The results indicate that the traditional continuum assumption is not valid for describing the macroscale behavior of the surrogate and that boundary-induced size effects are present.

  20. Atrial fibrillation.

    PubMed

    Essential facts Atrial fibrillation (AF) causes an abnormal, sometimes fast pulse, and is the most common heart rhythm disturbance. It occurs when electrical impulses controlling the heart's natural rhythm lose co-ordination. People with AF have a four or five times higher risk of stroke because it increases the risk of a blood clot forming in the chambers of the heart. The condition is responsible for 22,500 strokes a year in the UK, according to the British Heart Foundation (BHF). PMID:24593083

  1. Atrial fibrillation

    PubMed Central

    Munger, Thomas M.; Wu, Li-Qun; Shen, Win K.

    2014-01-01

    Atrial fibrillation is the most common arrhythmia affecting patients today. Disease prevalence is increasing at an alarming rate worldwide, and is associated with often catastrophic and costly consequences, including heart failure, syncope, dementia, and stroke. Therapies including anticoagulants, anti-arrhythmic medications, devices, and non-pharmacologic procedures in the last 30 years have improved patients' functionality with the disease. Nonetheless, it remains imperative that further research into AF epidemiology, genetics, detection, and treatments continues to push forward rapidly as the worldwide population ages dramatically over the next 20 years. PMID:24474959

  2. Feasibility study of the natural derived chitosan dialdehyde for chemical modification of collagen.

    PubMed

    Liu, Xinhua; Dan, Nianhua; Dan, Weihua; Gong, Juxia

    2016-01-01

    The aim of this study is to evaluate the chemical crosslinking effects of the natural derived chitosan dialdehyde (OCS) on collagen. Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC) and circular dichroism (CD) measurements suggest that introducing OCS might not destroy the natural triple helix conformation of collagen but enhance the thermal-stability of collagen. Meanwhile, a denser fibrous network of cross-linked collagen is observed by atomic force microscopy. Further, scanning electron microscopy (SEM) and aggregation kinetics analysis confirm that the fibrillation process of collagen advances successfully and OCS could lengthen the completion time of collagen fibrillogenesis but raise the reconstitution rate of collagen fibrils or microfibrils. Besides, the cytocompatibility analysis implies that when the dosage of OCS is less than 15%, introducing OCS into collagen might be favorable for the cell's adhesion, growth and proliferation. Taken as a whole, the present study demonstrates that OCS might be an ideal crosslinker for the chemical fixation of collagen.

  3. Collagenous gastritis.

    PubMed

    Colletti, R B; Trainer, T D

    1989-12-01

    Subepithelial fibrosis has previously been reported in the small intestine (collagenous sprue) and colon (collagenous colitis). We report a 15-yr-old girl with chronic gastritis and subepithelial fibrosis of the gastric corpus who presented with recurrent abdominal pain and acute upper gastrointestinal bleeding. Nodularity and erythema of the gastric corpus were persistent endoscopic findings. Biopsies revealed patchy chronic active gastritis with a striking focal thick band of collagen immediately beneath the surface epithelial cells that did not extend to deeper portions of the lamina propria. Contrast radiography demonstrated an abnormal mucosa of the gastric corpus with a mosaiclike surface pattern. Numerous studies have failed to elucidate the etiology. Despite treatment with ranitidine, sucralfate, and furazolidone, there has been no clinical or pathologic improvement. The pathogenesis and prognosis of collagenous gastritis, and its relationship to collagenous sprue and collagenous colitis, remain to be defined. PMID:2583419

  4. Microstructural Characterization of Vocal Folds toward a Strain-Energy Model of Collagen Remodeling

    PubMed Central

    Miri, Amir K.; Heris, Hossein K.; Tripathy, Umakanta; Wiseman, Paul W.; Mongeau, Luc

    2013-01-01

    Collagen fibrils are believed to control the immediate deformation of soft tissues under biomechanical load. Most extracellular matrix proteins remain intact during frozen sectioning, which allows them to be scanned using atomic force microscopy (AFM). Collagen fibrils are distinguishable because of their helical shape. In the present study, the shape and organization of collagen fibrils in dissected porcine vocal folds were quantified using nonlinear laser scanning microscopy data at the micrometer scale and AFM data at the nanometer scale. Rope-shape collagen fibrils were observed. Geometric characteristics for the fibrils were fed to a hyperelastic model to predict the biomechanical response of the tissue. The model simulates the micrometer-scale unlocking behavior of collagen bundles when extended from their unloaded configuration. Force spectroscopy using AFM was used to estimate the stiffness of collagen fibrils (1 ± 0.5 MPa). The presence of rope-shape fibrils is postulated to change the slope of the force-deflection response near the onset of nonlinearity. The proposed model could ultimately be used to evaluate changes in elasticity of soft tissues that result from the collagen remodeling. PMID:23643604

  5. Collagenous gastritis.

    PubMed

    Jain, Richa; Chetty, Runjan

    2010-12-01

    A 25-year-old patient presented with epigastric pain, which on gastric biopsy revealed the characteristic appearance of collagenous gastritis. There was a thick prominent subepithelial band that was confirmed to be collagen with a Masson's trichrome stain. There was associated Helicobacter pylori gastritis but no evidence of a lymphocytic gastritis. The patient did not have watery diarrhea. Collagenous gastritis can occur in young patients, be restricted to the stomach, and can be associated with celiac disease. PMID:19103610

  6. Nonlinear microscopy of collagen fibers

    NASA Astrophysics Data System (ADS)

    Strupler, M.; Pena, A.-M.; Hernest, M.; Tharaux, P.-L.; Fabre, A.; Marchal-Somme, J.; Crestani, B.; Débarre, D.; Martin, J.-L.; Beaurepaire, E.; Schanne-Klein, M.-C.

    2007-02-01

    We used intrinsic Second Harmonic Generation (SHG) by fibrillar collagen to visualize the three-dimensional architecture of collagen fibrosis at the micrometer scale using laser scanning nonlinear microscopy. We showed that SHG signals are highly specific to fibrillar collagen and provide a sensitive probe of the micrometer-scale structural organization of collagen in tissues. Moreover, recording simultaneously other nonlinear optical signals in a multimodal setup, we visualized the tissue morphology using Two-Photon Excited Fluorescence (2PEF) signals from endogenous chromophores such as NADH or elastin. We then compared different methods to determine accurate indexes of collagen fibrosis using nonlinear microscopy, given that most collagen fibrils are smaller than the microscope resolution and that second harmonic generation is a coherent process. In order to define a robust method to process our three-dimensional images, we either calculated the fraction of the images occupied by a significant SHG signal, or averaged SHG signal intensities. We showed that these scores provide an estimation of the extension of renal and pulmonary fibrosis in murine models, and that they clearly sort out the fibrotic mice.

  7. Human collagen produced in plants

    PubMed Central

    Shoseyov, Oded; Posen, Yehudit; Grynspan, Frida

    2014-01-01

    Consequential to its essential role as a mechanical support and affinity regulator in extracellular matrices, collagen constitutes a highly sought after scaffolding material for regeneration and healing applications. However, substantiated concerns have been raised with regard to quality and safety of animal tissue-extracted collagen, particularly in relation to its immunogenicity, risk of disease transmission and overall quality and consistency. In parallel, contamination with undesirable cellular factors can significantly impair its bioactivity, vis-a-vis its impact on cell recruitment, proliferation and differentiation. High-scale production of recombinant human collagen Type I (rhCOL1) in the tobacco plant provides a source of an homogenic, heterotrimeric, thermally stable “virgin” collagen which self assembles to fine homogenous fibrils displaying intact binding sites and has been applied to form numerous functional scaffolds for tissue engineering and regenerative medicine. In addition, rhCOL1 can form liquid crystal structures, yielding a well-organized and mechanically strong membrane, two properties indispensable to extracellular matrix (ECM) mimicry. Overall, the shortcomings of animal- and cadaver-derived collagens arising from their source diversity and recycled nature are fully overcome in the plant setting, constituting a collagen source ideal for tissue engineering and regenerative medicine applications. PMID:23941988

  8. Chondrogenic differentiation of human mesenchymal stem cells on fish scale collagen.

    PubMed

    Hsu, Han-Hsiu; Uemura, Toshimasa; Yamaguchi, Isamu; Ikoma, Toshiyuki; Tanaka, Junzo

    2016-08-01

    Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro. PMID:26829997

  9. Probing interactions between collagen proteins via microrheology

    NASA Astrophysics Data System (ADS)

    Shayegan, Marjan; Forde, Nancy R.

    2012-10-01

    Collagen is the major structural protein of our connective tissues. It provides integrity and mechanical strength through its hierarchical organization. Defects in collagen can lead to serious connective tissue diseases. Collagen is also widely used as a biomaterial. Given that mechanical properties are related to the structure of materials, the main goal of our research is to understand how molecular structure correlates with microscale mechanical properties of collagen solutions and networks. We use optical tweezers to trap and monitor thermal fluctuations of an embedded probe particle, from which viscoelastic properties of the solution are extracted. We find that elasticity becomes comparable to viscous behavior at collagen concentrations of 5mg/ml. Furthermore, by simultaneously neutralizing pH and adding salt, we observe changes in viscosity and elasticity of the solution over time. We attribute this to the self-assembly process of collagen molecules into fibrils with different mechanical properties. Self-assembly of collagen under these conditions is verified by turbidity measurements as well as electron microscopy. By comparing results from these local studies of viscoelasticity, we can detect spatial heterogeneity of fibril formation throughout the solution.

  10. Microscale Mechanical Testing of Individual Collagen Fibers

    NASA Astrophysics Data System (ADS)

    Poissant, Jeffrey

    Collagen is a key constituent for a large number of biological materials including bone, tendon, cartilage, skin and fish scales. Understanding the mechanical behavior of collagen's microscale structural components (fibers and fibrils) is therefore of utmost importance for fields such as biomimetics and biomedical engineering. However, the mechanics of collagen fibers and fibrils remain largely unexplored. The main research challenges are the small sample sizes (diameters less than 1 im) and the need to maintain physiologically relevant conditions. In this work, a microscale mechanical testing device (MMTD) capable of measuring the stress-strain response of individual collagen fibers and fibrils was developed. The MMTD consists of: (i) a transducer from a commercial nanoindenter to measure load and displacement, (ii) an optical microscope to observe the deformation of the sample in-situ and (iii) micromanipulators to isolate, position and fix samples. Collagen fibers and fibrils were extracted from fish scales using a novel dissection procedure and tested using the MMTD. A variety of tensile tests were performed including monotonic loading and cyclic tests with increasing loading rate or maximum displacement. The monotonic test results found that the elastic modulus, ultimate tensile strength and strain at failure range from 0.5 to 1.3 GPa, 100 to 200 MPa and 20% to 60%, respectively. The cyclic tests revealed that the largest increase in damage accumulation occurs at strains between 10% and 20%, when hydrogen bonds at the molecular level are ruptured. Further straining the fibril causes little additional damage accumulation and signals the approach of failure. The addition of water is shown to increase damage tolerance and strain to failure.

  11. Probing multiscale mechanics of collagen with optical tweezers

    NASA Astrophysics Data System (ADS)

    Shayegan, Marjan; Rezaei, Naghmeh; Lam, Norman H.; Altindal, Tuba; Wieczorek, Andrew; Forde, Nancy R.

    2013-09-01

    How the molecular structure of the structural, extracellular matrix protein collagen correlates with its mechanical properties at different hierarchical structural levels is not known. We demonstrate the utility of optical tweezers to probe collagen's mechanical response throughout its assembly hierarchy, from single molecule force-extension measurements through microrheology measurements on solutions of collagen molecules, collagen fibrillar gels and gelatin. These experiments enable the determination of collagen's flexibility, mechanics, and timescales and strengths of interaction at different levels of hierarchy, information critical to developing models of how collagen's physiological function and stability are influenced by its chemical composition. By investigating how the viscoelastic properties of collagen are affected by the presence of telopeptides, protein domains that strongly influence fibril formation, we demonstrate that these play a role in conferring transient elasticity to collagen solutions.

  12. Viscoelastic properties of model segments of collagen molecules.

    PubMed

    Gautieri, Alfonso; Vesentini, Simone; Redaelli, Alberto; Buehler, Markus J

    2012-03-01

    Collagen is the prime construction material in vertebrate biology, determining the mechanical behavior of connective tissues such as tendon, bone and skin. Despite extensive efforts in the investigation of the origin of collagen unique mechanical properties, a deep understanding of the relationship between molecular structure and mechanical properties remains elusive, hindered by the complex hierarchical structure of collagen-based tissues. In particular, although extensive studies of viscoelastic properties have been pursued at the macroscopic (fiber/tissue) level, fewer investigations have been performed at the smaller scales, including in particular collagen molecules and fibrils. These scales are, however, important for a complete understanding of the role of collagen as an important constituent in the extracellular matrix. Here, using an atomistic modeling approach, we perform in silico creep tests of a collagen-like peptide, monitoring the strain-time response for different values of applied external load. The results show that individual collagen molecules exhibit a nonlinear viscoelastic behavior, with a Young's modulus increasing from 6 to 16GPa (for strains up to 20%), a viscosity of 3.84.±0.38Pa·s, and a relaxation time in the range of 0.24-0.64ns. The single molecule viscosity, for the first time reported here, is several orders of magnitude lower than the viscosity found for larger-scale single collagen fibrils, suggesting that the viscous behavior of collagen fibrils and fibers involves additional mechanisms, such as molecular sliding between collagen molecules within the fibril or the effect of relaxation of larger volumes of solvent. Based on our molecular modeling results we propose a simple structural model that describes collagen tissue as a hierarchical structure, providing a bottom-up description of elastic and viscous properties form the properties of the tissue basic building blocks. PMID:22204879

  13. Bioengineered collagens

    PubMed Central

    Ramshaw, John AM; Werkmeister, Jerome A; Dumsday, Geoff J

    2014-01-01

    Mammalian collagen has been widely used as a biomedical material. Nevertheless, there are still concerns about the variability between preparations, particularly with the possibility that the products may transmit animal-based diseases. Many groups have examined the possible application of bioengineered mammalian collagens. However, translating laboratory studies into large-scale manufacturing has often proved difficult, although certain yeast and plant systems seem effective. Production of full-length mammalian collagens, with the required secondary modification to give proline hydroxylation, has proved difficult in E. coli. However, recently, a new group of collagens, which have the characteristic triple helical structure of collagen, has been identified in bacteria. These proteins are stable without the need for hydroxyproline and are able to be produced and purified from E. coli in high yield. Initial studies indicate that they would be suitable for biomedical applications. PMID:24717980

  14. Amyloid fibrils

    PubMed Central

    Rambaran, Roma N

    2008-01-01

    Amyloid refers to the abnormal fibrous, extracellular, proteinaceous deposits found in organs and tissues. Amyloid is insoluble and is structurally dominated by β-sheet structure. Unlike other fibrous proteins it does not commonly have a structural, supportive or motility role but is associated with the pathology seen in a range of diseases known as the amyloidoses. These diseases include Alzheimer's, the spongiform encephalopathies and type II diabetes, all of which are progressive disorders with associated high morbidity and mortality. Not surprisingly, research into the physicochemical properties of amyloid and its formation is currently intensely pursued. In this chapter we will highlight the key scientific findings and discuss how the stability of amyloid fibrils impacts on bionanotechnology. PMID:19158505

  15. Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells.

    PubMed

    Herchenhan, Andreas; Uhlenbrock, Franziska; Eliasson, Pernilla; Weis, MaryAnn; Eyre, David; Kadler, Karl E; Magnusson, S Peter; Kjaer, Michael

    2015-06-26

    Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used β-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation. PMID:25979340

  16. MORPHOLOGICAL AND CHEMICAL STUDIES OF COLLAGEN FORMATION

    PubMed Central

    Lowther, D. A.; Green, N. M.; Chapman, J. A.

    1961-01-01

    Electron micrographs of thin sections of nuclear, microsomal, and mitochondrial fractions obtained from a carrageenin-induced granuloma showed considerable contamination of the heavier by the lighter fractions. Striated collagen fibrils could be identified in the nuclei + debris fraction. Only a few striated fibrils occurred in the mitochondrial fraction; very fine filaments (diameter 50 A) could be seen in this fraction, but could not be distinguished with certainty from fibrillar material derived from broken nuclei. 35 per cent of the mitochondrial and 80 per cent of the microsomal collagen was extractable by 0.2 M NaCl and could be purified by the standard methods of solution and reprecipitation. The amino acid composition of these collagen fractions determined by ion exchange chromatography was within the range normally found for collagen and gelatin from other mammalian species, allowing for 10 to 20 per cent of some non-collagenous contaminant of the microsomal collagen. Hydroxyproline and proline were isolated by chromatography on paper from hydrolysates of the nuclear, mitochondrial, and microsomal collagen fractions, after incubation of tissue slices with L-14C-proline. The specific activities of the hydroxyproline from these collagens were in the approximate ratio 1:2:6, while that of bound hydroxyproline derived from the supernatant was only 1, indicating primary synthesis of collagen in the microsomes. Attempts to demonstrate incorporation of L-14C-proline into collagen or into free hydroxyproline in cell free systems were unsuccessful, nor was it possible to demonstrate non-specific incorporation of L-14C-valine into TCA-insoluble material by various combinations of subcellular fractions. PMID:13763869

  17. Collagenous gastroduodenitis on collagenous colitis.

    PubMed

    Stolte, M; Ritter, M; Borchard, F; Koch-Scherrer, G

    1990-07-01

    We report on a case of collagenous gastroduodenitis with concomitant collagenous colitis in a 75-year-old woman with watery diarrhea of approximately six months' standing. The step biopsy material obtained from the colon revealed continuous collagenous colitis with thickening of the basal membrane to 30 microns. The biopsies taken from the stomach and duodenum also revealed a band-like deposition of collagen in the duodenum (bulb and proximal portion of the descending portion) along the basal membrane of the lining epithelium, associated with partial atrophy of the villi. In the stomach, this band of collagen was located, parallel to the mucosal surface, at the level of the floor of the foveolae. PMID:2209504

  18. Three-dimensional collagen architecture in bovine articular cartilage.

    PubMed

    Jeffery, A K; Blunn, G W; Archer, C W; Bentley, G

    1991-09-01

    The three-dimensional architecture of bovine articular cartilage collagen and its relationship to split lines has been studied with scanning electron microscopy. In the middle and superficial zones, collagen was organised in a layered or leaf-like manner. The orientation was vertical in the intermediate zone, curving to become horizontal and parallel to the articular surface in the superficial zone. Each leaf consisted of a fine network of collagen fibrils. Adjacent leaves merged or were closely linked by bridging fibrils and were arranged according to the split-line pattern. The surface layer (lamina splendens) was morphologically distinct. Although ordered, the overall collagen structure was different in each plane (anisotropic) a property described in previous morphological and biophysical studies. As all components of the articular cartilage matrix interact closely, the three-dimensional organisation of collagen is important when considering cartilage function and the processes of cartilage growth, injury and repair. PMID:1894669

  19. Generation of 3D Collagen Gels with Controlled Diverse Architectures.

    PubMed

    Doyle, Andrew D

    2016-01-01

    Rat tail collagen solutions have been used as polymerizable in vitro three dimensional (3D) extracellular matrix (ECM) gels for single and collective cell migration assays as well as spheroid formation. Factors such as ECM concentration, pH, ionic concentration, and temperature can alter collagen polymerization and ECM architecture. This unit describes how to generate 3D collagen gels that have distinct architectures ranging from a highly reticular meshwork of short thin fibrils with small pores to a loose matrix consisting of stiff, parallel-bundled long fibrils by changing collagen polymerization temperature. This permits analysis of 3D cell migration in different ECM architectures found in vivo while maintaining a similar ECM concentration. Also included are collagen labeling techniques helpful for ECM visualization during live fluorescence imaging. © 2016 by John Wiley & Sons, Inc. PMID:27580704

  20. In vitro mineralization of dense collagen substrates: a biomimetic approach toward the development of bone-graft materials.

    PubMed

    Thula, Taili T; Rodriguez, Douglas E; Lee, Myong Hwa; Pendi, Laura; Podschun, Jacob; Gower, Laurie B

    2011-08-01

    Bone is an organic-inorganic composite which has hierarchical structuring that leads to high strength and toughness. The nanostructure of bone consists of nanocrystals of hydroxyapatite embedded and aligned within the interstices of collagen fibrils. This unique nanostructure leads to exceptional properties, both mechanical and biological, making it difficult to emulate bone properties without having a bone-like nanostructured material. A primary goal of our group's work is to use biomimetic processing techniques that lead to bone-like structures. In our prior studies, we demonstrated that intrafibrillar mineralization of porous collagen sponges, leading to a bone-like nanostructure, can be achieved using a polymer-induced liquid precursor (PILP) mineralization process. The objective of this study was to investigate the use of this polymer-directed crystallization process to mineralize dense collagen substrates. To examine collagen scaffolds that truly represent the dense-packed matrix of bone, manatee bone was demineralized to isolate its collagen matrix, consisting of a dense, lamellar osteonal microstructure. This biogenic collagen scaffold was then remineralized using polyaspartate to direct the mineralization process through an amorphous precursor pathway. The various conditions investigated included polymer molecular weight, substrate dimension and mineralization time. Mineral penetration depths of up to 100 μms were achieved using this PILP process, compared to no penetration with only surface precipitates observed for the conventional crystallization process. Electron microscopy, wide-angle X-ray diffraction and thermal analysis were used to characterize the resulting hydroxyapatite/collagen composites. These studies demonstrate that the original interpenetrating bone nanostructure and osteonal microstructure could be recovered in a biogenic matrix using the PILP process.

  1. In Vitro Mineralization of Dense Collagen Substrates: A Biomimetic Approach Toward the Development of Bone-Graft Materials

    PubMed Central

    Thula, Taili T.; Rodriguez, Douglas E.; Lee, Myong Hwa; Pendi, Laura; Podschun, Jacob; Gower, Laurie B.

    2012-01-01

    Bone is an organic-inorganic composite which has hierarchical structuring that leads to high strength and toughness. The nanostructure of bone consists of nanocrystals of hydroxyapatite embedded and aligned within the interstices of collagen fibrils. This unique nanostructure leads to exceptional properties, both mechanical and biological, making it difficult to emulate bone properties without having a bone-like nanostructured material. A primary goal of our group’s work is to use biomimetic processing techniques that lead to bone-like structures. In our prior studies, we demonstrated that intrafibrillar mineralization of porous collagen sponges, leading to a bone-like nanostructure, can be achieved using a polymer-induced liquid-precursor (PILP) mineralization process. The objective of this study was to investigate the use of this polymer-directed crystallization process to mineralize dense collagen substrates. To examine collagen scaffolds that truly represent the dense-packed matrix of bone, manatee bone was demineralized to isolate its collagen matrix, consisting of a dense, lamellar osteonal microstructure. This biogenic collagen scaffold was then remineralized using polyaspartate to direct the mineralization process through an amorphous precursor pathway. Various conditions investigated included polymer molecular weight, substrate dimension and mineralization time. Mineral penetration depths of up to 100 μms were achieved using this PILP process, compared to no penetration with only surface precipitates observed for the conventional crystallization process. Electron microscopy, wide-angle X-ray diffraction, and thermal analysis were used to characterize the resulting hydroxyapatite/collagen composites. These studies demonstrate that the original interpenetrating bone nanostructure and osteonal microstructure could be recovered in a biogenic matrix using the PILP process. PMID:21550424

  2. In vitro mineralization of dense collagen substrates: a biomimetic approach toward the development of bone-graft materials.

    PubMed

    Thula, Taili T; Rodriguez, Douglas E; Lee, Myong Hwa; Pendi, Laura; Podschun, Jacob; Gower, Laurie B

    2011-08-01

    Bone is an organic-inorganic composite which has hierarchical structuring that leads to high strength and toughness. The nanostructure of bone consists of nanocrystals of hydroxyapatite embedded and aligned within the interstices of collagen fibrils. This unique nanostructure leads to exceptional properties, both mechanical and biological, making it difficult to emulate bone properties without having a bone-like nanostructured material. A primary goal of our group's work is to use biomimetic processing techniques that lead to bone-like structures. In our prior studies, we demonstrated that intrafibrillar mineralization of porous collagen sponges, leading to a bone-like nanostructure, can be achieved using a polymer-induced liquid precursor (PILP) mineralization process. The objective of this study was to investigate the use of this polymer-directed crystallization process to mineralize dense collagen substrates. To examine collagen scaffolds that truly represent the dense-packed matrix of bone, manatee bone was demineralized to isolate its collagen matrix, consisting of a dense, lamellar osteonal microstructure. This biogenic collagen scaffold was then remineralized using polyaspartate to direct the mineralization process through an amorphous precursor pathway. The various conditions investigated included polymer molecular weight, substrate dimension and mineralization time. Mineral penetration depths of up to 100 μms were achieved using this PILP process, compared to no penetration with only surface precipitates observed for the conventional crystallization process. Electron microscopy, wide-angle X-ray diffraction and thermal analysis were used to characterize the resulting hydroxyapatite/collagen composites. These studies demonstrate that the original interpenetrating bone nanostructure and osteonal microstructure could be recovered in a biogenic matrix using the PILP process. PMID:21550424

  3. Mineralization by inhibitor exclusion: the calcification of collagen with fetuin.

    PubMed

    Price, Paul A; Toroian, Damon; Lim, Joo Eun

    2009-06-19

    One of our goals is to understand the mechanisms that deposit mineral within collagen fibrils, and as a first step we recently determined the size exclusion characteristics of the fibril. This study revealed that apatite crystals up to 12 unit cells in size can access the water within the fibril, whereas molecules larger than a 40-kDa protein are excluded. Based on these observations, we proposed a novel mechanism for fibril mineralization: that macromolecular inhibitors of apatite growth favor fibril mineralization by selectively inhibiting crystal growth in the solution outside of the fibril. To test this mechanism, we developed a system in which crystal formation is driven by homogeneous nucleation at high calcium phosphate concentration and the only macromolecule in solution is fetuin, a 48-kDa inhibitor of apatite growth. Our experiments with this system demonstrated that fetuin determines the location of mineral growth; in the presence of fetuin mineral grows exclusively within the fibril, whereas in its absence mineral grows in solution outside the fibril. Additional experiments showed that fetuin is also able to localize calcification to the interior of synthetic matrices that have size exclusion characteristics similar to those of collagen and that it does so by selectively inhibiting mineral growth outside of these matrices. We termed this new calcification mechanism "mineralization by inhibitor exclusion," the selective mineralization of a matrix using a macromolecular inhibitor of mineral growth that is excluded from that matrix. Future studies will be needed to evaluate the possible role of this mechanism in bone mineralization.

  4. The Role of Collagen Organization on the Properties of Bone.

    PubMed

    Garnero, Patrick

    2015-09-01

    Bone is a complex tissue constituted by a collagen matrix filled in with crystal of hydroxyapatite (HAP). Bone mechanical properties are influenced by the collagen matrix which is organized into hierarchical structures from the individual type I collagen heterotrimer flanked by linear telopeptides at each end to the collagen fibrils that are interconnected by enzymatic and non-enzymatic cross-links. Although most studies focused on the role of collagen cross-links in bone strength, other organizational features may also play a role. At the molecular level it has been shown that homotrimer of type I collagen found in bone tissue of some patients with osteogenesis imperfecta (OI) is characterized by decreased mechanical competence compared to the regular heterotrimer. The state of C-telopeptide isomerization-which can be estimated by the measurement in body fluids of the native and isomerized isoforms-has also been shown to be associated with bone strength, particularly the post-yield properties independent of bone size and bone mineral density. Other higher hierarchical features of collagen organization have shown to be associated with changes in bone mechanical behavior in ex vivo models and may also be relevant to explain bone fragility in diseases characterized by collagen abnormalities e.g., OI and Paget's disease. These include the orientation of collagen fibrils in a regular longitudinal direction, the D-spacing period between collagen fibrils and the collagen-HAP interfacial bonding. Preliminary data indicate that some of these organizational features can change during treatment with bisphosphonate, raloxifene, and PTH suggesting that they may contribute to their anti-fracture efficacy. It remains however to be determined which of these parameters play a specific and independent role in bone matrix properties, what is the magnitude of mechanical strength explained by collagen organization, whether they are relevant to explain osteoporosis-induced bone

  5. Collagen in Human Tissues: Structure, Function, and Biomedical Implications from a Tissue Engineering Perspective

    NASA Astrophysics Data System (ADS)

    Balasubramanian, Preethi; Prabhakaran, Molamma P.; Sireesha, Merum; Ramakrishna, Seeram

    The extracellular matrix is a complex biological structure encoded with various proteins, among which the collagen family is the most significant and abundant of all, contributing 30-35% of the whole-body protein. "Collagen" is a generic term for proteins that forms a triple-helical structure with three polypeptide chains, and around 29 types of collagen have been identified up to now. Although most of the members of the collagen family form such supramolecular structures, extensive diversity exists between each type of collagen. The diversity is not only based on the molecular assembly and supramolecular structures of collagen types but is also observed within its tissue distribution, function, and pathology. Collagens possess complex hierarchical structures and are present in various forms such as collagen fibrils (1.5-3.5 nm wide), collagen fibers (50-70 nm wide), and collagen bundles (150-250 nm wide), with distinct properties characteristic of each tissue providing elasticity to skin, softness of the cartilage, stiffness of the bone and tendon, transparency of the cornea, opaqueness of the sclera, etc. There exists an exclusive relation between the structural features of collagen in human tissues (such as the collagen composition, collagen fibril length and diameter, collagen distribution, and collagen fiber orientation) and its tissue-specific mechanical properties. In bone, a transverse collagen fiber orientation prevails in regions of higher compressive stress whereas longitudinally oriented collagen fibers correlate to higher tensile stress. The immense versatility of collagen compels a thorough understanding of the collagen types and this review discusses the major types of collagen found in different human tissues, highlighting their tissue-specific uniqueness based on their structure and mechanical function. The changes in collagen during a specific tissue damage or injury are discussed further, focusing on the many tissue engineering applications for

  6. Amyloid Fibril Solubility.

    PubMed

    Rizzi, L G; Auer, S

    2015-11-19

    It is well established that amyloid fibril solubility is protein specific, but how solubility depends on the interactions between the fibril building blocks is not clear. Here we use a simple protein model and perform Monte Carlo simulations to directly measure the solubility of amyloid fibrils as a function of the interaction between the fibril building blocks. Our simulations confirms that the fibril solubility depends on the fibril thickness and that the relationship between the interactions and the solubility can be described by a simple analytical formula. The results presented in this study reveal general rules how side-chain-side-chain interactions, backbone hydrogen bonding, and temperature affect amyloid fibril solubility, which might prove to be a powerful tool to design protein fibrils with desired solubility and aggregation properties in general. PMID:26496385

  7. Atrial Fibrillation Medications

    MedlinePlus

    ... think you are pregnant If you notice red, dark brown or black urine or stools If you ... Fibrillation • Introduction • What is Atrial Fibrillation? • Why AFib Matters • Understand your Risk for AFib Children • Symptoms of ...

  8. Amyloid Fibril Solubility.

    PubMed

    Rizzi, L G; Auer, S

    2015-11-19

    It is well established that amyloid fibril solubility is protein specific, but how solubility depends on the interactions between the fibril building blocks is not clear. Here we use a simple protein model and perform Monte Carlo simulations to directly measure the solubility of amyloid fibrils as a function of the interaction between the fibril building blocks. Our simulations confirms that the fibril solubility depends on the fibril thickness and that the relationship between the interactions and the solubility can be described by a simple analytical formula. The results presented in this study reveal general rules how side-chain-side-chain interactions, backbone hydrogen bonding, and temperature affect amyloid fibril solubility, which might prove to be a powerful tool to design protein fibrils with desired solubility and aggregation properties in general.

  9. The organization of collagen in cryofractured rabbit articular cartilage: a scanning electron microscopic study.

    PubMed

    Clark, J M

    1985-01-01

    Adult rabbit articular cartilage was prepared for scanning electron microscopy using, in order, glutaraldehyde fixation, enzymatic removal of proteoglycan, dehydration in ethanol, cryofracture in liquid nitrogen, and critical-point drying. Enzymes were effective in fixed material. Fixation, cryofracture, alignment of fracture surfaces with "split lines," and retention of subchondral bone were found to be necessary steps for the preservation of collagen detail. The fibrous framework was found to be similar to that proposed by Benninghoff and favored by more recent phase-contrast microscopic studies. Vertical fibers extending from subchondral bone and a network of tangentially oriented superficial fibrils converge in the transitional zone. No random layer is seen. Pericellular capsules interdigitate with the vertical fibers. When cartilage is prepared in a manner that minimizes tissue damage, scanning electron microscopy provides useful, unique information. PMID:3981292

  10. Collagen Gel Contraction by Fibroblasts: The Role of Myosin 2 and Gravity Effects

    NASA Technical Reports Server (NTRS)

    Johnson-Wint, Barbara P.; Malouvier, Alexandre; Holton, Emily

    1996-01-01

    Several lines of evidence suggest that collagen organization by connective tissue cells is sensitive to force. For instance, in flight experiments on rats the collagen fibrils which were produced under weightlessness and which were immediately next to the tendon fibroblasts were shown to be oriented randomly around the cells while the older fibrils right next to these and which were produced under 1 G, were highly organized.

  11. Trimerization and Triple Helix Stabilization of the Collagen XIX NC2 Domain*

    PubMed Central

    Boudko, Sergei P.; Engel, Jürgen; Bächinger, Hans Peter

    2008-01-01

    The mechanisms of chain selection and assembly of fibril-associated collagens with interrupted triple helices (FACITs) must differ from that of fibrillar collagens, since they lack the characteristic C-propeptide. We analyzed two carboxyl-terminal noncollagenous domains, NC2 and NC1, of collagen XIX as potential trimerization units and found that NC2 forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. In contrast, the NC1 domain requires formation of an adjacent collagen triple helix to form interchain disulfide bridges. The NC2 domain of collagen XIX and probably of other FACITs is responsible for chain selection and trimerization. PMID:18845531

  12. Measurement of Elastic Modulus of Collagen Type I Single Fiber.

    PubMed

    Dutov, Pavel; Antipova, Olga; Varma, Sameer; Orgel, Joseph P R O; Schieber, Jay D

    2016-01-01

    Collagen fibers are the main components of the extra cellular matrix and the primary contributors to the mechanical properties of tissues. Here we report a novel approach to measure the longitudinal component of the elastic moduli of biological fibers under conditions close to those found in vivo and apply it to type I collagen from rat tail tendon. This approach combines optical tweezers, atomic force microscopy, and exploits Euler-Bernoulli elasticity theory for data analysis. This approach also avoids drying for measurements or visualization, since samples are freshly extracted. Importantly, strains are kept below 0.5%, which appear consistent with the linear elastic regime. We find, surprisingly, that the longitudinal elastic modulus of type I collagen cannot be represented by a single quantity but rather is a distribution that is broader than the uncertainty of our experimental technique. The longitudinal component of the single-fiber elastic modulus is between 100 MPa and 360 MPa for samples extracted from different rats and/or different parts of a single tail. Variations are also observed in the fibril-bundle/fibril diameter with an average of 325±40 nm. Since bending forces depend on the diameter to the fourth power, this variation in diameter is important for estimating the range of elastic moduli. The remaining variations in the modulus may be due to differences in composition of the fibril-bundles, or the extent of the proteoglycans constituting fibril-bundles, or that some single fibrils may be of fibril-bundle size.

  13. Atrial fibrillation.

    PubMed

    Bang, Casper N

    2013-10-01

    Atrial fibrillation (AF) is a common complication after myocardial infarction (MI) and new-onset AF has been demonstrated to be associated with adverse outcome and a large excess risk of death in both MI and aortic stenosis (AS) patients. Prevention of new-onset AF is therefore a potential therapeutic target in AS and MI patients. Lipid-lowering drugs, particularly statins, have anti-inflammatory and antioxidant properties that may prevent AF. Accordingly, statins are recommended as a class IIa recommendation for prevention of new-onset AF after coronary artery bypass grafting (CABG). However, this preventive effect has not been investigated on new-onset AF in asymptomatic patients with AS or a large scale first-time MI patient sample and data in patients not undergoing invasive cardiac interventions are limited. This PhD thesis was conducted at the Heart Centre, Rigshospitalet, Denmark, with the aim to investigate the three aforementioned questions and to add to the existing evidence of AF prevention with statins. This was done using three different settings: 1) a randomized patients sample of 1,873 from the Simvastatin and Ezetimibe in Aortic Stenosis (SEAS) study, 2) a register patient sample of 97,499 with first-time MI, and 3) all published studies until beginning of June 2011 examining statin treatment on new-onset and recurrent AF in patients not undergoing cardiac surgery. This thesis revealed that statins did not lower the incidence or the time to new-onset AF in patients with asymptomatic AS. However, statin treatment showed an independently preventive effect on new-onset AF, including type-dependent effect and a trend to dosage-dependent effect. In addition, this thesis showed that good compliance to statin treatment was important to prevent new-onset AF. Finally, the meta-analysis in this PhD thesis showed a preventive effect in the observational studies although this effect was absent in the randomized controlled trials. Based on this PhD thesis

  14. Ultrastructural and tissue-culture studies on the role of fibronectin, collagen and glycosaminoglycans in the migration of neural crest cells in the fowl embryo.

    PubMed

    Newgreen, D F; Gibbins, I L; Sauter, J; Wallenfels, B; Wütz, R

    1982-01-01

    The initial migration of neural crest (NC) cells into cell-free space was studied by transmission electron microscopy at trunk levels of fowl embryos, some of which were fixed in the presence of ruthenium red. Migrating NC cells occurred in zones which contained fewer ruthenium-red stained 15-40nm diameter granules than other regions. The ruthenium-red stained granules were linked by similarly stained thin (greater than 3nm diameter) microfibrils. The granules resemble proteoglycan and the microfibrils may be hyaluronate. NC cells contacted thicker (greater than 10 nm diameter) fibrils and interstitial bodies, which did not require ruthenium red for visualization. Cytoplasmic microfilaments were sometimes aligned at the point of contact with the extracellular fibrils, which may be fibronectin and collagen. Phase-contrast time-lapse videotaping and scanning electron microscopy showed that NC cells of the fowl embryo in vitro migrated earlier and more extensively on glass coated with fibronectin-rich fibrous material and adsorbed fibronectin molecules than on glass coated with collagen type I (fibres and adsorbed molecules). NC cells became completely enmeshed in fibronectin-rich fibres, but generally remained on the surface of collagen-fibre gels. When given a choice, NC cells strongly preferred fibronectin coatings to plain glass, and plain glass to dried collagen gels. NC cells showed a slight preference for plain glass over glass to which collagen was adsorbed. Addition to the culture medium of hyaluronate (initial conc. 20 mg/ml), chondroitin (5 mg/ml) and fully sulphated chondroitin sulphate and dermatan sulphate (up to 10 mg/ml) did not drastically alter NC cell migration on fibronectin-rich fibrous substrates. PMID:7034954

  15. Ultrastructural and tissue-culture studies on the role of fibronectin, collagen and glycosaminoglycans in the migration of neural crest cells in the fowl embryo.

    PubMed

    Newgreen, D F; Gibbins, I L; Sauter, J; Wallenfels, B; Wütz, R

    1982-01-01

    The initial migration of neural crest (NC) cells into cell-free space was studied by transmission electron microscopy at trunk levels of fowl embryos, some of which were fixed in the presence of ruthenium red. Migrating NC cells occurred in zones which contained fewer ruthenium-red stained 15-40nm diameter granules than other regions. The ruthenium-red stained granules were linked by similarly stained thin (greater than 3nm diameter) microfibrils. The granules resemble proteoglycan and the microfibrils may be hyaluronate. NC cells contacted thicker (greater than 10 nm diameter) fibrils and interstitial bodies, which did not require ruthenium red for visualization. Cytoplasmic microfilaments were sometimes aligned at the point of contact with the extracellular fibrils, which may be fibronectin and collagen. Phase-contrast time-lapse videotaping and scanning electron microscopy showed that NC cells of the fowl embryo in vitro migrated earlier and more extensively on glass coated with fibronectin-rich fibrous material and adsorbed fibronectin molecules than on glass coated with collagen type I (fibres and adsorbed molecules). NC cells became completely enmeshed in fibronectin-rich fibres, but generally remained on the surface of collagen-fibre gels. When given a choice, NC cells strongly preferred fibronectin coatings to plain glass, and plain glass to dried collagen gels. NC cells showed a slight preference for plain glass over glass to which collagen was adsorbed. Addition to the culture medium of hyaluronate (initial conc. 20 mg/ml), chondroitin (5 mg/ml) and fully sulphated chondroitin sulphate and dermatan sulphate (up to 10 mg/ml) did not drastically alter NC cell migration on fibronectin-rich fibrous substrates.

  16. Collagenous gastritis.

    PubMed

    Jin, Xiaoyi; Koike, Tomoyuki; Chiba, Takashi; Kondo, Yutaka; Ara, Nobuyuki; Uno, Kaname; Asano, Naoki; Iijima, Katsunori; Imatani, Akira; Watanabe, Mika; Shirane, Akio; Shimosegawa, Tooru

    2013-09-01

    In the present paper, we report a case of rare collagenous gastritis. The patient was a 25-year-old man who had experienced nausea, abdominal distention and epigastralgia since 2005. Esophagogastroduodenoscopy (EGD) carried out at initial examination by the patient's local doctor revealed an extensively discolored depression from the upper gastric body to the lower gastric body, mainly including the greater curvature, accompanied by residual mucosa with multiple islands and nodularity with a cobblestone appearance. Initial biopsies sampled from the nodules and accompanying atrophic mucosa were diagnosed as chronic gastritis. In August, 2011, the patient was referred to Tohoku University Hospital for observation and treatment. EGD at our hospital showed the same findings as those by the patient's local doctor. Pathological findings included a membranous collagen band in the superficial layer area of the gastric mucosa, which led to a diagnosis of collagenous gastritis. Collagenous gastritis is an extremely rare disease, but it is important to recognize its characteristic endoscopic findings to make a diagnosis. PMID:23363075

  17. Techniques for Type I Collagen Organization

    NASA Astrophysics Data System (ADS)

    Anderson-Jackson, LaTecia Diamond

    Tissue Engineering is a process in which cells, engineering, and material methods are used in amalgamation to improve biological functions. The purpose of tissue engineering is to develop alternative solutions to treat or cure tissues and organs that have been severely altered or damaged by diseases, congenital defects, trauma, or cancer. One of the most common and most promising biological materials for tissue engineering to develop scaffolds is Type I collagen. A major challenge in biomedical research is aligning Type I collagen to mimic biological structures, such as ligaments, tendons, bones, and other hierarchal aligned structures within the human body. The intent of this research is to examine possible techniques for organizing Type I collagen and to assess which of the techniques is effective for potential biological applications. The techniques used in this research to organize collagen are soft lithography with solution-assisted sonication embossing, directional freezing, and direct poling. The final concentration used for both soft lithography with solution-assisted sonication embossing and direct poling was 1 mg/ml, whereas for directional freezing the final concentration varied between 4mg/ml, 2mg/ml, and 1 mg/ml. These techniques were characterized using the Atomic Force Microscope (AFM) and Helium Ion Microscope (HIM). In this study, we have found that out of the three techniques, the soft lithography and directional freezing techniques have been successful in organizing collagen in a particular pattern, but not alignment. We concluded alignment may be dependent on the pH of collagen and the amount of acetic acid used in collagen solution. However, experiments are still being conducted to optimize all three techniques to align collagen in a unidirectional arrangement.

  18. Study of the collagen structure in the superficial zone and physiological state of articular cartilage using a 3D confocal imaging technique

    PubMed Central

    Wu, Jian P; Kirk, Thomas B; Zheng, Ming H

    2008-01-01

    Introduction The collagen structure in the superficial zone of articular cartilage is critical to the tissue's durability. Early osteoarthritis is often characterized with fissures on the articular surface. This is closely related to the disruption of the collagen network. However, the traditional histology can not offer visualization of the collagen structure in articular cartilage because it uses conventional optical microscopy that does not have insufficient imaging resolution to resolve collagen from proteoglycans in hyaline articular cartilage. This study examines the 3D collagen network of articular cartilage scored from 0 to 2 in the scoring system of International Cartilage Repair Society, and aims to develop a 3D histology for assessing early osteoarthritis. Methods Articular cartilage was visually classified into five physiological groups: normal cartilage, aged cartilage, cartilage with artificial and natural surface disruption, and fibrillated. The 3D collagen matrix of the cartilage was acquired using a 3D imaging technique developed previously. Traditional histology was followed to grade the physiological status of the cartilage in the scoring system of International Cartilage Repair Society. Results Normal articular cartilage contains interwoven collagen bundles near the articular surface, approximately within the lamina splendens. However, its collagen fibres in the superficial zone orient predominantly in a direction spatially oblique to the articular surface. With age and disruption of the articular surface, the interwoven collagen bundles are gradually disappeared, and obliquely oriented collagen fibres change to align predominantly in a direction spatially perpendicular to the articular surface. Disruption of the articular surface is well related to the disappearance of the interwoven collagen bundles. Conclusion A 3D histology has been developed to supplement the traditional histology and study the subtle changes in the collagen network in the

  19. Immunogold labelling of human von Willebrand factor adsorbed to collagen.

    PubMed

    Furlan, M; Robles, R; Lämmle, B; Zimmermann, J; Hunziker, E

    1991-06-01

    von Willebrand factor (vWF) mediates adhesion of platelets to the exposed subendothelium at sites of vascular injury. This function is expressed through binding of vWF to both collagen and receptors on the platelet membrane. We have developed a new method using immunogold staining and electron microscopy, permitting visualization of human vWF adsorbed to collagen fibrils. The electron micrographs revealed strings of gold beads reflecting the polymeric structure of vWF. Our data showed dramatic differences in the binding of vWF to collagens of different sources: high binding density was observed using a collagen preparation isolated from aortic tissue whereas colloidal gold was virtually absent from tendon collagen. Using the immunogold labelling method we demonstrated that high shear rate enhanced vWF binding to aortic collagen.

  20. Alternating potentials assisted electrochemical deposition of mineralized collagen coatings.

    PubMed

    Zhuang, Junjun; Lin, Jun; Li, Juan; Weng, Wenjian; Cheng, Kui; Wang, Huiming

    2015-12-01

    Mineralized collagen coatings were synthesized by electrochemical deposition with alternating negative and positive potentials. The obtained coatings demonstrated a multi-layer structure alternating consisting of weakly and highly mineralized collagen layers and the proportion of each layer could be controlled by adjusting the deposition time. The coatings deposited using alternating potentials assisted electrochemical deposition (AP-ECD) showed significantly enhanced osteoblasts proliferation, and rhBMP-2 loading capability compared to those of the coatings deposited using constant potential electrochemical deposition (CP-ECD). The enhanced cytocompatibility and rhBMP-2 loading capability of the coatings might be attributed to their high proportion of weakly mineralized collagen layer. Furthermore, the deposition mechanism for alternating potentials is proposed as that positive potential induces deposition of negatively charged collagen fibrils to form a weakly mineralized collagen layer. Our results suggest that the present deposition method could be a promising approach to engineer mineralized collagen coating with better biological performances.

  1. Development and utilization of a bovine type I collagen microfibril model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The structure of fibrous collagen, a long triple helix that self-associates in a staggered array to form a matrix of fibrils, fibers and fiber bundles, makes it uniquely suitable as a scaffold for biomaterial engineering. A major challenge for this application is to stabilize collagen structure by m...

  2. Pathways of tau fibrillization.

    PubMed

    Kuret, Jeff; Chirita, Carmen N; Congdon, Erin E; Kannanayakal, Theresa; Li, Guibin; Necula, Mihaela; Yin, Haishan; Zhong, Qi

    2005-01-01

    New methods for analyzing tau fibrillization have yielded insights into the biochemical transitions involved in the process. Here we review the parallels between the sequential progression of tau fibrillization observed macroscopically in Alzheimer's disease (AD) lesions and the pathway of tau aggregation observed in vitro with purified tau preparations. In addition, pharmacological agents for further dissection of fibrillization mechanism and lesion formation are discussed. PMID:15615636

  3. Development of a recombinant human collagen-type III based hemostat.

    PubMed

    Yang, C; Hillas, P; Tang, J; Balan, J; Notbohm, H; Polarek, J

    2004-04-15

    Animal-tissue-derived collagen, containing mostly type I collagen with a minor amount of type III collagen, has been widely used in the production of hemostats for many decades, although it has been known for a long time that type III collagen is more likely to induce platelet aggregation in vitro. Because it is hard to purify type III from animal tissue, it has not been possible to correlate this finding with in vivo data. In this report, it is demonstrated that recombinant human collagen III fibrils are more capable of inducing platelet aggregation in vitro than those comprised of bovine collagen I, in agreement with previously published data on tissue-derived type III collagen. When formed into three-dimensional matrices, the use of type III collagen results in formulations with better mechanical integrity, larger surface area, and higher hemostatic activity in a rabbit spleen injury model as compared with commercially available hemostats formed from bovine type I collagen.

  4. Northern pike (Esox lucius) collagen: Extraction, characterization and potential application.

    PubMed

    Kozlowska, J; Sionkowska, A; Skopinska-Wisniewska, J; Piechowicz, K

    2015-11-01

    Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the scales of northern pike (Esox lucius) were extracted and characterized. It was the first time that this species was used as sources of collagen. FT-IR and amino acid analysis results revealed the presence of collagen. Glycine accounts for one-third of its amino acid residues and specific for collagen amino acid - hydroxyproline - is present in isolated protein. The content of imino acid: proline and hydroxyproline in ASC and PSC was similar (12.5% Pro and 6.5% Hyp). Both ASC and PSC were type I collagen. The denaturation temperature of ASC and PSC were 28.5 and 27°C, respectively. Thin collagen films were obtained by casting of collagen solution onto glass plates. The surface properties of ASC and PSC films were different - the surface of ASC collagen film was more polar and less rough than PSC and we can observe the formation of collagen fibrils after solvent evaporation. ASC films showed much higher tensile properties than PSC. The obtained results suggest that northern pike scales have potential as an alternative source of collagen for use in various fields.

  5. Highly nonlinear stress-relaxation response of articular cartilage in indentation: Importance of collagen nonlinearity.

    PubMed

    Mäkelä, J T A; Korhonen, R K

    2016-06-14

    Modern fibril-reinforced computational models of articular cartilage can include inhomogeneous tissue composition and structure, and nonlinear mechanical behavior of collagen, proteoglycans and fluid. These models can capture well experimental single step creep and stress-relaxation tests or measurements under small strains in unconfined and confined compression. Yet, it is known that in indentation, especially at high strain velocities, cartilage can express highly nonlinear response. Different fibril reinforced poroelastic and poroviscoelastic models were used to assess measured highly nonlinear stress-relaxation response of rabbit articular cartilage in indentation. Experimentally measured depth-dependent volume fractions of different tissue constituents and their mechanical nonlinearities were taken into account in the models. In particular, the collagen fibril network was modeled using eight separate models that implemented five different constitutive equations to describe the nonlinearity. These consisted of linear elastic, nonlinear viscoelastic and multiple nonlinear elastic representations. The model incorporating the most nonlinearly increasing Young׳s modulus of collagen fibrils as a function of strain captured best the experimental data. Relative difference between the model and experiment was ~3%. Surprisingly, the difference in the peak forces between the experiment and the model with viscoelastic collagen fibrils was almost 20%. Implementation of the measured volume fractions did not improve the ability of the model to capture the measured mechanical data. These results suggest that a highly nonlinear formulation for collagen fibrils is needed to replicate multi-step stress-relaxation response of rabbit articular cartilage in indentation with high strain rates. PMID:27130474

  6. Highly nonlinear stress-relaxation response of articular cartilage in indentation: Importance of collagen nonlinearity.

    PubMed

    Mäkelä, J T A; Korhonen, R K

    2016-06-14

    Modern fibril-reinforced computational models of articular cartilage can include inhomogeneous tissue composition and structure, and nonlinear mechanical behavior of collagen, proteoglycans and fluid. These models can capture well experimental single step creep and stress-relaxation tests or measurements under small strains in unconfined and confined compression. Yet, it is known that in indentation, especially at high strain velocities, cartilage can express highly nonlinear response. Different fibril reinforced poroelastic and poroviscoelastic models were used to assess measured highly nonlinear stress-relaxation response of rabbit articular cartilage in indentation. Experimentally measured depth-dependent volume fractions of different tissue constituents and their mechanical nonlinearities were taken into account in the models. In particular, the collagen fibril network was modeled using eight separate models that implemented five different constitutive equations to describe the nonlinearity. These consisted of linear elastic, nonlinear viscoelastic and multiple nonlinear elastic representations. The model incorporating the most nonlinearly increasing Young׳s modulus of collagen fibrils as a function of strain captured best the experimental data. Relative difference between the model and experiment was ~3%. Surprisingly, the difference in the peak forces between the experiment and the model with viscoelastic collagen fibrils was almost 20%. Implementation of the measured volume fractions did not improve the ability of the model to capture the measured mechanical data. These results suggest that a highly nonlinear formulation for collagen fibrils is needed to replicate multi-step stress-relaxation response of rabbit articular cartilage in indentation with high strain rates.

  7. Engineering scale development of the vapor-liquid-solid (VLS) process for the production of silicon carbide fibrils and linear fibril assemblies

    SciTech Connect

    Tenhover, M.; Biernacki, J.; Schatz, K.; Ko, F.

    1995-08-01

    In order to exploit the superior thermomechanical properties of the VLS fibril, the feasibility of scaled-up production of the SiC fibril is demonstrated in this study. Through time series study and computer simulation, the parameters affecting the growth process and properties of the fibrils were examined. To facilitate translation of the superior mechanical properties into higher level preform structures, conventional and unconventional processing methods were evaluated. As revealed by scanning electron microscopic examination and X-ray diffractometry, high level alignment of the fibrils was achieved by the wet-laid process.

  8. Internal strain drives spontaneous periodic buckling in collagen and regulates remodeling

    PubMed Central

    Dittmore, Andrew; Silver, Jonathan; Sarkar, Susanta K.; Marmer, Barry; Goldberg, Gregory I.; Neuman, Keir C.

    2016-01-01

    Fibrillar collagen, an essential structural component of the extracellular matrix, is remarkably resistant to proteolysis, requiring specialized matrix metalloproteinases (MMPs) to initiate its remodeling. In the context of native fibrils, remodeling is poorly understood; MMPs have limited access to cleavage sites and are inhibited by tension on the fibril. Here, single-molecule recordings of fluorescently labeled MMPs reveal cleavage-vulnerable binding regions arrayed periodically at ∼1-µm intervals along collagen fibrils. Binding regions remain periodic even as they migrate on the fibril, indicating a collective process of thermally activated and self-healing defect formation. An internal strain relief model involving reversible structural rearrangements quantitatively reproduces the observed spatial patterning and fluctuations of defects and provides a mechanism for tension-dependent stabilization of fibrillar collagen. This work identifies internal–strain-driven defects that may have general and widespread regulatory functions in self-assembled biological filaments. PMID:27402741

  9. Estrogen Depletion Results in Nanoscale Morphology Changes in Dermal Collagen

    PubMed Central

    Fang, Ming; Liroff, Kaitlin G.; Turner, A. Simon; Les, Clifford M.; Orr, Bradford G.; Holl, Mark M. Banaszak

    2012-01-01

    Tissue cryo-sectioning combined with Atomic Force Microscopy (AFM) imaging reveals that the nanoscale morphology of dermis collagen fibrils, quantified using the metric of D-periodic spacing, changes under the condition of estrogen depletion. Specifically, a new subpopulation of fibrils with D-spacings in the region between 56 and 59 nm is present two years following ovariectomy in ovine dermal samples. In addition, the overall width of the distribution, both values above and below the mean, has increased. The change in width due to an increase in lower values of D-spacings was previously reported for ovine bone; however, this report demonstrates that the effect is also present in non-mineralized collagen fibrils. A non-parametric Kolmogrov-Smirnov test of the cumulative density function indicates a statistical difference in the sham and OVX D-spacing distributions (p < 0.01). PMID:22437310

  10. Biomimetic silicification of demineralized hierarchical collagenous tissues

    PubMed Central

    Ryou, Heonjune; Diogenes, Anibal; Yiu, Cynthia K.Y.; Mazzoni, Annalisa; Chen, Ji-hua; Arola, Dwayne D.; Hargreaves, Kenneth M.; Pashley, David H.; Tay, Franklin R.

    2013-01-01

    Unlike man-made composite materials, natural biominerals containing composites usually demonstrate different levels of sophisticated hierarchical structures which are responsible for their mechanical properties and other metabolic functions. However, the complex spatial organizations of the organic-inorganic phases are far beyond what they be achieved by contemporary engineering techniques. Here, we demonstrate that carbonated apatite present in collagen matrices derived from fish scale and bovine bone may be replaced by amorphous silica, using an approach that simulates what is utilized by phylogenetically ancient glass sponges. The structural hierarchy of these collagen-based biomaterials is replicated by the infiltration and condensation of fluidic polymer-stabilized silicic acid precursors within the intrafibrillar milieu of type I collagen fibrils. This facile biomimetic silicification strategy may be used for fabricating silica-based, three-dimensional functional materials with specific morphological and hierarchical requirements. PMID:23586938

  11. Biomimetic silicification of demineralized hierarchical collagenous tissues.

    PubMed

    Niu, Li-na; Jiao, Kai; Ryou, Heonjune; Diogenes, Anibal; Yiu, Cynthia K Y; Mazzoni, Annalisa; Chen, Ji-hua; Arola, Dwayne D; Hargreaves, Kenneth M; Pashley, David H; Tay, Franklin R

    2013-05-13

    Unlike man-made composite materials, natural biominerals containing composites usually demonstrate different levels of sophisticated hierarchical structures which are responsible for their mechanical properties and other metabolic functions. However, the complex spatial organizations of the organic-inorganic phases are far beyond what they achieved by contemporary engineering techniques. Here, we demonstrate that carbonated apatite present in collagen matrices derived from fish scale and bovine bone may be replaced by amorphous silica, using an approach that simulates what is utilized by phylogenetically ancient glass sponges. The structural hierarchy of these collagen-based biomaterials is replicated by the infiltration and condensation of fluidic polymer-stabilized silicic acid precursors within the intrafibrillar milieu of type I collagen fibrils. This facile biomimetic silicification strategy may be used for fabricating silica-based, three-dimensional functional materials with specific morphological and hierarchical requirements.

  12. Post-translational control of collagen fibrillogenesis in mineralizing cultures of chick osteoblasts

    NASA Technical Reports Server (NTRS)

    Gerstenfeld, L. C.; Riva, A.; Hodgens, K.; Eyre, D. R.; Landis, W. J.

    1993-01-01

    Cultured osteoblasts from chick embryo calvaria were used as a model system to investigate the post-translational extracellular mechanisms controlling the macroassembly of collagen fibrils. The results of these studies demonstrated that cultured osteoblasts secreted a collagenous extracellular matrix that assembled and mineralized in a defined temporal and spatial sequence. The assembly of collagen occurred in a polarized fashion, such that successive orthogonal arrays of fibrils formed between successive cell layers proceeding from the culture surface toward the media. Mineralization followed in the same manner, being observed first in the deepest and oldest fibril layers. Collagen fibrillogenesis, the kinetics of cross-link formation, and collagen stability in the extracellular matrix of the cultures were examined over a 30 day culture period. Between days 8 and 12 in culture, collagen fibril diameters increased from < 30 nm to an average of 30-45 nm. Thereafter, diameters ranged in size from 20 to 200 nm. Quantitation of the collagen cross-linking residues, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP), showed that these mature cross-links increased from undetectable levels to concentrations found in normal chick bone. Analysis of the kinetics of their formation by pulse-chase labeling the cultures with [3H]lysine showed a doubling time of approximately 5 days. The relationships between cross-link formation, fibrillogenesis, and collagen stability were examined in cultures treated with beta-aminopropionitrile (beta-APN), a potent inhibitor of lysyl oxidase and cross-link formation. In beta-APN-treated cultures, total collagen synthesis was increased twofold, with no change in mRNA levels for type I collagen, whereas the amount of collagen accumulated in the cell layer was decreased by 50% and mineral deposition was reduced. The rate of collagen retention in the matrix was assessed by pulse-chase analysis of [3H]proline over a 16 day period in

  13. Advanced glycation end products suppress lysyl oxidase and induce bone collagen degradation in a rat model of renal osteodystrophy.

    PubMed

    Aoki, Chiharu; Uto, Kenta; Honda, Kazuho; Kato, Yoshiharu; Oda, Hideaki

    2013-11-01

    Renal osteodystrophy (ROD) is a major problem in patients with renal insufficiency. The present study was designed to elucidate the role of bone collagen changes and osteoblast differentiation in a rat model of ROD pathogenesis induced by adenine. Typical characteristics of renal failure, including increased serum urea nitrogen, creatinine, inorganic phosphorus, and intact parathyroid hormone levels, and decreased serum calcium and 1,25(OH)2D3 levels, were observed in adenine-induced rats. Micro-computed tomography analysis of the femur in adenine-induced rats showed decreased bone mineral density and osteoporotic changes, confirmed by the three-point bending test. The cancellous bone histomorphometric parameters of the tibia showed increased osteoblast number, decreased osteoclast surface with peritrabecular fibrosis, and increased osteoid tissue, indicating a severe mineralization disorder similar to clinical ROD. Scanning and transmission electron microscopy revealed irregular alignment and increased diameter of bone collagen fibrils in adenine-induced rats. Protein expression analysis showed greater accumulation of advanced glycation end products (AGEs) in peritrabecular osteoblasts of adenine-induced rats than in the controls. In contrast, suppressed expression of runt-related transcription factor 2, alkaline phosphatase, secreted phosphoprotein 1 (Spp1), and lysyl oxidase (Lox) mRNA levels, particularly the amount of active LOX protein, were observed. In in-vitro experiments, mineralizing MC3T3-E1 osteoblastic cells stimulated with AGE-modified bovine serum albumin had attenuated the expression of Spp1 mRNA levels and active LOX protein, with a decrease in extracellular nodules of mineralization. These observations provide clues to ROD pathogenesis, as they indicate that the suppression of osteoblast differentiation and decreased active LOX protein associated with accumulation of AGEs in osteoblasts caused structural abnormalities of bone collagen fibrils and

  14. Vulnerability to ventricular fibrillation

    NASA Astrophysics Data System (ADS)

    Janse, Michiel J.

    1998-03-01

    One of the factors that favors the development of ventricular fibrillation is an increase in the dispersion of refractoriness. Experiments will be described in which an increase in dispersion in the recovery of excitability was determined during brief episodes of enhanced sympathetic nerve activity, known to increase the risk of fibrillation. Whereas in the normal heart ventricular fibrillation can be induced by a strong electrical shock, a premature stimulus of moderate intensity only induces fibrillation in the presence of regional ischemia, which greatly increases the dispersion of refractoriness. One factor that is of importance for the transition of reentrant ventricular tachycardia to ventricular fibrillation during acute regional ischemia is the subendocardial Purkinje system. After selective destruction of the Purkinje network by lugol, reentrant tachycardias still develop in the ischemic region, but they do not degenerate into fibrillation. Finally, attempts were made to determine the minimal mass of thin ventricular myocardium required to sustain fibrillation induced by burst pacing. This was done by freezing of subendocardial and midmural layers. The rim of surviving epicardial muscle had to be larger than 20 g. Extracellular electrograms during fibrillation in both the intact and the "frozen" left ventricle were indistinguishable, but activation patterns were markedly different. In the intact ventricle epicardial activation was compatible with multiple wavelet reentry, in the "frozen" heart a single, or at most two wandering reentrant waves were seen.

  15. Biology of collagen-proteoglycan interaction.

    PubMed

    Junqueira, L C; Montes, G S

    1983-12-01

    The purpose of this article is to review our knowledge to date of collagen-proteoglycan interaction. Many topics have been taken into account in order to provide a reasonably complete picture of this highly complex subject. Basic information about collagen biology, and an overview of the current concepts and advances regarding proteoglycans, have served as a basis to elucidate collagen-proteoglycan interaction. The bases of some methods of study have been reviewed in order to provide a fuller understanding of the results that are cited in this article. The experimental models and biological examples discussed herein demonstrate that collagen-proteoglycan interaction is essential to the extracellular matrix resiliency. The organization of these macromolecules is critical: collagen molecules become assembled into fibrils, fibrils aggregate to form fibers, fibers associate into bundles of fibers, and proteoglycans in the ground substance play a major role in the ordering process; on the other hand, glycosaminoglycans (GAGs) are composed of repeating monomers--GAGs linked to a same protein core form a proteoglycan--which, in turn, may bind to a hyaluronic acid molecule to form a proteoglycan aggregate together with other proteoglycans. Further growth of these complex macromolecules at higher hierarchical levels occurs by interaction of collagen with proteoglycans. A striking correlation between the tissue distribution of the genetically-distinct types of interstitial collagen and the occurrence of the different GAGs (which argues strongly in favour of a specific interaction) is demonstrated comprehensively in this review. Tissues composed of collagen type I possess small amounts of proteoglycans which contain almost exclusively dermatan sulfate; while tissues containing only collagen type II have high amounts of chondroitin sulfates. Collagen type III is the major fibrillary constituent of tissues that possess intermediate levels of proteoglycans, which contain great

  16. Role of decorin on in vitro fibrillogenesis of type I collagen.

    PubMed

    Sini, P; Denti, A; Tira, M E; Balduini, C

    1997-11-01

    Tendon and corneal decorins are differently iduronated dermatan sulphate/proteoglycan (DS/PG) and the biochemical parameter that differentiates type I collagens is the hydroxylysine glycoside content. We have examined the effect of tendon and corneal decorins on the individual phases (tlag, dA/dt) of differently glycosylated type I collagens fibril formation, at molar ratios PG:collagen monomer ranging from 0.15:1 to 0.45:1. The results obtained indicate that decorins exert a different effect on the individual phases of fibril formation, correlated to the degree of glycosylation of collagen: at the same PG:collagen ratio the fibril formation of highly glycosylated corneal collagen is more efficiently inhibited than that of the poorly glycosylated one (tendon). Moreover tendon and corneal decorins exert a higher control on the fibrillogenesis of homologous collagen with respect to the heterologous one. These data suggest a possible tissue-specificity of the interaction decorin/type I collagen correlated to the structure of the PG and collagen present in extracellular matrices. PMID:9511994

  17. The role of the amorphous phase on the biomimetic mineralization of collagen

    PubMed Central

    Nudelman, Fabio; Bomans, Paul H. H.; George, Anne; de With, Gijsbertus

    2012-01-01

    Bone is a hierarchically structured composite material whose basic building block is the mineralized collagen fibril, where the collagen is the scaffold into which the hydroxyapatite (HA) crystals nucleate and grow. Understanding the mechanisms of hydroxyapatite formation inside the collagen is key to unravelling osteogenesis. In this work, we employed a biomimetic in vitro mineralization system to investigate the role of the amorphous precursor calcium phosphate phase in the mineralization of collagen. We observed that the rate of collagen mineralization is highly dependent on the concentration of polyaspartic acid, an inhibitor of hydroxyapatite nucleation and inducer of intrafibrillar mineralization. The lower the concentration of the polymer, the faster the mineralization and crystallization. Addition of the non-collagenous protein C-DMP1, a nucleator of hydroxyapatite, substantially accelerates mineral infiltration as well as HA nucleation. We have also demonstrated that Cu ions interfere with the mineralization process first by inhibiting the entry of the calcium phosphate into the collagen, and secondly by stabilizing the ACP, such that it does not convert into HA. Interestingly, under these conditions mineralization happens preferentially in the overlap regions of the collagen fibril. Our results show that the interactions between the amorphous precursor phase and the collagen fibril play an important role in the control over mineralization. PMID:25383016

  18. Dense tissue-like collagen matrices formed in cell-free conditions.

    PubMed

    Mosser, Gervaise; Anglo, Anny; Helary, Christophe; Bouligand, Yves; Giraud-Guille, Marie-Madeleine

    2006-01-01

    A new protocol was developed to produce dense organized collagen matrices hierarchically ordered on a large scale. It consists of a two stage process: (1) the organization of a collagen solution and (2) the stabilization of the organizations by a sol-gel transition that leads to the formation of collagen fibrils. This new protocol relies on the continuous injection of an acid-soluble collagen solution into glass microchambers. It leads to extended concentration gradients of collagen, ranging from 5 to 1000 mg/ml. The self-organization of collagen solutions into a wide array of spatial organizations was investigated. The final matrices obtained by this procedure varied in concentration, structure and density. Changes in the liquid state of the samples were followed by polarized light microscopy, and the final stabilized gel states obtained after fibrillogenesis were analyzed by both light and electron microscopy. Typical organizations extended homogeneously by up to three centimetres in one direction and several hundreds of micrometers in other directions. Fibrillogenesis of collagen solutions of high and low concentrations led to fibrils spatially arranged as has been described in bone and derm, respectively. Moreover, a relationship was revealed between the collagen concentration and the aggregation of and rotational angles between lateral fibrils. These results constitute a strong base from which to further develop highly enriched collagen matrices that could lead to substitutes that mimic connective tissues. The matrices thus obtained may also be good candidates for the study of the three-dimensional migration of cells.

  19. Gel filtration chromatography of triple-helical calf skin collagen.

    PubMed

    Noelken, M E; Bettin, B D

    1983-10-15

    Gel filtration of type I collagen has been of limited use, because at low pH where the protein is not associated it binds to agarose gels, and at neutrality collagen has a tendency to form fibrils. The more porous polyacrylamide-based gels do not interact with collagen but cannot be used at very high flow rates because they are compressible. It was found that these difficulties are surmounted by use of Fractogel TSK HW-65F, a spherical gel made from a weakly hydrophilic vinyl polymer, and use of the buffer system 0.5 M urea, 0.117 M Tris-HCl, pH 7.3, which prevents fibril formation. The solvent has only a slight effect on the thermal stability of collagen, as determined by circular dichroism measurements. The recovery of native collagen, at 25 degrees C, was at least 88% and that of partially unfolded collagen, at 35 degrees C where it is about one-third unfolded, was 98%. The Fractogel TSK gels and the urea, Tris solvent system should be useful for both preparative work and for studies involving interaction of unaggregated type I collagen with smaller molecules at physiological pH.

  20. Thermal Destabilization of Collagen Matrix Hierarchical Structure by Freeze/Thaw

    PubMed Central

    Ozcelikkale, Altug; Han, Bumsoo

    2016-01-01

    This study aims to characterize and understand the effects of freezing on collagen structures and functionality. Specifically, thermodynamic destabilization of collagen at molecular- and fibril-levels by combination of low temperatures and freezing were experimentally characterized using modulated differential scanning calorimetry. In order to delineate the effects of sub-zero temperature and water-ice phase change, we hypothesized that the extent of destabilization can be determined based on post-thaw heat induced thermal denaturation of collagen. It is found that thermal denaturation temperature of collagen in hydrogel decreases by 1.4–1.6°C after freeze/thaw while no such decrease is observed in the case of molecular solution. The destabilization is predominantly due to ice formation. Exposure to low temperatures in the absence of ice has only minimal effect. Calorimetry measurements combined with morphological examination of collagen matrices by scanning electron microscopy suggest that freezing results in destabilization of collagen fibrils due to expansion of intrafibrillar space by ice formation. This fibril-level damage can be alleviated by use of cryoprotectant DMSO at concentrations as low as 0.5 M. A theoretical model explaining the change in collagen post-thaw thermal stability by freezing-induced fibril expansion is also proposed. PMID:26765741

  1. The effects of UV irradiation on collagen D-band revealed by atomic force microscopy.

    PubMed

    Kontomaris, Stylianos V; Yova, Dido; Stylianou, Andreas; Balogiannis, Giorgos

    2015-01-01

    The objective of this paper was to investigate the influence of UV irradiation on collagen D-band periodicity by using the AFM imaging and nanoindentation methods. It is well known than UV irradiation is one of the main factors inducing destabilization of collagen molecules. Due to the human's skin chronic exposure to sun light, the research concerning the influence of UV radiation on collagen is of great interest. The impact of UV irradiation on collagen can be studied in nanoscale using Atomic Force Microscopy (AFM). AFM is a powerful tool as far as surface characterization is concerned, due to its ability to relate high resolution imaging with mechanical properties. Hence, high resolution images of individual collagen fibrils and load-displacement curves on the overlapping and gap regions, under various time intervals of UV exposure, were obtained. The results demonstrated that the UV rays affect the height level differences between the overlapping and gap regions. Under various time intervals of UV exposure, the height difference between overlaps and gaps reduced from ~3.7 nm to ~0.8 nm and the fibril diameters showed an average of 8-10% reduction. In addition, the irradiation influenced the mechanical properties of collagen fibrils. The Young's modulus values were reduced per 66% (overlaps) and 61% (gaps) compared to their initial values. The observed alterations on the structural and the mechanical properties of collagen fibrils are probably a consequence of the polypeptide chain scission due to the impact of the UV irradiation.

  2. Nanostructure and mechanics of mummified type I collagen from the 5300-year-old Tyrolean Iceman

    PubMed Central

    Janko, Marek; Zink, Albert; Gigler, Alexander M.; Heckl, Wolfgang M.; Stark, Robert W.

    2010-01-01

    Skin protects the body from pathogens and degradation. Mummified skin in particular is extremely resistant to decomposition. External influences or the action of micro-organisms, however, can degrade the connective tissue and lay the subjacent tissue open. To determine the degree of tissue preservation in mummified human skin and, in particular, the reason for its durability, we investigated the structural integrity of its main protein, type I collagen. We extracted samples from the Neolithic glacier mummy known as ‘the Iceman’. Atomic force microscopy (AFM) revealed collagen fibrils that had characteristic banding patterns of 69 ± 5 nm periodicity. Both the microstructure and the ultrastructure of dermal collagen bundles and fibrils were largely unaltered and extremely well preserved by the natural conservation process. Raman spectra of the ancient collagen indicated that there were no significant modifications in the molecular structure. However, AFM nanoindentation measurements showed slight changes in the mechanical behaviour of the fibrils. Young's modulus of single mummified fibrils was 4.1 ± 1.1 GPa, whereas the elasticity of recent collagen averages 3.2 ± 1.0 GPa. The excellent preservation of the collagen indicates that dehydration owing to freeze-drying of the collagen is the main process in mummification and that the influence of the degradation processes can be addressed, even after 5300 years. PMID:20356896

  3. Nanostructure and mechanics of mummified type I collagen from the 5300-year-old Tyrolean Iceman.

    PubMed

    Janko, Marek; Zink, Albert; Gigler, Alexander M; Heckl, Wolfgang M; Stark, Robert W

    2010-08-01

    Skin protects the body from pathogens and degradation. Mummified skin in particular is extremely resistant to decomposition. External influences or the action of micro-organisms, however, can degrade the connective tissue and lay the subjacent tissue open. To determine the degree of tissue preservation in mummified human skin and, in particular, the reason for its durability, we investigated the structural integrity of its main protein, type I collagen. We extracted samples from the Neolithic glacier mummy known as 'the Iceman'. Atomic force microscopy (AFM) revealed collagen fibrils that had characteristic banding patterns of 69 +/- 5 nm periodicity. Both the microstructure and the ultrastructure of dermal collagen bundles and fibrils were largely unaltered and extremely well preserved by the natural conservation process. Raman spectra of the ancient collagen indicated that there were no significant modifications in the molecular structure. However, AFM nanoindentation measurements showed slight changes in the mechanical behaviour of the fibrils. Young's modulus of single mummified fibrils was 4.1 +/- 1.1 GPa, whereas the elasticity of recent collagen averages 3.2 +/- 1.0 GPa. The excellent preservation of the collagen indicates that dehydration owing to freeze-drying of the collagen is the main process in mummification and that the influence of the degradation processes can be addressed, even after 5300 years.

  4. Collagen type IX from human cartilage: a structural profile of intermolecular cross-linking sites.

    PubMed Central

    Diab, M; Wu, J J; Eyre, D R

    1996-01-01

    Type IX collagen, a quantitatively minor collagenous component of cartilage, is known to be associated with and covalently cross-linked to type II collagen fibrils in chick and bovine cartilage. Type IX collagen molecules have also been shown to form covalent cross-links with each other in bovine cartilage. In the present study we demonstrate by structural analysis and location of cross-linking sites that, in human cartilage, type IX collagen is covalently cross-linked to type II collagen and to other molecules of type IX collagen. We also present evidence that, if the proteoglycan form of type IX collagen is present in human cartilage, it can only be a minor component of the matrix, similar to findings with bovine cartilage. PMID:8660302

  5. In vitro phagocytosis of exogenous collagen by fibroblasts from the periodontal ligament: an electron microscopic study.

    PubMed Central

    Svoboda, E L; Brunette, D M; Melcher, A H

    1979-01-01

    There have been numerous electron microscopic reports of apparent phagocytosis of collagen by fibroblasts and other cells in vivo. We have developed an in vitro system which, to the best of our knowledge, will permit for the first time the study of regulatory mechanisms governing phagocytosis and digestion of collagen fibres. Cells were cultured from explants of monkey periodontal ligament, subcultured, and grown to confluence in alpha-MEM plus 15% fetal calf serum plus antibiotics. The confluent cells were then cultured together with minced rat tail tendon collagen in alpha-MEM lacking proline, lysine, glycine and fetal calf serum for up to 7 days, after which they were processed for electron microscopy. Intracellular collagen profiles could be seen in cultured cells that were associated with exogenous collagen fibrils as early as 24 hours after addition of the collagen. Through electron microscopic examination of serial sections of the culture, we have demonstrated: (1) that fibroblasts can phagocytose collagen; (2) that the observed intracellular collagen is not the result of aggregation of endogenous synthesized collagen; (3) that it is not possible to base a decision as to whether a collagen fibril has been phagocytosed in whole or in part by the type of vesicle with which it is associated; (4) that cleavage of collagen into small pieces may not be a necessary prelude to its phagocytosis. Images Fig. 1 Fig. 2 Fig. 4 (cont.) Fig. 4 Fig. 6 (cont.) Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:108237

  6. Shear-induced amyloid fibrillization: the role of inertia.

    PubMed

    McBride, Samantha A; Sanford, Sean P; Lopez, Juan M; Hirsa, Amir H

    2016-04-14

    Agitation of protein is known to induce deleterious effects on protein stability and structure, with extreme agitation sometimes resulting in complete aggregation into amyloid fibrils. Many mechanisms have been proposed to explain how protein becomes unstable when subjected to flow, including alignment of protein species, shear-induced unfolding, simple mixing, or fragmentation of existing fibrils to create new seeds. Here a shearing flow was imposed on a solution of monomeric human insulin via a rotating Couette device with a small hydrophobic fluid interface. The results indicate that even very low levels of shear are capable of accelerating amyloid fibril formation. Simulations of the flow suggest that the shear enhances fibrillization kinetics when flow inertia is non-negligible and the resulting meridional circulation allows for advection of bulk protein to the hydrophobic interface.

  7. Shear-induced amyloid fibrillization: the role of inertia.

    PubMed

    McBride, Samantha A; Sanford, Sean P; Lopez, Juan M; Hirsa, Amir H

    2016-04-14

    Agitation of protein is known to induce deleterious effects on protein stability and structure, with extreme agitation sometimes resulting in complete aggregation into amyloid fibrils. Many mechanisms have been proposed to explain how protein becomes unstable when subjected to flow, including alignment of protein species, shear-induced unfolding, simple mixing, or fragmentation of existing fibrils to create new seeds. Here a shearing flow was imposed on a solution of monomeric human insulin via a rotating Couette device with a small hydrophobic fluid interface. The results indicate that even very low levels of shear are capable of accelerating amyloid fibril formation. Simulations of the flow suggest that the shear enhances fibrillization kinetics when flow inertia is non-negligible and the resulting meridional circulation allows for advection of bulk protein to the hydrophobic interface. PMID:26956731

  8. In situ fibril stretch and sliding is location-dependent in mouse supraspinatus tendons.

    PubMed

    Connizzo, Brianne K; Sarver, Joseph J; Han, Lin; Soslowsky, Louis J

    2014-12-18

    Tendons are able to transmit high loads efficiently due to their finely optimized hierarchical collagen structure. Two mechanisms by which tendons respond to load are collagen fibril sliding and deformation (stretch). Although many studies have demonstrated that regional variations in tendon structure, composition, and organization contribute to the full tendon׳s mechanical response, the location-dependent response to loading at the fibril level has not been investigated. In addition, the instantaneous response of fibrils to loading, which is clinically relevant for repetitive stretch or fatigue injuries, has also not been studied. Therefore, the purpose of this study was to quantify the instantaneous response of collagen fibrils throughout a mechanical loading protocol, both in the insertion site and in the midsubstance of the mouse supraspinatus tendon. Utilizing a novel atomic force microscopy-based imaging technique, tendons at various strain levels were directly visualized and analyzed for changes in fibril d-period with increasing tendon strain. At the insertion site, d-period significantly increased from 0% to 1% tendon strain, increased again from 3% to 5% strain, and decreased after 5% strain. At the midsubstance, d-period increased from 0% to 1% strain and then decreased after 7% strain. In addition, fibril d-period heterogeneity (fibril sliding) was present, primarily at 3% strain with a large majority occurring in the tendon midsubstance. This study builds upon previous work by adding information on the instantaneous and regional-dependent fibrillar response to mechanical loading and presents data proposing that collagen fibril sliding and stretch are directly related to tissue organization and function.

  9. Type V Collagen in Health, Disease, and Fibrosis.

    PubMed

    Mak, Ki M; Png, Chien Yi M; Lee, Danielle J

    2016-05-01

    Type V collagen (COLV) is a regulatory fibril-forming collagen. It has at least three different molecular isoforms-α1(V)2 α2(V), α1(V)3, and α1(V)α2(V)α3(V)-formed by combinations of three different polypeptide α chains-α1(V), α2(V), and α3(V). COL V is a relatively minor collagen of the extracellular matrix (ECM). Morphologically, COLV occurs as heterotypic fibrils with type I collagen (COLI), microfilaments, or 12-nm-thick fibrils. COLV is synthesized in various mesenchymal cells and its gene expression is modulated by TGF-β and growth factors. While resistant to digestion by interstitial collagenases, native and denatured COLV are degraded by metalloproteinases and gelatinases, thereby promoting ECM remodeling. COLV interacts with matrix collagens and structural proteins, conferring structural integrity to tissue scaffolds. It binds matrix macromolecules, modulating cellular behavior, and functions. COLV co-assembles with COLI into heterotypic fibrils in the cornea and skin dermis, acting as a dominant regulator of collagen fibrillogenesis. COLV deficiency is associated with loss of corneal transparency and classic Ehlers-Danlos syndrome, while COLV overexpression is found in cancer, granulation tissue, inflammation, atherosclerosis, and fibrosis of lungs, skin, kidneys, adipose tissue, and liver. COLV isoform containing the α3(V) chain is involved in mediating pancreatic islet cell functions. In the liver, COLV is a minor but regular component of the ECM. Increases in COLV are associated with both early and advanced hepatic fibrosis. The neoepitopes of COLV have been shown to be a useful noninvasive serum biomarker for assessing fibrotic progression and resolution in experimental hepatic fibrosis. COLV is multifunctional in health, disease, and fibrosis.

  10. Type V Collagen in Health, Disease, and Fibrosis.

    PubMed

    Mak, Ki M; Png, Chien Yi M; Lee, Danielle J

    2016-05-01

    Type V collagen (COLV) is a regulatory fibril-forming collagen. It has at least three different molecular isoforms-α1(V)2 α2(V), α1(V)3, and α1(V)α2(V)α3(V)-formed by combinations of three different polypeptide α chains-α1(V), α2(V), and α3(V). COL V is a relatively minor collagen of the extracellular matrix (ECM). Morphologically, COLV occurs as heterotypic fibrils with type I collagen (COLI), microfilaments, or 12-nm-thick fibrils. COLV is synthesized in various mesenchymal cells and its gene expression is modulated by TGF-β and growth factors. While resistant to digestion by interstitial collagenases, native and denatured COLV are degraded by metalloproteinases and gelatinases, thereby promoting ECM remodeling. COLV interacts with matrix collagens and structural proteins, conferring structural integrity to tissue scaffolds. It binds matrix macromolecules, modulating cellular behavior, and functions. COLV co-assembles with COLI into heterotypic fibrils in the cornea and skin dermis, acting as a dominant regulator of collagen fibrillogenesis. COLV deficiency is associated with loss of corneal transparency and classic Ehlers-Danlos syndrome, while COLV overexpression is found in cancer, granulation tissue, inflammation, atherosclerosis, and fibrosis of lungs, skin, kidneys, adipose tissue, and liver. COLV isoform containing the α3(V) chain is involved in mediating pancreatic islet cell functions. In the liver, COLV is a minor but regular component of the ECM. Increases in COLV are associated with both early and advanced hepatic fibrosis. The neoepitopes of COLV have been shown to be a useful noninvasive serum biomarker for assessing fibrotic progression and resolution in experimental hepatic fibrosis. COLV is multifunctional in health, disease, and fibrosis. PMID:26910848

  11. Deuterium nuclear magnetic resonance of specifically labeled native collagen. Investigation of protein molecular dynamics using the quadrupolar echo technique.

    PubMed Central

    Jelinski, L W; Sullivan, C E; Batchelder, L S; Torchia, D A

    1980-01-01

    Collagen was labeled with [3,3,3-d3]alanine and with [d10]leucine via tissue culture. 2H nuclear magnetic resonance (NMR) spectra were obtained of collagen in solution and as fibrils using the quadrupolar echo technique. The 2H NMR data for [3,3,3-d3]alanine-labeled collagen fibrils were analyzed in terms of a model for motion in which the molecule is considered to jump between two sites, separated azimuthally by an angle 2 delta, in a time which is rapid compared with the residence time in both sites. The data suggest that the molecule undergoes reorientation over an angle, 2 delta, of approximately 30 degrees in the fibrils, and that the average angle between the alanine C alpha--C beta bond axis and the long axis of the helix is approximately 75 degrees. Reorientation is possibly segmental. The T2 for [3,3,3-d3]alanine-labeled collagen fibrils was estimated to be 105 mus. The 2H NMR data for the methyl groups of [d10]leucine-labeled collagen were analyzed qualitatively. These data established that for collagen in solution and as fibrils, rotation occurs about the leucine side-chain bonds, in addition to threefold methyl rotation and reorientation of the peptide backbone. The T2 for the methyl groups of leucine-labeled collagen is estimated to be approximately 130 mus. Taken together, these data provide strong evidence that both polypeptide backbone reorientation and amino acid side-chain motion occur in collagen molecules in the fibrils. Stabilizing interactions that determine fibril structure must therefore depend upon at least two sets of contacts in any given local region. PMID:7248459

  12. Deuterium nuclear magnetic resonance of specifically labeled native collagen: investigation of protein molecular dynamics using quadrupolar echo technique

    SciTech Connect

    Jelinski, L.W.; Sullivan, C.E.; Batchelder, L.S.; Torchia, D.A.

    1980-10-01

    Collagen was labeled with )3,3,3-d/sub 3/) alanine and with (d/sub 10/) eucine via tissue culture. /sup 2/H nuclear magnetic resonance (NMR) spectra were obtained of collagen in solution and as fibrils using the quadrupolar echo techniqe. The /sup 2/H NMR data for (3,3,3-d/sub 3/)alanine-labeled collagen fibrils were analyzed in terms of a model for motion in which the molecule is considered to ump between two sites, separated azimuthally by an angle 2delta, in a time which is rapid compared with the residence time in both sites. The data suggest that the molecule undergoes reorientation over an angle, 2 delta, of approx. 30/sup 0/ in the fibrils, and that the average angle between the alanine C/sup ..cap alpha../-C/sup ..beta../ bond axis and the long axis of the helix is approx. 75/sup 0/. Reorientation is possibly segmental. The T/sub 2/ for (3,3,3-d/sub 3/)alanine-labeled collagen fibrils was estimated to be 105 ..mu..s. The /sup 2/H NMR data for the methyl groups of (d/sub 10/)leucine-labeled collagen were analyzed qualitatively. These data established that for collagen in solution and as fibrils, rotation occurs about the leucine side-chain bonds, in addition to threefold methyl rotation and reorientation of the peptide backbone. The T/sub 2/ for the methyl groups of leucine-labeled collagen is estimated to be approx. 130 ..mu..s. Taken together, these data provide strong evidence that both polypeptide backbone reorientation and amino acid side-chain motion occur in collagen molecules in the fibrils. Stabilizing interactions that determine fibril structure must therefore depend upon at least two sets of contacts in any given local region.

  13. What Is Atrial Fibrillation?

    MedlinePlus

    ... regular beat. Certain cells in your heart make electric signals that cause the heart to contract and ... read your ECG to find out if the electric signals are normal. In atrial fibrillation (AFib), the ...

  14. Fabrication of homobifunctional crosslinker stabilized collagen for biomedical application.

    PubMed

    Lakra, Rachita; Kiran, Manikantan Syamala; Sai, Korrapati Purna

    2015-12-01

    Collagen biopolymer has found widespread application in the field of tissue engineering owing to its excellent tissue compatibility and negligible immunogenicity. Mechanical strength and enzymatic degradation of the collagen necessitates the physical and chemical strength enhancement. One such attempt deals with the understanding of crosslinking behaviour of EGS (ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester)) with collagen to improve the physico-chemical properties. The incorporation of a crosslinker during fibril formation enhanced the thermal and mechanical stability of collagen. EGS crosslinked collagen films exhibited higher denaturation temperature (T d) and the residue left after thermogravimetric analysis was about 16 ± 5.2%. Mechanical properties determined by uniaxial tensile tests showed a threefold increase in tensile strength and Young's modulus at higher concentration (100 μM). Water uptake capacity reduced up to a moderate extent upon crosslinking which is essential for the transport of nutrients to the cells. Cell viability was found to be 100% upon treatment with 100 μM EGS whereas only 30% viability could be observed with glutaraldehyde. Rheological studies of crosslinked collagen showed an increase in shear stress and shear viscosity at 37 °C. Crosslinking with EGS resulted in the formation of a uniform fibrillar network. Trinitrobenzene sulfonate (TNBS) assay confirmed that EGS crosslinked collagen by forming a covalent interaction with ε-amino acids of collagen. The homobifunctional crosslinker used in this study enhanced the effectiveness of collagen as a biomaterial for biomedical application. PMID:26610606

  15. Mechanisms of lamellar collagen formation in connective tissues.

    PubMed

    Ghazanfari, Samaneh; Khademhosseini, Ali; Smit, Theodoor H

    2016-08-01

    The objective of tissue engineering is to regenerate functional tissues. Engineering functional tissues requires an understanding of the mechanisms that guide the formation and evolution of structure in the extracellular matrix (ECM). In particular, the three-dimensional (3D) collagen fiber arrangement is important as it is the key structural determinant that provides mechanical integrity and biological function. In this review, we survey the current knowledge on collagen organization mechanisms that can be applied to create well-structured functional lamellar tissues and in particular intervertebral disc and cornea. Thus far, the mechanisms behind the formation of cross-aligned collagen fibers in the lamellar structures is not fully understood. We start with cell-induced collagen alignment and strain-stabilization behavior mechanisms which can explain a single anisotropically aligned collagen fiber layer. These mechanisms may explain why there is anisotropy in a single layer in the first place. However, they cannot explain why a consecutive collagen layer is laid down with an alternating alignment. Therefore, we explored another mechanism, called liquid crystal phasing. While dense concentrations of collagen show such behavior, there is little evidence that the conditions for liquid crystal phasing are actually met in vivo. Instead, lysyl aldehyde-derived collagen cross-links have been found essential for correct lamellar matrix deposition. Furthermore, we suggest that supra-cellular (tissue-level) shear stress may be instrumental in the alignment of collagen fibers. Understanding the potential mechanisms behind the lamellar collagen structure in connective tissues will lead to further improvement of the regeneration strategies of functional complex lamellar tissues.

  16. Changes to collagen structure during leather processing.

    PubMed

    Sizeland, Katie H; Edmonds, Richard L; Basil-Jones, Melissa M; Kirby, Nigel; Hawley, Adrian; Mudie, Stephen; Haverkamp, Richard G

    2015-03-11

    As hides and skins are processed to produce leather, chemical and physical changes take place that affect the strength and other physical properties of the material. The structural basis of these changes at the level of the collagen fibrils is not fully understood and forms the basis of this investigation. Synchrotron-based small-angle X-ray scattering (SAXS) is used to quantify fibril orientation and D-spacing through eight stages of processing from fresh green ovine skins to staked dry crust leather. Both the D-spacing and fibril orientation change with processing. The changes in thickness of the leather during processing affect the fibril orientation index (OI) and account for much of the OI differences between process stages. After thickness is accounted for, the main difference in OI is due to the hydration state of the material, with dry materials being less oriented than wet. Similarly significant differences in D-spacing are found at different process stages. These are due also to the moisture content, with dry samples having a smaller D-spacing. This understanding is useful for relating structural changes that occur during different stages of processing to the development of the final physical characteristics of leather.

  17. Microstructural and Mechanical Differences Between Digested Collagen-Fibrin Co-Gels and Pure Collagen and Fibrin Gels

    PubMed Central

    Lai, Victor K.; Frey, Christina R.; Kerandi, Allan M.; Lake, Spencer P.; Tranquillo, Robert T.; Barocas, Victor H.

    2012-01-01

    Collagen and fibrin are important extra-cellular matrix (ECM) components in the body, providing structural integrity to various tissues. These biopolymers are also common scaffolds used in tissue engineering. This study investigated how co-gelation of collagen and fibrin affected the properties of each individual protein network. Collagen-fibrin co-gels were cast and subsequently digested using either plasmin or collagenase; the microstructure and mechanical behavior of the resulting networks were then compared with respective pure collagen or fibrin gels of the same protein concentration. The morphologies of the collagen networks were further analyzed via 3-D network reconstruction from confocal image z-stacks. Both collagen and fibrin exhibited a decrease in mean fiber diameter when formed in the co-gels compared to the pure gels; this microstructural change was accompanied by increased failure strain and decreased tangent modulus for both collagen and fibrin following selected digestion of the co-gels. In addition, analysis of the reconstructed collagen networks indicated presence of very long fibers and clustering of fibrils, resulting in very high connectivities for collagen networks formed in co-gels. PMID:22828381

  18. Tenascin-x deficiency mimics ehlers-danlos syndrome in mice through alteration of collagen deposition

    SciTech Connect

    Mao, J.R.; Taylor, G.; Dean, W.B.; Wagner, D.R.; Afzal, V.; Lotz, J.C.; Rubin, E.M.; Bristow, J.

    2002-03-01

    Tenascin-X is a large extracellular matrix protein of unknown function1-3. Tenascin-X deficiency in humans is associated with Ehlers-Danlos syndrome4,5, a generalized connective tissue disorder resulting from altered metabolism of the fibrillar collagens6. Because TNXB is the first Ehlers-Danlos syndrome gene that does not encode a fibrillar collagen or collagen-modifying enzyme7-14, we suggested that tenascin-X might regulate collagen synthesis or deposition15. To test this hypothesis, we inactivated Tnxb in mice. Tnxb-/- mice showed progressive skin hyperextensibility, similar to individuals with Ehlers-Danlos syndrome. Biomechanical testing confirmed increased deformability and reduced tensile strength of their skin. The skin of Tnxb-/- mice was histologically normal, but its collagen content was significantly reduced. At the ultrastructural level, collagen fibrils of Tnxb-/- mice were of normal size and shape, but the density of fibrils in their skin was reduced, commensurate with the reduction in collagen content. Studies of cultured dermal fibroblasts showed that although synthesis of collagen I by Tnxb-/- and wildtype cells was similar, Tnxb-/- fibroblasts failed to deposit collagen I into cell-associated matrix. This study confirms a causative role for TNXB in human Ehlers-Danlos syndrome and suggests that tenascin-X is an essential regulator of collagen deposition by dermal fibroblasts.

  19. Measurement of Elastic Modulus of Collagen Type I Single Fiber.

    PubMed

    Dutov, Pavel; Antipova, Olga; Varma, Sameer; Orgel, Joseph P R O; Schieber, Jay D

    2016-01-01

    Collagen fibers are the main components of the extra cellular matrix and the primary contributors to the mechanical properties of tissues. Here we report a novel approach to measure the longitudinal component of the elastic moduli of biological fibers under conditions close to those found in vivo and apply it to type I collagen from rat tail tendon. This approach combines optical tweezers, atomic force microscopy, and exploits Euler-Bernoulli elasticity theory for data analysis. This approach also avoids drying for measurements or visualization, since samples are freshly extracted. Importantly, strains are kept below 0.5%, which appear consistent with the linear elastic regime. We find, surprisingly, that the longitudinal elastic modulus of type I collagen cannot be represented by a single quantity but rather is a distribution that is broader than the uncertainty of our experimental technique. The longitudinal component of the single-fiber elastic modulus is between 100 MPa and 360 MPa for samples extracted from different rats and/or different parts of a single tail. Variations are also observed in the fibril-bundle/fibril diameter with an average of 325±40 nm. Since bending forces depend on the diameter to the fourth power, this variation in diameter is important for estimating the range of elastic moduli. The remaining variations in the modulus may be due to differences in composition of the fibril-bundles, or the extent of the proteoglycans constituting fibril-bundles, or that some single fibrils may be of fibril-bundle size. PMID:26800120

  20. Cross-linking and the molecular packing of corneal collagen

    NASA Technical Reports Server (NTRS)

    Yamauchi, M.; Chandler, G. S.; Tanzawa, H.; Katz, E. P.

    1996-01-01

    We have quantitatively characterized, for the first time, the cross-linking in bovine cornea collagen as a function of age. The major iminium reducible cross-links were dehydro-hydroxylysinonorleucine (deH-HLNL) and dehydro-histidinohydroxymerodesmosine (deH-HHMD). The former rapidly diminished after birth; however, the latter persisted in mature animals at a level of 0.3 - 0.4 moles/mole of collagen. A nonreducible cross-link, histidinohydroxylysinonorleucine (HHL), previously found only in skin, was also found to be a major mature cross-link in cornea. The presence of HHL indicates that cornea fibrils have a molecular packing similar to skin collagen. However, like deH-HHMD, the HHL content in corneal fibrils only reaches a maximum value with time about half that of skin. These data suggest that the corneal fibrils are comprised of discrete filaments that are internally stabilized by HHL and deH-HHMD cross-links. This pattern of intermolecular cross-linking would facilitate the special collagen swelling property required for corneal transparency.

  1. Disentangling the multifactorial contributions of fibronectin, collagen and cyclic strain on MMP expression and extracellular matrix remodeling by fibroblasts.

    PubMed

    Zhang, Yang; Lin, Zhe; Foolen, Jasper; Schoen, Ingmar; Santoro, Alberto; Zenobi-Wong, Marcy; Vogel, Viola

    2014-11-01

    Early wound healing is associated with fibroblasts assembling a provisional fibronectin-rich extracellular matrix (ECM), which is subsequently remodeled and interlaced by type I collagen. This exposes fibroblasts to time-variant sets of matrices during different stages of wound healing. Our goal was thus to gain insight into the ECM-driven functional regulation of human foreskin fibroblasts (HFFs) being either anchored to a fibronectin (Fn) or to a collagen-decorated matrix, in the absence or presence of cyclic mechanical strain. While the cells reoriented in response to the onset of uniaxial cyclic strain, cells assembled exogenously added Fn with a preferential Fn-fiber alignment along their new orientation. Exposure of HFFs to exogenous Fn resulted in an increase in matrix metalloproteinase (MMP) expression levels, i.e. MMP-15 (RT-qPCR), and MMP-9 activity (zymography), while subsequent exposure to collagen slightly reduced MMP-15 expression and MMP-9 activity compared to Fn-exposure alone. Cyclic strain upregulated Fn fibrillogenesis and actin stress fiber formation, but had comparatively little effect on MMP activity. We thus propose that the appearance of collagen might start to steer HFFs towards homeostasis, as it decreased both MMP secretion and the tension of Fn matrix fibrils as assessed by Fluorescence Resonance Energy Transfer. These results suggest that HFFs might have a high ECM remodeling or repair capacity in contact with Fn alone (early event), which is reduced in the presence of Col1 (later event), thereby down-tuning HFF activity, a processes which would be required in a tissue repair process to finally reach tissue homeostasis.

  2. Type IX collagen knock-out mouse shows progressive hearing loss.

    PubMed

    Suzuki, Nobuyoshi; Asamura, Kenji; Kikuchi, Yasutake; Takumi, Yutaka; Abe, Satoko; Imamura, Yasutada; Hayashi, Toshihiko; Aszodi, Attila; Fässler, Reinhard; Usami, Shin-ichi

    2005-03-01

    Type IX collagen is one of the important components, together with type II, V, and XI collagens, in the tectorial membrane of the organ of Corti. To confirm the significance of type IX collagen for normal hearing, we assessed the detailed morphological and electrophysiological features of type IX collagen knock-out mice, which have recently been reported as a deafness model. Through assessment by auditory brainstem response (ABR), knock-out mice were shown to have progressive hearing loss. At the light microscopic level, the tectorial membrane of knock-out mice was found to be abnormal in shape. These morphological changes started in the basal turn and were progressive toward the apical turn. Electron microscopy confirmed disturbance of organization of the collagen fibrils. These results suggest that mutations in type IX collagen genes may lead to abnormal integrity of collagen fibers in the tectorial membrane.

  3. Anticoagulation in atrial fibrillation

    PubMed Central

    Piccini, Jonathan P

    2014-01-01

    Atrial fibrillation increases the risk of stroke, which is a leading cause of death and disability worldwide. The use of oral anticoagulation in patients with atrial fibrillation at moderate or high risk of stroke, estimated by established criteria, improves outcomes. However, to ensure that the benefits exceed the risks of bleeding, appropriate patient selection is essential. Vitamin K antagonism has been the mainstay of treatment; however, newer drugs with novel mechanisms are also available. These novel oral anticoagulants (direct thrombin inhibitors and factor Xa inhibitors) obviate many of warfarin’s shortcomings, and they have demonstrated safety and efficacy in large randomized trials of patients with non-valvular atrial fibrillation. However, the management of patients taking warfarin or novel agents remains a clinical challenge. There are several important considerations when selecting anticoagulant therapy for patients with atrial fibrillation. This review will discuss the rationale for anticoagulation in patients with atrial fibrillation; risk stratification for treatment; available agents; the appropriate implementation of these agents; and additional, specific clinical considerations for treatment. PMID:24733535

  4. Getting to the heart of cardiac remodeling; how collagen subtypes may contribute to phenotype.

    PubMed

    Collier, P; Watson, C J; van Es, M H; Phelan, D; McGorrian, C; Tolan, M; Ledwidge, M T; McDonald, K M; Baugh, J A

    2012-01-01

    The objective of this study was to investigate the nature and biomechanical properties of collagen fibers within the human myocardium. Targeting cardiac interstitial abnormalities will likely become a major focus of future preventative strategies with regard to the management of cardiac dysfunction. Current knowledge regarding the component structures of myocardial collagen networks is limited, further delineation of which will require application of more innovative technologies. We applied a novel methodology involving combined confocal laser scanning and atomic force microscopy to investigate myocardial collagen within ex-vivo right atrial tissue from 10 patients undergoing elective coronary bypass surgery. Immuno-fluorescent co-staining revealed discrete collagen I and III fibers. During single fiber deformation, overall median values of stiffness recorded in collagen III were 37±16% lower than in collagen I [p<0.001]. On fiber retraction, collagen I exhibited greater degrees of elastic recoil [p<0.001; relative percentage increase in elastic recoil 7±3%] and less energy dissipation than collagen III [p<0.001; relative percentage increase in work recovered 7±2%]. In atrial biopsies taken from patients in permanent atrial fibrillation (n=5) versus sinus rhythm (n=5), stiffness of both collagen fiber subtypes was augmented (p<0.008). Myocardial fibrillar collagen fibers organize in a discrete manner and possess distinct biomechanical differences; specifically, collagen I fibers exhibit relatively higher stiffness, contrasting with higher susceptibility to plastic deformation and less energy efficiency on deformation with collagen III fibers. Augmented stiffness of both collagen fiber subtypes in tissue samples from patients with atrial fibrillation compared to those in sinus rhythm are consistent with recent published findings of increased collagen cross-linking in this setting.

  5. The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors

    PubMed Central

    Nudelman, Fabio; Pieterse, Koen; George, Anne; Bomans, Paul H. H.; Friedrich, Heiner; Brylka, Laura J.; Hilbers, Peter A. J.; de With, Gijsbertus; Sommerdijk, Nico A. J. M.

    2011-01-01

    Bone is a composite material, in which collagen fibrils form a scaffold for a highly organized arrangement of uniaxially oriented apatite crystals1,2. In the periodic 67 nm cross-striated pattern of the collagen fibril3–5, the less dense 40-nm-long gap zone has been implicated as the place where apatite crystals nucleate from an amorphous phase, and subsequently grow6–9. This process is believed to be directed by highly acidic non-collagenous proteins6,7,9–11; however, the role of the collagen matrix12–14 during bone apatite mineralization remains unknown. Here, combining nanometre-scale resolution cryogenic transmission electron microscopy and cryogenic electron tomography15 with molecular modelling, we show that collagen functions in synergy with inhibitors of hydroxyapatite nucleation to actively control mineralization. The positive net charge close to the C-terminal end of the collagen molecules promotes the infiltration of the fibrils with amorphous calcium phosphate (ACP). Furthermore, the clusters of charged amino acids, both in gap and overlap regions, form nucleation sites controlling the conversion of ACP into a parallel array of oriented apatite crystals. We developed a model describing the mechanisms through which the structure, supramolecular assembly and charge distribution of collagen can control mineralization in the presence of inhibitors of hydroxyapatite nucleation. PMID:20972429

  6. Thermal Denaturation Studies of Collagen by Microthermal Analysis and Atomic Force Microscopy

    PubMed Central

    Bozec, Laurent; Odlyha, Marianne

    2011-01-01

    The structural properties of collagen have been the subject of numerous studies over past decades, but with the arrival of new technologies, such as the atomic force microscope and related techniques, a new era of research has emerged. Using microthermal analysis, it is now possible to image samples as well as performing localized thermal measurements without damaging or destroying the sample itself. This technique was successfully applied to characterize the thermal response between native collagen fibrils and their denatured form, gelatin. Thermal transitions identified at (150 ± 10)°C and (220 ± 10)°C can be related to the process of gelatinization of the collagen fibrils, whereas at higher temperatures, both the gelatin and collagen samples underwent two-stage transitions with a common initial degradation temperature at (300 ± 10)°C and a secondary degradation temperature of (340 ± 10)°C for the collagen and of (420 ± 10)°C for the gelatin, respectively. The broadening and shift in the secondary degradation temperature was linked to the spread of thermal degradation within the gelatin and collagen fibrils matrix further away from the point of contact between probe and sample. Finally, similar measurements were performed inside a bone resorption lacuna, suggesting that microthermal analysis is a viable technique for investigating the thermomechanical response of collagen for in situ samples that would be, otherwise, too challenging or not possible using bulk techniques. PMID:21723833

  7. Topologically defined composites of collagen types I and V as in vitro cell culture scaffolds.

    PubMed

    Franke, Katja; Sapudom, Jiranuwat; Kalbitzer, Liv; Anderegg, Ulf; Pompe, Tilo

    2014-06-01

    Cell fate is known to be triggered by cues from the extracellular matrix, including its chemical, biological and physical characteristics. Specifically, mechanical and topological properties are increasingly recognized as important signals. The aim of this work was to provide an easily accessible biomimetic in vitro platform of topologically defined collagen I matrices to dissect cell behaviour under various conditions in vitro. We reconstituted covalently bound layers of three-dimensional (3-D) networks of collagen type I and collagen type V with a defined network topology. A new erosion algorithm enabled us to analyse the mean pore diameter and fibril content, while the mean fibril diameter was examined by an autocorrelation method. Different concentrations and ratios of collagen I and V resulted in pore diameters from 2.4 to 4.5μm and fibril diameters from 0.6 to 0.8μm. A comparison of telopeptide intact collagen I to telopeptide deficient collagen I revealed obvious differences in network structure. The good correlation of the topological data to measurements of network stiffness as well as invasion of human dermal fibroblasts proves that the topological analysis provides meaningful measures of the functional characteristics of the reconstituted 3-D collagen matrices.

  8. The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors.

    PubMed

    Nudelman, Fabio; Pieterse, Koen; George, Anne; Bomans, Paul H H; Friedrich, Heiner; Brylka, Laura J; Hilbers, Peter A J; de With, Gijsbertus; Sommerdijk, Nico A J M

    2010-12-01

    Bone is a composite material in which collagen fibrils form a scaffold for a highly organized arrangement of uniaxially oriented apatite crystals. In the periodic 67 nm cross-striated pattern of the collagen fibril, the less dense 40-nm-long gap zone has been implicated as the place where apatite crystals nucleate from an amorphous phase, and subsequently grow. This process is believed to be directed by highly acidic non-collagenous proteins; however, the role of the collagen matrix during bone apatite mineralization remains unknown. Here, combining nanometre-scale resolution cryogenic transmission electron microscopy and cryogenic electron tomography with molecular modelling, we show that collagen functions in synergy with inhibitors of hydroxyapatite nucleation to actively control mineralization. The positive net charge close to the C-terminal end of the collagen molecules promotes the infiltration of the fibrils with amorphous calcium phosphate (ACP). Furthermore, the clusters of charged amino acids, both in gap and overlap regions, form nucleation sites controlling the conversion of ACP into a parallel array of oriented apatite crystals. We developed a model describing the mechanisms through which the structure, supramolecular assembly and charge distribution of collagen can control mineralization in the presence of inhibitors of hydroxyapatite nucleation.

  9. Modification by UV radiation of the surface of thin films based on collagen extracted from fish scales.

    PubMed

    Sionkowska, Alina; Kozłowska, Justyna; Lazare, Sylvain

    2014-06-01

    Collagen was extracted from fish scales (Esox lucius) through demineralization process. Thin films by solvent evaporation from collagen extracted from fish scales were prepared. The surface of thin films made of fish scales collagen was modified by ultraviolet (UV)-irradiation with the wavelength λ = 254 nm. The amino acid composition of the Esox lucius scale collagen was analyzed before and after UV-irradiation by means of high-pressure liquid chromatography. The surface properties of films were investigated using the technique of atomic force microscopy (AFM) and by means of contact angle measurements allowing the calculation of surface free energy. Measurements of the contact angle for diiodomethane (D) and glycerol (G) on the surface of fish collagen films were made and surface free energy was calculated. The structure of collagen before and after UV-irradiation was studied using Fourier-transform infrared spectroscopy. It was found that after UV-irradiation the amount of all amino acids present in collagen molecule decreased. It was found also that the contact angle and the surface free energy were altered by UV-irradiation of collagen film. AFM showed that the surface roughness of collagen films was also altered by UV-irradiation. UV-irradiation caused the decrease of surface roughness due to photochemical processes, which occurred in the top layer of collagen film. The formation of collagen fibrils after solvent evaporation was observed using AFM. The diameter of collagen fibrils was bigger for irradiated collagen film than the diameter of collagen fibrils before UV-irradiation.

  10. Collagen morphology and texture analysis: from statistics to classification

    NASA Astrophysics Data System (ADS)

    Mostaço-Guidolin, Leila B.; Ko, Alex C.-T.; Wang, Fei; Xiang, Bo; Hewko, Mark; Tian, Ganghong; Major, Arkady; Shiomi, Masashi; Sowa, Michael G.

    2013-07-01

    In this study we present an image analysis methodology capable of quantifying morphological changes in tissue collagen fibril organization caused by pathological conditions. Texture analysis based on first-order statistics (FOS) and second-order statistics such as gray level co-occurrence matrix (GLCM) was explored to extract second-harmonic generation (SHG) image features that are associated with the structural and biochemical changes of tissue collagen networks. Based on these extracted quantitative parameters, multi-group classification of SHG images was performed. With combined FOS and GLCM texture values, we achieved reliable classification of SHG collagen images acquired from atherosclerosis arteries with >90% accuracy, sensitivity and specificity. The proposed methodology can be applied to a wide range of conditions involving collagen re-modeling, such as in skin disorders, different types of fibrosis and muscular-skeletal diseases affecting ligaments and cartilage.

  11. Hierarchical and non-hierarchical mineralisation of collagen

    PubMed Central

    Liu, Yan; Kim, Young-Kyung; Dai, Lin; Li, Nan; Khan, Sara; Pashley, David H.; Tay, Franklin R.

    2010-01-01

    Biomineralisation of collagen involves functional motifs incorporated in extracellular matrix protein molecules to accomplish the objectives of stabilising amorphous calcium phosphate into nanoprecursors and directing the nucleation and growth of apatite within collagen fibrils. Here we report the use of small inorganic polyphosphate molecules to template hierarchical intrafibrillar apatite assembly in reconstituted collagen in the presence of polyacrylic acid to sequester calcium and phosphate into transient amorphous nanophases. The use of polyphosphate without a sequestration analogue resulted only in randomly-oriented extrafibrillar precipitations along the fibrillar surface. Conversely, the use of polyacrylic acid without a templating analogue resulted only in non-hierarchical intrafibrillar mineralisation with continuous apatite strands instead of discrete crystallites. The ability of using simple non-protein molecules to recapitulate different levels of structural hierarchy in mineralised collagen signifies the ultimate simplicity in Nature’s biomineralisation design principles and challenges the need for using more complex recombinant matrix proteins in bioengineering applications. PMID:21040969

  12. Targeted deletion of collagen V in tendons and ligaments results in a classic Ehlers-Danlos syndrome joint phenotype.

    PubMed

    Sun, Mei; Connizzo, Brianne K; Adams, Sheila M; Freedman, Benjamin R; Wenstrup, Richard J; Soslowsky, Louis J; Birk, David E

    2015-05-01

    Collagen V mutations underlie classic Ehlers-Danlos syndrome, and joint hypermobility is an important clinical manifestation. We define the function of collagen V in tendons and ligaments, as well as the role of alterations in collagen V expression in the pathobiology in classic Ehlers-Danlos syndrome. A conditional Col5a1(flox/flox) mouse model was bred with Scleraxis-Cre mice to create a targeted tendon and ligament Col5a1-null mouse model, Col5a1(Δten/Δten). Targeting was specific, resulting in collagen V-null tendons and ligaments. Col5a1(Δten/Δten) mice demonstrated decreased body size, grip weakness, abnormal gait, joint laxity, and early-onset osteoarthritis. These gross changes were associated with abnormal fiber organization, as well as altered collagen fibril structure with increased fibril diameters and decreased fibril number that was more severe in a major joint stabilizing ligament, the anterior cruciate ligament (ACL), than in the flexor digitorum longus tendon. The ACL also had a higher collagen V content than did the flexor digitorum longus tendon. The collagen V-null ACL and flexor digitorum longus tendon both had significant alterations in mechanical properties, with ACL exhibiting more severe changes. The data demonstrate critical differential regulatory roles for collagen V in tendon and ligament structure and function and suggest that collagen V regulatory dysfunction is associated with an abnormal joint phenotype, similar to the hypermobility phenotype in classic Ehlers-Danlos syndrome.

  13. Targeted deletion of collagen V in tendons and ligaments results in a classic Ehlers-Danlos syndrome joint phenotype.

    PubMed

    Sun, Mei; Connizzo, Brianne K; Adams, Sheila M; Freedman, Benjamin R; Wenstrup, Richard J; Soslowsky, Louis J; Birk, David E

    2015-05-01

    Collagen V mutations underlie classic Ehlers-Danlos syndrome, and joint hypermobility is an important clinical manifestation. We define the function of collagen V in tendons and ligaments, as well as the role of alterations in collagen V expression in the pathobiology in classic Ehlers-Danlos syndrome. A conditional Col5a1(flox/flox) mouse model was bred with Scleraxis-Cre mice to create a targeted tendon and ligament Col5a1-null mouse model, Col5a1(Δten/Δten). Targeting was specific, resulting in collagen V-null tendons and ligaments. Col5a1(Δten/Δten) mice demonstrated decreased body size, grip weakness, abnormal gait, joint laxity, and early-onset osteoarthritis. These gross changes were associated with abnormal fiber organization, as well as altered collagen fibril structure with increased fibril diameters and decreased fibril number that was more severe in a major joint stabilizing ligament, the anterior cruciate ligament (ACL), than in the flexor digitorum longus tendon. The ACL also had a higher collagen V content than did the flexor digitorum longus tendon. The collagen V-null ACL and flexor digitorum longus tendon both had significant alterations in mechanical properties, with ACL exhibiting more severe changes. The data demonstrate critical differential regulatory roles for collagen V in tendon and ligament structure and function and suggest that collagen V regulatory dysfunction is associated with an abnormal joint phenotype, similar to the hypermobility phenotype in classic Ehlers-Danlos syndrome. PMID:25797646

  14. Electrospun bio-composite P(LLA-CL)/collagen I/collagen III scaffolds for nerve tissue engineering.

    PubMed

    Kijeńska, Ewa; Prabhakaran, Molamma P; Swieszkowski, Wojciech; Kurzydlowski, Krzysztof J; Ramakrishna, Seeram

    2012-05-01

    One of the biggest challenges in peripheral nerve tissue engineering is to create an artificial nerve graft that could mimic the extracellular matrix (ECM) and assist in nerve regeneration. Bio-composite nanofibrous scaffolds made from synthetic and natural polymeric blends provide suitable substrate for tissue engineering and it can be used as nerve guides eliminating the need of autologous nerve grafts. Nanotopography or orientation of the fibers within the scaffolds greatly influences the nerve cell morphology and outgrowth, and the alignment of the fibers ensures better contact guidance of the cells. In this study, poly (L-lactic acid)-co-poly(ε-caprolactone) or P(LLA-CL), collagen I and collagen III are utilized for the fabrication of nanofibers of different compositions and orientations (random and aligned) by electrospinning. The morphology, mechanical, physical, and chemical properties of the electrospun scaffolds along with their biocompatibility using C17.2 nerve stem cells are studied to identify the suitable material compositions and topography of the electrospun scaffolds required for peripheral nerve regeneration. Aligned P(LLA-CL)/collagen I/collagen III nanofibrous scaffolds with average diameter of 253 ± 102 nm were fabricated and characterized with a tensile strength of 11.59 ± 1.68 MPa. Cell proliferation studies showed 22% increase in cell proliferation on aligned P(LLA-CL)/collagen I/collagen III scaffolds compared with aligned pure P(LLA-CL) scaffolds. Results of our in vitro cell proliferation, cell-scaffold interaction, and neurofilament protein expression studies demonstrated that the electrospun aligned P(LLA-CL)/collagen I/collagen III nanofibrous scaffolds mimic more closely towards the ECM of nerve and have great potential as a substrate for accelerated regeneration of the nerve.

  15. Evaluation of bioprosthetic heart valve failure using a matrix-fibril shear stress transfer approach.

    PubMed

    Anssari-Benam, Afshin; Barber, Asa H; Bucchi, Andrea

    2016-02-01

    A matrix-fibril shear stress transfer approach is devised and developed in this paper to analyse the primary biomechanical factors which initiate the structural degeneration of the bioprosthetic heart valves (BHVs). Using this approach, the critical length of the collagen fibrils l c and the interface shear acting on the fibrils in both BHV and natural aortic valve (AV) tissues under physiological loading conditions are calculated and presented. It is shown that the required critical fibril length to provide effective reinforcement to the natural AV and the BHV tissue is l c  = 25.36 µm and l c  = 66.81 µm, respectively. Furthermore, the magnitude of the required shear force acting on fibril interface to break a cross-linked fibril in the BHV tissue is shown to be 38 µN, while the required interfacial force to break the bonds between the fibril and the surrounding extracellular matrix is 31 µN. Direct correlations are underpinned between these values and the ultimate failure strength and the failure mode of the BHV tissue compared with the natural AV, and are verified against the existing experimental data. The analyses presented in this paper explain the role of fibril interface shear and critical length in regulating the biomechanics of the structural failure of the BHVs, for the first time. This insight facilitates further understanding into the underlying causes of the structural degeneration of the BHVs in vivo. PMID:26715134

  16. Evaluation of bioprosthetic heart valve failure using a matrix-fibril shear stress transfer approach.

    PubMed

    Anssari-Benam, Afshin; Barber, Asa H; Bucchi, Andrea

    2016-02-01

    A matrix-fibril shear stress transfer approach is devised and developed in this paper to analyse the primary biomechanical factors which initiate the structural degeneration of the bioprosthetic heart valves (BHVs). Using this approach, the critical length of the collagen fibrils l c and the interface shear acting on the fibrils in both BHV and natural aortic valve (AV) tissues under physiological loading conditions are calculated and presented. It is shown that the required critical fibril length to provide effective reinforcement to the natural AV and the BHV tissue is l c  = 25.36 µm and l c  = 66.81 µm, respectively. Furthermore, the magnitude of the required shear force acting on fibril interface to break a cross-linked fibril in the BHV tissue is shown to be 38 µN, while the required interfacial force to break the bonds between the fibril and the surrounding extracellular matrix is 31 µN. Direct correlations are underpinned between these values and the ultimate failure strength and the failure mode of the BHV tissue compared with the natural AV, and are verified against the existing experimental data. The analyses presented in this paper explain the role of fibril interface shear and critical length in regulating the biomechanics of the structural failure of the BHVs, for the first time. This insight facilitates further understanding into the underlying causes of the structural degeneration of the BHVs in vivo.

  17. Lifestyle and atrial fibrillation.

    PubMed

    Mattioli, Anna Vittoria

    2011-07-01

    Lifestyle factors, in particular dietary intake, have been recognized as important, modifiable risk factors for cardiovascular disease. Consuming a heart-healthy diet lowers the individual's risk for cardiovascular disease. Data on the relationship between lifestyle and atrial fibrillation are controversial; however, the strong association between obesity, atrial/ventricular dysfunction and a nonhealthy lifestyle and atrial fibrillation, suggests that a correction of nutritional habits could prevent the development of arrhythmias through a reduction of underlying cardiac diseases. Today, the Mediterranean diet is considered one of the most effective in terms of its prevention of cardiovascular disease.

  18. Microrheological Characterization of Collagen Systems: From Molecular Solutions to Fibrillar Gels

    PubMed Central

    Shayegan, Marjan; Forde, Nancy R.

    2013-01-01

    Collagen is the most abundant protein in the extracellular matrix (ECM), where its structural organization conveys mechanical information to cells. Using optical-tweezers-based microrheology, we investigated mechanical properties both of collagen molecules at a range of concentrations in acidic solution where fibrils cannot form and of gels of collagen fibrils formed at neutral pH, as well as the development of microscale mechanical heterogeneity during the self-assembly process. The frequency scaling of the complex shear modulus even at frequencies of ∼10 kHz was not able to resolve the flexibility of collagen molecules in acidic solution. In these solutions, molecular interactions cause significant transient elasticity, as we observed for 5 mg/ml solutions at frequencies above ∼200 Hz. We found the viscoelasticity of solutions of collagen molecules to be spatially homogeneous, in sharp contrast to the heterogeneity of self-assembled fibrillar collagen systems, whose elasticity varied by more than an order of magnitude and in power-law behavior at different locations within the sample. By probing changes in the complex shear modulus over 100-minute timescales as collagen self-assembled into fibrils, we conclude that microscale heterogeneity appears during early phases of fibrillar growth and continues to develop further during this growth phase. Experiments in which growing fibrils dislodge microspheres from an optical trap suggest that fibril growth is a force-generating process. These data contribute to understanding how heterogeneities develop during self-assembly, which in turn can help synthesis of new materials for cellular engineering. PMID:23936454

  19. Collagen vascular disease

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/001223.htm Collagen vascular disease To use the sharing features on ... were previously said to have "connective tissue" or "collagen vascular" disease. We now have names for many ...

  20. Biochemical and biophysical characterization of collagens of marine sponge, Ircinia fusca (Porifera: Demospongiae: Irciniidae).

    PubMed

    Pallela, Ramjee; Bojja, Sreedhar; Janapala, Venkateswara Rao

    2011-07-01

    Collagens were isolated and partially characterized from the marine demosponge, Ircinia fusca from Gulf of Mannar (GoM), India, with an aim to develop potentially applicable collagens from unused and under-used resources. The yield of insoluble, salt soluble and acid soluble forms of collagens was 31.71 ± 1.59, 20.69 ± 1.03, and 17.38 ± 0.87 mg/g dry weight, respectively. Trichrome staining, Scanning & Transmission Electron microscopic (SEM & TEM) studies confirmed the presence of collagen in the isolated, terminally globular irciniid filaments. The partially purified (gel filtration chromatography), non-fibrillar collagens appeared as basement type collagenous sheets under light microscopy whereas the purified fibrillar collagens appeared as fibrils with a repeated band periodicity of 67 nm under Atomic Force Microscope (AFM). The non-fibrillar and fibrillar collagens were seen to have affinity for anti-collagen type IV and type I antibodies raised against human collagens, respectively. The macromolecules, i.e., total protein, carbohydrate and lipid contents within the tissues were also quantified. The present information on the three characteristic irciniid collagens (filamentous, fibrillar and non-fibrillar) could assist the future attempts to unravel the therapeutically important, safer collagens from marine sponges for their use in pharmaceutical and cosmeceutical industries.

  1. Collagen XII Contributes to Epicardial and Connective Tissues in the Zebrafish Heart during Ontogenesis and Regeneration

    PubMed Central

    Marro, Jan; Pfefferli, Catherine; de Preux Charles, Anne-Sophie; Bise, Thomas

    2016-01-01

    Zebrafish heart regeneration depends on cardiac cell proliferation, epicardium activation and transient reparative tissue deposition. The contribution and the regulation of specific collagen types during the regenerative process, however, remain poorly characterized. Here, we identified that the non-fibrillar type XII collagen, which serves as a matrix-bridging component, is expressed in the epicardium of the zebrafish heart, and is boosted after cryoinjury-induced ventricular damage. During heart regeneration, an intense deposition of Collagen XII covers the outer epicardial cap and the interstitial reparative tissue. Analysis of the activated epicardium and fibroblast markers revealed a heterogeneous cellular origin of Collagen XII. Interestingly, this matrix-bridging collagen co-localized with fibrillar type I collagen and several glycoproteins in the post-injury zone, suggesting its role in tissue cohesion. Using SB431542, a selective inhibitor of the TGF-β receptor, we showed that while the inhibitor treatment did not affect the expression of collagen 12 and collagen 1a2 in the epicardium, it completely suppressed the induction of both genes in the fibrotic tissue. This suggests that distinct mechanisms might regulate collagen expression in the outer heart layer and the inner injury zone. On the basis of this study, we postulate that the TGF-β signaling pathway induces and coordinates formation of a transient collagenous network that comprises fibril-forming Collagen I and fiber-associated Collagen XII, both of which contribute to the reparative matrix of the regenerating zebrafish heart. PMID:27783651

  2. Rapid Fabrication of Living Tissue Models by Collagen Plastic Compression: Understanding Three-Dimensional Cell Matrix Repair In Vitro

    PubMed Central

    Cheema, Umber; Brown, Robert A.

    2013-01-01

    Objective To produce biomimetic collagen scaffolds for tissue modeling and as tissue-engineered implants. Approach Control of collagen fibril material parameters in collagen hydrogel scaffolds by using plastic compression (PC), resulting in direct control of cell proliferation, cell migration, and cell–cell interaction. Results We were able to control the density of collagen in such scaffolds from between 0.2% and 30%, and controllably layer the fibrils in the Z-plane. Cell migration was observed in gels where a gradient of collagen density was present. In these gels, cells preferentially migrated toward the collagen-dense areas. Cell proliferation rates were measurably higher in dense collagen gels. Innovation The use of PC to control material properties of collagen hydrogels results in collagen scaffolds that are biomimetic. These collagen gels reproduce the relevant matrix-mechanical environment in which behavior is more representative of that found in vivo. Conclusion The material properties of native collagen type I gels can be engineered to match those found in tissues in vivo to elicit more biomimetic cell behavior. PMID:24527341

  3. Distributions of types I, II and III collagen by region in the human supraspinatus tendon.

    PubMed

    Buckley, Mark R; Evans, Elisabeth B; Matuszewski, Paul E; Chen, Yi-Ling; Satchel, Lauren N; Elliott, Dawn M; Soslowsky, Louis J; Dodge, George R

    2013-01-01

    The mechanical properties of the human supraspinatus tendon (SST) are highly heterogeneous and may reflect an important adaptive response to its complex, multiaxial loading environment. However, these functional properties are associated with a location-dependent structure and composition that have not been fully elucidated. Therefore, the objective of this study was to determine the concentrations of types I, II and III collagen in six distinct regions of the SST and compare changes in collagen concentration across regions with local changes in mechanical properties. We hypothesized that type I collagen content would be high throughout the tendon, type II collagen would be restricted to regions of compressive loading and type III collagen content would be high in regions associated with damage. We further hypothesized that regions of high type III collagen content would correspond to regions with low tensile modulus and a low degree of collagen alignment. Although type III collagen content was not significantly higher in regions that are frequently damaged, all other hypotheses were supported by our results. In particular, type II collagen content was highest near the insertion while type III collagen was inversely correlated with tendon modulus and collagen alignment. The measured increase in type II collagen under the coracoacromial arch provides evidence of adaptation to compressive loading in the SST. Moreover, the structure-function relationship between type III collagen content and tendon mechanics established in this study demonstrates a mechanism for altered mechanical properties in pathological tendons and provides a guideline for identifying therapeutic targets and pathology-specific biomarkers.

  4. Type VI Collagen Regulates Dermal Matrix Assembly and Fibroblast Motility.

    PubMed

    Theocharidis, Georgios; Drymoussi, Zoe; Kao, Alexander P; Barber, Asa H; Lee, David A; Braun, Kristin M; Connelly, John T

    2016-01-01

    Type VI collagen is a nonfibrillar collagen expressed in many connective tissues and implicated in extracellular matrix (ECM) organization. We hypothesized that type VI collagen regulates matrix assembly and cell function within the dermis of the skin. In the present study we examined the expression pattern of type VI collagen in normal and wounded skin and investigated its specific function in new matrix deposition by human dermal fibroblasts. Type VI collagen was expressed throughout the dermis of intact human skin, at the expanding margins of human keloid samples, and in the granulation tissue of newly deposited ECM in a mouse model of wound healing. Generation of cell-derived matrices (CDMs) by human dermal fibroblasts with stable knockdown of COL6A1 revealed that type VI collagen-deficient matrices were significantly thinner and contained more aligned, thicker, and widely spaced fibers than CDMs produced by normal fibroblasts. In addition, there was significantly less total collagen and sulfated proteoglycans present in the type VI collagen-depleted matrices. Normal fibroblasts cultured on de-cellularized CDMs lacking type VI collagen displayed increased cell spreading, migration speed, and persistence. Taken together, these findings indicate that type VI collagen is a key regulator of dermal matrix assembly, composition, and fibroblast behavior and may play an important role in wound healing and tissue regeneration. PMID:26763426

  5. NONPOTENTIALITY OF CHROMOSPHERIC FIBRILS IN NOAA ACTIVE REGIONS 11092 AND 9661

    SciTech Connect

    Jing Ju; Yuan Yuan; Xu Yan; Wang Haimin; Reardon, Kevin; Wiegelmann, Thomas E-mail: yy46@njit.edu E-mail: haimin@flare.njit.edu E-mail: wiegelmann@linmpi.mpg.de

    2011-10-01

    In this paper, we present a method to automatically segment chromospheric fibrils from H{alpha} observations and further identify their orientation. We assume that chromospheric fibrils are aligned with the magnetic field. By comparing the orientation of the fibrils with the azimuth of the embedding chromospheric magnetic field extrapolated from a potential field model, the shear angle, a measure of nonpotentiality, along the fibrils is readily deduced. Following this approach, we make a quantitative assessment of the nonpotentiality of fibrils in two NOAA active regions (ARs): (1) the relatively simple AR 11092, observed with very high resolution by Interferometric Bidimensional Spectrometer, and (2) a {beta}-{gamma}-{delta} AR 9661, observed with median resolution by Big Bear Solar Observatory before and after an X1.6 flare.

  6. Are the polarization colors of picrosirius red-stained collagen determined only by the diameter of the fibers?

    PubMed

    Dayan, D; Hiss, Y; Hirshberg, A; Bubis, J J; Wolman, M

    1989-01-01

    Polarization colors of various purified collagens were studied in fibers of similar thickness. Three different soluble collagens of type I, insoluble collagen type I, lathyritic collagen type I, two p-N-collagens type I, pepsin extract collagen type II, two soluble collagens type III, p-N-collagen type III, and soluble collagen type V were submitted to a routine histopathologic procedure of fixation, preparation of 5-microns-thick sections, staining with Picrosirius red and examination under crossed polars. Polarization colors were determined for thin fibers (0.8 micron or less) an thick fibers, (1.6-2.4 microns). Most thin fibers of collagens and p-N-collagens showed green to yellowish-green polarization colors with no marked differences between the various samples. Thick fibers of all p-N-collagens, lathyritic and normal 0.15 M NaCl-soluble collagens showed green to greenish-yellow polarization colors, while in all other collagens, polarization colors of longer wavelengths (from yellowish-orange to red) were observed. These data suggested that fiber thickness was not the only factor involved in determining the polarization colors of Picrosirius red-stained collagens. Tightly packed and presumably, better aligned collagen molecules showed polarization colors of longer wavelengths. Thus, packing of collagen molecules and not only fiber thickness plays a role in the pattern of polarization colors of Picrosirius red-stained collagens.

  7. Alignment validation

    SciTech Connect

    ALICE; ATLAS; CMS; LHCb; Golling, Tobias

    2008-09-06

    The four experiments, ALICE, ATLAS, CMS and LHCb are currently under constructionat CERN. They will study the products of proton-proton collisions at the Large Hadron Collider. All experiments are equipped with sophisticated tracking systems, unprecedented in size and complexity. Full exploitation of both the inner detector andthe muon system requires an accurate alignment of all detector elements. Alignmentinformation is deduced from dedicated hardware alignment systems and the reconstruction of charged particles. However, the system is degenerate which means the data is insufficient to constrain all alignment degrees of freedom, so the techniques are prone to converging on wrong geometries. This deficiency necessitates validation and monitoring of the alignment. An exhaustive discussion of means to validate is subject to this document, including examples and plans from all four LHC experiments, as well as other high energy experiments.

  8. 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

  9. On the presence of affine fibril and fiber kinematics in the mitral valve anterior leaflet.

    PubMed

    Lee, Chung-Hao; Zhang, Will; Liao, Jun; Carruthers, Christopher A; Sacks, Jacob I; Sacks, Michael S

    2015-04-21

    In this study, we evaluated the hypothesis that the constituent fibers follow an affine deformation kinematic model for planar collagenous tissues. Results from two experimental datasets were utilized, taken at two scales (nanometer and micrometer), using mitral valve anterior leaflet (MVAL) tissues as the representative tissue. We simulated MVAL collagen fiber network as an ensemble of undulated fibers under a generalized two-dimensional deformation state, by representing the collagen fibrils based on a planar sinusoidally shaped geometric model. The proposed approach accounted for collagen fibril amplitude, crimp period, and rotation with applied macroscopic tissue-level deformation. When compared to the small angle x-ray scattering measurements, the model fit the data well, with an r(2) = 0.976. This important finding suggests that, at the homogenized tissue-level scale of ∼1 mm, the collagen fiber network in the MVAL deforms according to an affine kinematics model. Moreover, with respect to understanding its function, affine kinematics suggests that the constituent fibers are largely noninteracting and deform in accordance with the bulk tissue. It also suggests that the collagen fibrils are tightly bounded and deform as a single fiber-level unit. This greatly simplifies the modeling efforts at the tissue and organ levels, because affine kinematics allows a straightforward connection between the macroscopic and local fiber strains. It also suggests that the collagen and elastin fiber networks act independently of each other, with the collagen and elastin forming long fiber networks that allow for free rotations. Such freedom of rotation can greatly facilitate the observed high degree of mechanical anisotropy in the MVAL and other heart valves, which is essential to heart valve function. These apparently novel findings support modeling efforts directed toward improving our fundamental understanding of tissue biomechanics in healthy and diseased conditions.

  10. Micro-mechanical model for the tension-stabilized enzymatic degradation of collagen tissues

    NASA Astrophysics Data System (ADS)

    Nguyen, Thao; Ruberti, Jeffery

    We present a study of how the collagen fiber structure influences the enzymatic degradation of collagen tissues. Experiments of collagen fibrils and tissues show that mechanical tension can slow and halt enzymatic degradation. Tissue-level experiments also show that degradation rate is minimum at a stretch level coincident with the onset of strain-stiffening in the stress response. To understand these phenomena, we developed a micro-mechanical model of a fibrous collagen tissue undergoing enzymatic degradation. Collagen fibers are described as sinusoidal elastica beams, and the tissue is described as a distribution of fibers. We assumed that the degradation reaction is inhibited by the axial strain energy of the crimped collagen fibers. The degradation rate law was calibrated to experiments on isolated single fibrils from bovine sclera. The fiber crimp and properties were fit to uniaxial tension tests of tissue strips. The fibril-level kinetic and tissue-level structural parameters were used to predict tissue-level degradation-induced creep rate under a constant applied force. We showed that we could accurately predict the degradation-induce creep rate of the pericardium and cornea once we accounted for differences in the fiber crimp structure and properties.

  11. Collagen oligomers modulate physical and biological properties of three-dimensional self-assembled matrices.

    PubMed

    Bailey, J L; Critser, P J; Whittington, C; Kuske, J L; Yoder, M C; Voytik-Harbin, S L

    2011-02-01

    Elucidation of mechanisms underlying collagen fibril assembly and matrix-induced guidance of cell fate will contribute to the design and expanded use of this biopolymer for research and clinical applications. Here, we define how Type I collagen oligomers affect in-vitro polymerization kinetics as well as fibril microstructure and mechanical properties of formed matrices. Monomers and oligomers were fractionated from acid-solubilized pig skin collagen and used to generate formulations varying in monomer/oligomer content or average polymer molecular weight (AMW). Polymerization half-times decreased with increasing collagen AMW and closely paralleled lag times, indicating that oligomers effectively served as nucleation sites. Furthermore, increasing AMW yielded matrices with increased interfibril branching and had no correlative effect on fibril density or diameter. These microstructure changes increased the stiffness of matrices as evidenced by increases in both shear storage and compressive moduli. Finally, the biological relevance of modulating collagen AMW was evidenced by the ability of cultured endothelial colony forming cells to sense associated changes in matrix physical properties and alter vacuole and capillary-like network formation. This work documents the importance of oligomers as another physiologically-relevant design parameter for development and standardization of polymerizable collagen formulations to be used for cell culture, regenerative medicine, and engineered tissue applications. PMID:20740490

  12. Complications of collagenous colitis.

    PubMed

    Freeman, Hugh-James

    2008-03-21

    Microscopic forms of colitis have been described, including collagenous colitis. This disorder generally has an apparently benign clinical course. However, a number of gastric and intestinal complications, possibly coincidental, may develop with collagenous colitis. Distinctive inflammatory disorders of the gastric mucosa have been described, including lymphocytic gastritis and collagenous gastritis. Celiac disease and collagenous sprue (or collagenous enteritis) may occur. Colonic ulceration has been associated with use of nonsteroidal anti-inflammatory drugs, while other forms of inflammatory bowel disease, including ulcerative colitis and Crohn's disease, may evolve from collagenous colitis. Submucosal "dissection", colonic fractures or mucosal tears and perforation from air insufflation during colonoscopy may occur and has been hypothesized to be due to compromise of the colonic wall from submucosal collagen deposition. Similar changes may result from increased intraluminal pressure during barium enema contrast studies. Finally, malignant disorders have also been reported, including carcinoma and lymphoproliferative disease. PMID:18350593

  13. Lumican deficiency results in cardiomyocyte hypertrophy with altered collagen assembly.

    PubMed

    Dupuis, Loren E; Berger, Matthew G; Feldman, Samuel; Doucette, Lorna; Fowlkes, Vennece; Chakravarti, Shukti; Thibaudeau, Sarah; Alcala, Nicolas E; Bradshaw, Amy D; Kern, Christine B

    2015-07-01

    The ability of the heart to adapt to increased stress is dependent on the modification of its extracellular matrix (ECM) architecture that is established during postnatal development as cardiomyocytes differentiate, a process that is poorly understood. We hypothesized that the small leucine-rich proteoglycan (SLRP) lumican (LUM), which binds collagen and facilitates collagen assembly in other tissues, may play a critical role in establishing the postnatal murine myocardial ECM. Although previous studies suggest that LUM deficient mice (lum(-/-)) exhibit skin anomalies consistent with Ehlers-Danlos syndrome, lum(-/-) hearts have not been evaluated. These studies show that LUM was immunolocalized to non-cardiomyocytes of the cardiac ventricles and its expression increased throughout development. Lumican deficiency resulted in significant (50%) perinatal death and further examination of the lum(-/-) neonatal hearts revealed an increase in myocardial tissue without a significant increase in cell proliferation. However cardiomyocytes from surviving postnatal day 0 (P0), 1 month (1 mo) and adult (4 mo) lum(-/-) hearts were significantly larger than their wild type (WT) littermates. Immunohistochemistry revealed that the increased cardiomyocyte size in the lum(-/-) hearts correlated with alteration of the cardiomyocyte pericellular ECM components collagenα1(I) and the class I SLRP decorin (DCN). Western blot analysis demonstrated that the ratio of glycosaminoglycan (GAG) decorated DCN to core DCN was reduced in P0 and 1 mo lum(-/-) hearts. There was also a reduction in the β and γ forms of collagenα1(I) in lum(-/-) hearts. While the total insoluble collagen content was significantly reduced, the fibril size was increased in lum(-/-) hearts, indicating that LUM may play a role in collagen fiber stability and lateral fibril assembly. These results suggest that LUM controls cardiomyocyte growth by regulating the pericellular ECM and also indicates that LUM may coordinate

  14. Lumican deficiency results in cardiomyocyte hypertrophy with altered collagen assembly.

    PubMed

    Dupuis, Loren E; Berger, Matthew G; Feldman, Samuel; Doucette, Lorna; Fowlkes, Vennece; Chakravarti, Shukti; Thibaudeau, Sarah; Alcala, Nicolas E; Bradshaw, Amy D; Kern, Christine B

    2015-07-01

    The ability of the heart to adapt to increased stress is dependent on the modification of its extracellular matrix (ECM) architecture that is established during postnatal development as cardiomyocytes differentiate, a process that is poorly understood. We hypothesized that the small leucine-rich proteoglycan (SLRP) lumican (LUM), which binds collagen and facilitates collagen assembly in other tissues, may play a critical role in establishing the postnatal murine myocardial ECM. Although previous studies suggest that LUM deficient mice (lum(-/-)) exhibit skin anomalies consistent with Ehlers-Danlos syndrome, lum(-/-) hearts have not been evaluated. These studies show that LUM was immunolocalized to non-cardiomyocytes of the cardiac ventricles and its expression increased throughout development. Lumican deficiency resulted in significant (50%) perinatal death and further examination of the lum(-/-) neonatal hearts revealed an increase in myocardial tissue without a significant increase in cell proliferation. However cardiomyocytes from surviving postnatal day 0 (P0), 1 month (1 mo) and adult (4 mo) lum(-/-) hearts were significantly larger than their wild type (WT) littermates. Immunohistochemistry revealed that the increased cardiomyocyte size in the lum(-/-) hearts correlated with alteration of the cardiomyocyte pericellular ECM components collagenα1(I) and the class I SLRP decorin (DCN). Western blot analysis demonstrated that the ratio of glycosaminoglycan (GAG) decorated DCN to core DCN was reduced in P0 and 1 mo lum(-/-) hearts. There was also a reduction in the β and γ forms of collagenα1(I) in lum(-/-) hearts. While the total insoluble collagen content was significantly reduced, the fibril size was increased in lum(-/-) hearts, indicating that LUM may play a role in collagen fiber stability and lateral fibril assembly. These results suggest that LUM controls cardiomyocyte growth by regulating the pericellular ECM and also indicates that LUM may coordinate

  15. Developmental Stage-dependent Regulation of Prolyl 3-Hydroxylation in Tendon Type I Collagen.

    PubMed

    Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

    2016-01-01

    3-Hydroxyproline (3-Hyp), which is unique to collagen, is a fairly rare post-translational modification. Recent studies have suggested a function of prolyl 3-hydroxylation in fibril assembly and its relationships with certain disorders, including recessive osteogenesis imperfecta and high myopia. However, no direct evidence for the physiological and pathological roles of 3-Hyp has been presented. In this study, we first estimated the overall alterations in prolyl hydroxylation in collagens purified from skin, bone, and tail tendon of 0.5-18-month-old rats by LC-MS analysis with stable isotope-labeled collagen, which was recently developed as an internal standard for highly accurate collagen analyses. 3-Hyp was found to significantly increase in tendon collagen until 3 months after birth and then remain constant, whereas increased prolyl 3-hydroxylation was not observed in skin and bone collagen. Site-specific analysis further revealed that 3-Hyp was increased in tendon type I collagen in a specific sequence region, including a previously known modification site at Pro(707) and newly identified sites at Pro(716) and Pro(719), at the early ages. The site-specific alterations in prolyl 3-hydroxylation with aging were also observed in bovine Achilles tendon. We postulate that significant increases in 3-Hyp at the consecutive modification sites are correlated with tissue development in tendon. The present findings suggest that prolyl 3-hydroxylation incrementally regulates collagen fibril diameter in tendon.

  16. Polyvinyl alcohol-graft-polyethylene glycol hydrogels improve utility and biofunctionality of injectable collagen biomaterials.

    PubMed

    Hartwell, Ryan; Chan, Ben; Elliott, Keenan; Alnojeidi, Hatem; Ghahary, Aziz

    2016-06-08

    Collagen-based materials have become a staple in both research and the clinic. In wound care, collagen-based materials comprise a core gamut of biological dressings and therapeutic strategies. In research, collagen-based materials are employed in everything from 3D cultures to bioprinting. Soluble collagen is well characterized to undergo fibrillation at neutral pH and 37 °C. To remain stable, a neutralized collagen solution must be maintained at 4 °C. These physical characteristics of collagen impose limitations on its utility. In our previous work, we identified that the incorporation of a simple polyvinyl alcohol:borate hydrogel could improve the rate of collagen gel fibrillation. In this work we sought to further investigate the interactions of polyvinyl alcohol blend variants, as surfactant-like polymers, in comparison with known non-polymer surfactants. To conduct our investigations scaffold variants were created using increasing concentrations of polyvinyl alcohol, differing combinations of polymers, and non-polymer surfactants Tweens 20 and 80, and TritonX-100. Activation energy for collagen fibrillation was found to significantly decrease in the presence of polyvinyl alcohols (p  <  0.01) at and above 0.4%w/v concentration. Further, addition of polyvinyl alcohol-graft-polyethylene glycol had the greatest enhancement (2.02 fold) on the fibrillation kinetics (p  <  0.01), wetting properties and the stability of the collagen scaffolds post-freeze drying. Our results demonstrated that the addition of polyvinyl alcohol hydrogels to a collagen solution could stabilize collagen solution such that the solution could easily be lyophilized (at pH 7) and then reconstituted with water. Cells cultured in polyvinyl alcohol scaffolds also exhibited more organized F-actin, as well as a reduced abundance of pro-collagen and α-smooth actin. In conclusion, our results demonstrate for the first time that polyvinyl alcohol, preferably polyvinyl alcohol

  17. The nanometre-scale physiology of bone: steric modelling and scanning transmission electron microscopy of collagen-mineral structure.

    PubMed

    Alexander, Benjamin; Daulton, Tyrone L; Genin, Guy M; Lipner, Justin; Pasteris, Jill D; Wopenka, Brigitte; Thomopoulos, Stavros

    2012-08-01

    The nanometre-scale structure of collagen and bioapatite within bone establishes bone's physical properties, including strength and toughness. However, the nanostructural organization within bone is not well known and is debated. Widely accepted models hypothesize that apatite mineral ('bioapatite') is present predominantly inside collagen fibrils: in 'gap channels' between abutting collagen molecules, and in 'intermolecular spaces' between adjacent collagen molecules. However, recent studies report evidence of substantial extrafibrillar bioapatite, challenging this hypothesis. We studied the nanostructure of bioapatite and collagen in mouse bones by scanning transmission electron microscopy (STEM) using electron energy loss spectroscopy and high-angle annular dark-field imaging. Additionally, we developed a steric model to estimate the packing density of bioapatite within gap channels. Our steric model and STEM results constrain the fraction of total bioapatite in bone that is distributed within fibrils at less than or equal to 0.42 inside gap channels and less than or equal to 0.28 inside intermolecular overlap regions. Therefore, a significant fraction of bone's bioapatite (greater than or equal to 0.3) must be external to the fibrils. Furthermore, we observe extrafibrillar bioapatite between non-mineralized collagen fibrils, suggesting that initial bioapatite nucleation and growth are not confined to the gap channels as hypothesized in some models. These results have important implications for the mechanics of partially mineralized and developing tissues.

  18. Impact of temperature and electrical potentials on the stability and structure of collagen adsorbed on the gold electrode

    NASA Astrophysics Data System (ADS)

    Meiners, Frank; Ahlers, Michael; Brand, Izabella; Wittstock, Gunther

    2015-01-01

    The morphology and structure of collagen type I adsorbed on gold electrodes were studied as a function of electrode potential and temperature by means of capacitance measurements, polarization modulation infrared reflection-absorption spectroscopy and scanning force microscopy at temperatures of 37 °C, 43 °C and 50 °C. The selected temperatures corresponded to the normal body temperature, temperature of denaturation of collagen molecules and denaturation of collagen fibrils, respectively. Independently of the solution temperature, collagen was adsorbed on gold electrodes in the potential range - 0.7 V < E < 0.4 V vs. Ag/AgCl, where the protein film was very stable. Fragments of collagen molecules made a direct contact to the gold surface and water was present in the film. Protein molecules were oriented preferentially with their long axis towards the gold surface. Collagen molecules in the adsorbed state preserved their native triple helical structure even at temperatures corresponding to collagen denaturation in aqueous solutions. Application of E < - 0.75 V vs. Ag/AgCl leads to the swelling of the protein film by water and desorption from the electrode surface. IR spectra provided no evidence of the thermal denaturation of adsorbed collagen molecules. A temperature increase to 50 °C leads to a distortion of the collagen film. The processes of aggregation and fibrilization were preferred over thermal denaturation for collagen adsorbed on the electrode surface and exposed to changing potentials.

  19. Lung response to ultrafine Kevlar aramid synthetic fibrils following 2-year inhalation exposure in rats.

    PubMed

    Lee, K P; Kelly, D P; O'Neal, F O; Stadler, J C; Kennedy, G L

    1988-07-01

    Four groups of 100 male and 100 female rats were exposed to ultrafine Kevlar fibrils at concentrations of 0, 2.5, 25, and 100 fibrils/cc for 6 hr/day, 5 days/week for 2 years. One group was exposed to 400 fibrils/cc for 1 year and allowed to recover for 1 year. At 2.5 fibrils/cc, the lungs had normal alveolar architecture with a few dust-laden macrophages (dust cell response) in the alveolar airspaces. At 25 fibrils/cc, the lungs showed a dust cell response, slight Type II pneumocyte hyperplasia, alveolar bronchiolarization, and a negligible amount of collagenized fibrosis in the alveolar duct region. At 100 fibrils/cc, the same pulmonary responses were seen as at 25 fibrils/cc. In addition, cystic keratinizing squamous cell carcinoma (CKSCC) was found in 4 female rats, but not in male rats. Female rats had more prominent foamy alveolar macrophages, cholesterol granulomas, and alveolar bronchiolarization. These pulmonary lesions were related to the development of CKSCC. The lung tumors were derived from metaplastic squamous cells in areas of alveolar bronchiolarization. At 400 fibrils/cc following 1 year of recovery, the lung dust content, average fiber length, and the pulmonary lesions were markedly reduced, but slight centriacinar emphysema and minimal collagenized fibrosis were found in the alveolar duct region. One male and 6 female rats developed CKSCC. The lung tumors were a unique type of experimentally induced tumors in the rats and have not been seen as spontaneous tumors in man or animals. Therefore, the relevance of this type of lung tumor to the human situation is minimal.

  20. Emergency management of atrial fibrillation

    PubMed Central

    Wakai, A; O'Neill, J

    2003-01-01

    Atrial fibrillation is the most common cardiac arrhythmia managed by emergency and acute general physicians. There is increasing evidence that selected patients with acute atrial fibrillation can be safely managed in the emergency department without the need for hospital admission. Meanwhile, there is significant variation in the current emergency management of acute atrial fibrillation. This review discusses evidence based emergency management of atrial fibrillation. The principles of emergency management of acute atrial fibrillation and the subset of patients who may not need hospital admission are reviewed. Finally, the need for evidence based guidelines before emergency department based clinical pathways for the management of acute atrial fibrillation becomes routine clinical practice is highlighted. PMID:12840118

  1. Rough fibrils provide a toughening mechanism in biological fibers.

    PubMed

    Brown, Cameron P; Harnagea, Catalin; Gill, Harinderjit S; Price, Andrew J; Traversa, Enrico; Licoccia, Silvia; Rosei, Federico

    2012-03-27

    Spider silk is a fascinating natural composite material. Its combination of strength and toughness is unrivalled in nature, and as a result, it has gained considerable interest from the medical, physics, and materials communities. Most of this attention has focused on the one to tens of nanometer scale: predominantly the primary (peptide sequences) and secondary (β sheets, helices, and amorphous domains) structure, with some insights into tertiary structure (the arrangement of these secondary structures) to describe the origins of the mechanical and biological performance. Starting with spider silk, and relating our findings to collagen fibrils, we describe toughening mechanisms at the hundreds of nanometer scale, namely, the fibril morphology and its consequences for mechanical behavior and the dissipation of energy. Under normal conditions, this morphology creates a nonslip fibril kinematics, restricting shearing between fibrils, yet allowing controlled local slipping under high shear stress, dissipating energy without bulk fracturing. This mechanism provides a relatively simple target for biomimicry and, thus, can potentially be used to increase fracture resistance in synthetic materials. PMID:22324287

  2. Genetics Home Reference: familial atrial fibrillation

    MedlinePlus

    ... fibrillation also increases the risk of stroke and sudden death. Complications of familial atrial fibrillation can occur at ... beats , increasing the risk of syncope, stroke, and sudden death. Most cases of atrial fibrillation are not caused ...

  3. Reinforcement of polymeric structures with asbestos fibrils

    NASA Technical Reports Server (NTRS)

    Rader, C. A.; Schwartz, A. M.

    1970-01-01

    Investigation determines structural potential of asbestos fibrils. Methods are developed for dispersing macrofibers of the asbestos into colloidal-sized ultimate fibrils and incorporating these fibrils in matrices without causing reagglomeration.

  4. Guidance of in vitro migration of human mesenchymal stem cells and in vivo guided bone regeneration using aligned electrospun fibers.

    PubMed

    Lee, Ji-hye; Lee, Young Jun; Cho, Hyeong-jin; Shin, Heungsoo

    2014-08-01

    Tissue regeneration is a complex process in which numerous chemical and physical signals are coordinated in a specific spatiotemporal pattern. In this study, we tested our hypothesis that cell migration and bone tissue formation can be guided and facilitated by microscale morphological cues presented from a scaffold. We prepared poly(l-lactic acid) (PLLA) electrospun fibers with random and aligned structures and investigated their effect on in vitro migration of human mesenchymal stem cells (hMSCs) and in vivo bone growth using a critical-sized defect model. Using a polydopamine coating on the fibers, we compared the synergistic effects of chemical signals. The adhesion morphology of hMSCs was consistent with the direction of fiber alignment, whereas the proliferation of hMSCs was not affected. The orientation of fibers profoundly affected cell migration, in which hMSCs cultured on aligned fibers migrated 10.46-fold faster along the parallel direction than along the perpendicular direction on polydopamine-coated PLLA nanofibers. We implanted each fiber type into a mouse calvarial defect model for 2 months. The micro-computed tomography (CT) imaging demonstrated that regenerated bone area was the highest when mice were implanted with aligned fibers with polydopamine coating, indicating a positive synergistic effect on bone regeneration. More importantly, scanning electron microscopy microphotographs revealed that the direction of regenerated bone tissue appeared to be consistent with the direction of the implanted fibers, and transmission electron microscopy images showed that the orientation of collagen fibrils appeared to be overlapped along the direction of nanofibers. Taken together, our results demonstrate that the aligned nanofibers can provide spatial guidance for in vitro cell migration as well as in vivo bone regeneration, which may be incorporated as major instructive cues for the stimulation of tissue regeneration.

  5. Both hyaluronan and collagen type II keep proteoglycan 4 (lubricin) at the cartilage surface in a condition that provides low friction during boundary lubrication.

    PubMed

    Majd, Sara Ehsani; Kuijer, Roel; Köwitsch, Alexander; Groth, Thomas; Schmidt, Tannin A; Sharma, Prashant K

    2014-12-01

    Wear resistant and ultralow friction in synovial joints is the outcome of a sophisticated synergy between the major macromolecules of the synovial fluid, e.g., hyaluronan (HA) and proteoglycan 4 (PRG4), with collagen type II fibrils and other non-collagenous macromolecules of the cartilage superficial zone (SZ). This study aimed at better understanding the mechanism of PRG4 localization at the cartilage surface. We show direct interactions between surface bound HA and freely floating PRG4 using the quartz crystal microbalance with dissipation (QCM-D). Freely floating PRG4 was also shown to bind with surface bound collagen type II fibrils. Albumin, the most abundant protein of the synovial fluid, effectively blocked the adsorption of PRG4 with HA, through interaction with C and N terminals on PRG4, but not that of PRG4 with collagen type II fibrils. The above results indicate that collagen type II fibrils strongly contribute in keeping PRG4 in the SZ during cartilage articulation in situ. Furthermore, PRG4 molecules adsorbed very well on mimicked SZ of absorbed HA molecules with entangled collagen type II fibrils and albumin was not able to block this interaction. In this last condition PRG4 adsorption resulted in a coefficient of friction (COF) of the same order of magnitude as the COF of natural cartilage, measured with an atomic force microscope in lateral mode.

  6. Cell Adhesion on Amyloid Fibrils Lacking Integrin Recognition Motif.

    PubMed

    Jacob, Reeba S; George, Edna; Singh, Pradeep K; Salot, Shimul; Anoop, Arunagiri; Jha, Narendra Nath; Sen, Shamik; Maji, Samir K

    2016-03-01

    Amyloids are highly ordered, cross-β-sheet-rich protein/peptide aggregates associated with both human diseases and native functions. Given the well established ability of amyloids in interacting with cell membranes, we hypothesize that amyloids can serve as universal cell-adhesive substrates. Here, we show that, similar to the extracellular matrix protein collagen, amyloids of various proteins/peptides support attachment and spreading of cells via robust stimulation of integrin expression and formation of integrin-based focal adhesions. Additionally, amyloid fibrils are also capable of immobilizing non-adherent red blood cells through charge-based interactions. Together, our results indicate that both active and passive mechanisms contribute to adhesion on amyloid fibrils. The present data may delineate the functional aspect of cell adhesion on amyloids by various organisms and its involvement in human diseases. Our results also raise the exciting possibility that cell adhesivity might be a generic property of amyloids. PMID:26742841

  7. Forward versus backward polarization-resolved SHG imaging of collagen structure in tissues

    NASA Astrophysics Data System (ADS)

    Teulon, Claire; Gusachenko, Ivan; Latour, Gaël.; Schanne-Klein, Marie-Claire

    2016-03-01

    Second harmonic generation (SHG) is a powerful technique to observe fibrillar collagen without any staining and with a good contrast. More information about the molecular structure of collagen fibrils in tissues and their 3D distribution can be gained with polarization-resolved SHG imaging. Nevertheless, strong focusing is required for effective imaging and light propagation in tissues is complex and not thoroughly understood yet, preventing accurate and reproducible measurements. Theoretical analysis, vectorial numerical simulations and experiments were implemented to understand how the SHG signal builds up and how geometrical parameters affect polarization-resolved measurements in homogeneous collagen-rich tissues.

  8. Hydroxyapatite reinforced collagen scaffolds with improved architecture and mechanical properties.

    PubMed

    Kane, Robert J; Weiss-Bilka, Holly E; Meagher, Matthew J; Liu, Yongxing; Gargac, Joshua A; Niebur, Glen L; Wagner, Diane R; Roeder, Ryan K

    2015-04-01

    Hydroxyapatite (HA) reinforced collagen scaffolds have shown promise for synthetic bone graft substitutes and tissue engineering scaffolds. Freeze-dried HA-collagen scaffolds are readily fabricated and have exhibited osteogenicity in vivo, but are limited by an inherent scaffold architecture that results in a relatively small pore size and weak mechanical properties. In order to overcome these limitations, HA-collagen scaffolds were prepared by compression molding HA reinforcements and paraffin microspheres within a suspension of concentrated collagen fibrils (∼ 180 mg/mL), cross-linking the collagen matrix, and leaching the paraffin porogen. HA-collagen scaffolds exhibited an architecture with high porosity (85-90%), interconnected pores ∼ 300-400 μm in size, and struts ∼ 3-100 μm in thickness containing 0-80 vol% HA whisker or powder reinforcements. HA reinforcement enabled a compressive modulus of up to ∼ 1 MPa, which was an order of magnitude greater than unreinforced collagen scaffolds. The compressive modulus was also at least one order of magnitude greater than comparable freeze-dried HA-collagen scaffolds and two orders of magnitude greater than absorbable collagen sponges used clinically. Moreover, scaffolds reinforced with up to 60 vol% HA exhibited fully recoverable elastic deformation upon loading to 50% compressive strain for at least 100,000 cycles. Thus, the scaffold mechanical properties were well-suited for surgical handling, fixation, and bearing osteogenic loads during bone regeneration. The scaffold architecture, permeability, and composition were shown to be conducive to the infiltration and differentiation of adipose-derive stromal cells in vitro. Acellular scaffolds were demonstrated to induce angiogenesis and osteogenesis after subcutaneous ectopic implantation by recruiting endogenous cell populations, suggesting that the scaffolds were osteoinductive.

  9. Lysyl Hydroxylase 3-mediated Glucosylation in Type I Collagen

    PubMed Central

    Sricholpech, Marnisa; Perdivara, Irina; Yokoyama, Megumi; Nagaoka, Hideaki; Terajima, Masahiko; Tomer, Kenneth B.; Yamauchi, Mitsuo

    2012-01-01

    Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing lysyl hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846–8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization. PMID:22573318

  10. MORPHOLOGICAL AND CHEMICAL STUDIES OF COLLAGEN FORMATION

    PubMed Central

    Chapman, J. A.

    1961-01-01

    This paper describes electron microscopic studies of developing connective tissue in granulomata induced by the subcutaneous injection of carrageenin into guinea pigs. Seven days after injection the granulomata contained many fibroblasts and exhibited rapid production of collagen. The fibroblasts were characterised by an extensively developed endoplasmic reticulum and showed numbers of fine, unstriated filaments in the outer regions of the cytoplasm. The filaments, about 50 A in diameter, tended to lie parallel to and closely adjacent to the cell boundary. The cytoplasmic membrane was frequently ill defined or disrupted, particularly bordering regions in which filaments occurred. In longitudinal sections of extended cell processes, filaments were abundant and, in some instances, the cytoplasmic membrane was barely detectable. In the extracellular space striated collagen fibrils were usually accompanied by filaments, 50 to 100 A in diameter, and these often exhibited the characteristic periodicity of collagen, particularly after intense electron bombardment. Much cellular debris was present in the extracellular space. These observations have led to the suggestion that connective tissue precursors are released from fibroblasts by the disintegration or dissolution of the cytoplasmic membrane and the shedding of cytoplasmic material, as in the apocrine gland cells. In some instances this release may take the form of the elongation from the cell of extended processes; disintegration of the cytoplasmic membrane surrounding these processes then leaves the contents in the extracellular phase. PMID:13692398

  11. Enigmatic insight into collagen.

    PubMed

    Deshmukh, Shrutal Narendra; Dive, Alka M; Moharil, Rohit; Munde, Prashant

    2016-01-01

    Collagen is a unique, triple helical molecule which forms the major part of extracellular matrix. It is the most abundant protein in the human body, representing 30% of its dry weight. It is the fibrous structural protein that makes up the white fibers (collagen fibers) of skin, tendons, bones, cartilage and all other connective tissues. Collagens are not only essential for the mechanical resistance and resilience of multicellular organisms, but are also signaling molecules defining cellular shape and behavior. The human body has at least 16 types of collagen, but the most prominent types are I, II and III. Collagens are produced by several cell types and are distinguishable by their molecular compositions, morphologic characteristics, distribution, functions and pathogenesis. This is the major fibrous glycoprotein present in the extracellular matrix and in connective tissue and helps in maintaining the structural integrity of these tissues. It has a triple helical structure. Various studies have proved that mutations that modify folding of the triple helix result in identifiable genetic disorders. Collagen diseases share certain similarities with autoimmune diseases, because autoantibodies specific to each collagen disease are produced. Therefore, this review highlights the role of collagen in normal health and also the disorders associated with structural and functional defects in collagen. PMID:27601823

  12. Enigmatic insight into collagen

    PubMed Central

    Deshmukh, Shrutal Narendra; Dive, Alka M; Moharil, Rohit; Munde, Prashant

    2016-01-01

    Collagen is a unique, triple helical molecule which forms the major part of extracellular matrix. It is the most abundant protein in the human body, representing 30% of its dry weight. It is the fibrous structural protein that makes up the white fibers (collagen fibers) of skin, tendons, bones, cartilage and all other connective tissues. Collagens are not only essential for the mechanical resistance and resilience of multicellular organisms, but are also signaling molecules defining cellular shape and behavior. The human body has at least 16 types of collagen, but the most prominent types are I, II and III. Collagens are produced by several cell types and are distinguishable by their molecular compositions, morphologic characteristics, distribution, functions and pathogenesis. This is the major fibrous glycoprotein present in the extracellular matrix and in connective tissue and helps in maintaining the structural integrity of these tissues. It has a triple helical structure. Various studies have proved that mutations that modify folding of the triple helix result in identifiable genetic disorders. Collagen diseases share certain similarities with autoimmune diseases, because autoantibodies specific to each collagen disease are produced. Therefore, this review highlights the role of collagen in normal health and also the disorders associated with structural and functional defects in collagen.

  13. Enigmatic insight into collagen

    PubMed Central

    Deshmukh, Shrutal Narendra; Dive, Alka M; Moharil, Rohit; Munde, Prashant

    2016-01-01

    Collagen is a unique, triple helical molecule which forms the major part of extracellular matrix. It is the most abundant protein in the human body, representing 30% of its dry weight. It is the fibrous structural protein that makes up the white fibers (collagen fibers) of skin, tendons, bones, cartilage and all other connective tissues. Collagens are not only essential for the mechanical resistance and resilience of multicellular organisms, but are also signaling molecules defining cellular shape and behavior. The human body has at least 16 types of collagen, but the most prominent types are I, II and III. Collagens are produced by several cell types and are distinguishable by their molecular compositions, morphologic characteristics, distribution, functions and pathogenesis. This is the major fibrous glycoprotein present in the extracellular matrix and in connective tissue and helps in maintaining the structural integrity of these tissues. It has a triple helical structure. Various studies have proved that mutations that modify folding of the triple helix result in identifiable genetic disorders. Collagen diseases share certain similarities with autoimmune diseases, because autoantibodies specific to each collagen disease are produced. Therefore, this review highlights the role of collagen in normal health and also the disorders associated with structural and functional defects in collagen. PMID:27601823

  14. Syndecan-1 Regulates Cell Migration and Fibronectin Fibril Assembly

    PubMed Central

    Stepp, Mary Ann; Daley, William P.; Bernstein, Audrey M.; Pal-Ghosh, Sonali; Tadvalkar, Gauri; Shashurin, Alexey; Palsen, Sarah; Jurjus, Rosalyn A.; Larsen, Melinda

    2011-01-01

    Corneal scarring is a major cause of blindness worldwide and can result from the deposition of abnormal amounts of collagen fibers lacking the correct size and spacing required to produce a clear cornea. Collagen fiber formation requires a preformed fibronectin (FN) matrix. We demonstrate that the loss of syndecan1 (sdc1) in corneal stromal cells (CSC) impacts cell migration rates, the sizes and composition of focal and fibrillar adhesions, the activation of integrins, and the assembly of fibronectin into fibrils. Integrin and fibronectin expression are not altered on sdc1 null CSCs. Cell adhesion, spreading, and migration studies using low compared to high concentrations of FN and collagen I (CNI) or vitronectin (VN) with and without activation of integrins by manganese chloride show that the impact of sdc1 depletion on integrin activation varies depending on the integrin-mediated activity evaluated. Differences in FN-fibrillogenesis and migration in sdc1 null CSCs are reversed by addition of manganese chloride but cell spreading differences remain. To determine if our findings on sdc1 were specific to the cornea, we compared the phenotypes of sdc1 null dermal fibroblasts with those of CSCs. We found that without sdc1, both cell types migrate faster; however, cell-type specific differences in FN expression and its assembly into fibrils exist between these two cell types. Together, our data demonstrate that sdc1 functions to regulate integrin activity in multiple cell types. Loss of sdc1-mediated integrin function results in cell-type specific differences in matrix assembly. A better understanding of how different cell types regulate FN fibril formation via syndecans and integrins will lead to better treatments for scarring and fibrosis. PMID:20580707

  15. Synthetic collagen fascicles for the regeneration of tendon tissue.

    PubMed

    Kew, S J; Gwynne, J H; Enea, D; Brookes, R; Rushton, N; Best, S M; Cameron, R E

    2012-10-01

    The structure of an ideal scaffold for tendon regeneration must be designed to provide a mechanical, structural and chemotactic microenvironment for native cellular activity to synthesize functional (i.e. load bearing) tissue. Collagen fibre scaffolds for this application have shown some promise to date, although the microstructural control required to mimic the native tendon environment has yet to be achieved allowing for minimal control of critical in vivo properties such as degradation rate and mass transport. In this report we describe the fabrication of a novel multi-fibre collagen fascicle structure, based on type-I collagen with failure stress of 25-49 MPa, approximating the strength and structure of native tendon tissue. We demonstrate a microscopic fabrication process based on the automated assembly of type-I collagen fibres with the ability to produce a controllable fascicle-like, structural motif allowing variable numbers of fibres per fascicle. We have confirmed that the resulting post-fabrication type-I collagen structure retains the essential phase behaviour, alignment and spectral characteristics of aligned native type-I collagen. We have also shown that both ovine tendon fibroblasts and human white blood cells in whole blood readily infiltrate the matrix on a macroscopic scale and that these cells adhere to the fibre surface after seven days in culture. The study has indicated that the synthetic collagen fascicle system may be a suitable biomaterial scaffold to provide a rationally designed implantable matrix material to mediate tendon repair and regeneration.

  16. Alignment fixture

    DOEpatents

    Bell, Grover C.; Gibson, O. Theodore

    1980-01-01

    A part alignment fixture is provided which may be used for precise variable lateral and tilt alignment relative to the fixture base of various shaped parts. The fixture may be used as a part holder for machining or inspection of parts or alignment of parts during assembly and the like. The fixture includes a precisely machined diameter disc-shaped hub adapted to receive the part to be aligned. The hub is nested in a guide plate which is adapted to carry two oppositely disposed pairs of positioning wedges so that the wedges may be reciprocatively positioned by means of respective micrometer screws. The sloping faces of the wedges contact the hub at respective quadrants of the hub periphery. The lateral position of the hub relative to the guide plate is adjusted by positioning the wedges with the associated micrometer screws. The tilt of the part is adjusted relative to a base plate, to which the guide plate is pivotally connected by means of a holding plate. Two pairs of oppositely disposed wedges are mounted for reciprocative lateral positioning by means of separate micrometer screws between flanges of the guide plate and the base plate. Once the wedges are positioned to achieve the proper tilt of the part or hub on which the part is mounted relative to the base plate, the fixture may be bolted to a machining, inspection, or assembly device.

  17. Curriculum Alignment.

    ERIC Educational Resources Information Center

    Crowell, Ronald; Tissot, Paula

    Curriculum alignment (CA) refers to the congruence of all the elements of a school's curriculum: curriculum goals; instructional program--what is taught and the materials used; and tests used to judge outcomes. CA can be a very powerful can be a very powerful factor in improving schools. Although further research is needed on CA, there is…

  18. Electroactive biomimetic collagen-silver nanowire composite scaffolds

    NASA Astrophysics Data System (ADS)

    Wickham, Abeni; Vagin, Mikhail; Khalaf, Hazem; Bertazzo, Sergio; Hodder, Peter; Dånmark, Staffan; Bengtsson, Torbjörn; Altimiras, Jordi; Aili, Daniel

    2016-07-01

    Electroactive biomaterials are widely explored as bioelectrodes and as scaffolds for neural and cardiac regeneration. Most electrodes and conductive scaffolds for tissue regeneration are based on synthetic materials that have limited biocompatibility and often display large discrepancies in mechanical properties with the surrounding tissue causing problems during tissue integration and regeneration. This work shows the development of a biomimetic nanocomposite material prepared from self-assembled collagen fibrils and silver nanowires (AgNW). Despite consisting of mostly type I collagen fibrils, the homogeneously embedded AgNWs provide these materials with a charge storage capacity of about 2.3 mC cm-2 and a charge injection capacity of 0.3 mC cm-2, which is on par with bioelectrodes used in the clinic. The mechanical properties of the materials are similar to soft tissues with a dynamic elastic modulus within the lower kPa range. The nanocomposites also support proliferation of embryonic cardiomyocytes while inhibiting the growth of both Gram-negative Escherichia coli and Gram-positive Staphylococcus epidermidis. The developed collagen/AgNW composites thus represent a highly attractive bioelectrode and scaffold material for a wide range of biomedical applications.Electroactive biomaterials are widely explored as bioelectrodes and as scaffolds for neural and cardiac regeneration. Most electrodes and conductive scaffolds for tissue regeneration are based on synthetic materials that have limited biocompatibility and often display large discrepancies in mechanical properties with the surrounding tissue causing problems during tissue integration and regeneration. This work shows the development of a biomimetic nanocomposite material prepared from self-assembled collagen fibrils and silver nanowires (AgNW). Despite consisting of mostly type I collagen fibrils, the homogeneously embedded AgNWs provide these materials with a charge storage capacity of about 2.3 mC cm-2

  19. Mechano-regulation of Collagen Biosynthesis in Periodontal Ligament

    PubMed Central

    Kaku, Masaru; Yamauchi, Mitsuo

    2014-01-01

    Purpose Periodontal ligament (PDL) plays critical roles in the development and maintenance of periodontium such as tooth eruption and dissipation of masticatory force. The mechanical properties of PDL are mainly derived from fibrillar type I collagen, the most abundant extracellular component. Study selection The biosynthesis of type I collagen is a long, complex process including a number of intra- and extracellular post-translational modifications. The final modification step is the formation of covalent intra- and intermolecular cross-links that provide collagen fibrils with stability and connectivity. Results It is now clear that collagen post-translational modifications are regulated by groups of specific enzymes and associated molecules in a tissue-specific manner; and these modifications appear to change in response to mechanical force. Conclusions This review focuses on the effect of mechanical loading on collagen biosynthesis and fibrillogenesis in PDL with emphasis on the post-translational modifications of collagens, which is an important molecular aspect to understand in the field of prosthetic dentistry. PMID:25311991

  20. Structural basis for collagen recognition by the immune receptor OSCAR.

    PubMed

    Zhou, Long; Hinerman, Jennifer M; Blaszczyk, Michal; Miller, Jeanette L C; Conrady, Deborah G; Barrow, Alexander D; Chirgadze, Dimitri Y; Bihan, Dominique; Farndale, Richard W; Herr, Andrew B

    2016-02-01

    The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2. PMID:26552697

  1. Human collagen produced in plants: more than just another molecule.

    PubMed

    Shoseyov, Oded; Posen, Yehudit; Grynspan, Frida

    2014-01-01

    Consequential to its essential role as a mechanical support and affinity regulator in extracellular matrices, collagen constitutes a highly sought after scaffolding material for regeneration and healing applications. However, substantiated concerns have been raised with regard to quality and safety of animal tissue-extracted collagen, particularly in relation to its immunogenicity, risk of disease transmission and overall quality and consistency. In parallel, contamination with undesirable cellular factors can significantly impair its bioactivity, vis-a-vis its impact on cell recruitment, proliferation and differentiation. High-scale production of recombinant human collagen Type I (rhCOL1) in the tobacco plant provides a source of an homogenic, heterotrimeric, thermally stable "virgin" collagen which self assembles to fine homogenous fibrils displaying intact binding sites and has been applied to form numerous functional scaffolds for tissue engineering and regenerative medicine. In addition, rhCOL1 can form liquid crystal structures, yielding a well-organized and mechanically strong membrane, two properties indispensable to extracellular matrix (ECM) mimicry. Overall, the shortcomings of animal- and cadaver-derived collagens arising from their source diversity and recycled nature are fully overcome in the plant setting, constituting a collagen source ideal for tissue engineering and regenerative medicine applications. PMID:23941988

  2. Effect of Silicone on the Collagen Fibrillogenesis and Stability

    PubMed Central

    Kadziński, Leszek; Prokopowicz, Magdalena; Jakóbkiewicz-Banecka, Joanna; Gabig-Cimińska, Magdalena; Łukasiak, Jerzy; Banecki, Bogdan

    2015-01-01

    Collagen, the most abundant protein in mammals, is able to form fibrils, which have central role in tissue repair, fibrosis, and tumor invasion. As a component of skin, tendons, and cartilages, this protein contacts with any implanted materials. An inherent problem associated with implanted prostheses is their propensity to be coated with host proteins shortly after implantation. Also, silicone implants undergoing relatively long periods of contact with blood can lead to formation of thrombi and emboli. In this paper, we demonstrate the existence of interactions between siloxanes and collagen. Low-molecular-weight cyclic siloxane (hexamethylcyclotrisiloxane—D3) and polydimethylsiloxanes (PDMS) forming linear chains, ranging in viscosity from 20 to 12,000 cSt, were analyzed. We show that D3 as well as short-chain PDMS interact with collagen, resulting in a decrease in fibrillogenesis. However, loss of collagen native structure does not occur because of these interactions. Rather, collagen seems to be sequestered in its native form in an interlayer formed by collagen–siloxane complexes. On the other hand, silicone molecules with longer chains (i.e., PDMS with viscosity of 1000 and 12,000 cSt, the highest viscosity analyzed here) demonstrate little interaction with this protein and do not seem to affect collagen activity. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1275–1281, 2015 PMID:25589402

  3. Structural basis for collagen recognition by the immune receptor OSCAR.

    PubMed

    Zhou, Long; Hinerman, Jennifer M; Blaszczyk, Michal; Miller, Jeanette L C; Conrady, Deborah G; Barrow, Alexander D; Chirgadze, Dimitri Y; Bihan, Dominique; Farndale, Richard W; Herr, Andrew B

    2016-02-01

    The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2.

  4. Human collagen produced in plants: more than just another molecule.

    PubMed

    Shoseyov, Oded; Posen, Yehudit; Grynspan, Frida

    2014-01-01

    Consequential to its essential role as a mechanical support and affinity regulator in extracellular matrices, collagen constitutes a highly sought after scaffolding material for regeneration and healing applications. However, substantiated concerns have been raised with regard to quality and safety of animal tissue-extracted collagen, particularly in relation to its immunogenicity, risk of disease transmission and overall quality and consistency. In parallel, contamination with undesirable cellular factors can significantly impair its bioactivity, vis-a-vis its impact on cell recruitment, proliferation and differentiation. High-scale production of recombinant human collagen Type I (rhCOL1) in the tobacco plant provides a source of an homogenic, heterotrimeric, thermally stable "virgin" collagen which self assembles to fine homogenous fibrils displaying intact binding sites and has been applied to form numerous functional scaffolds for tissue engineering and regenerative medicine. In addition, rhCOL1 can form liquid crystal structures, yielding a well-organized and mechanically strong membrane, two properties indispensable to extracellular matrix (ECM) mimicry. Overall, the shortcomings of animal- and cadaver-derived collagens arising from their source diversity and recycled nature are fully overcome in the plant setting, constituting a collagen source ideal for tissue engineering and regenerative medicine applications.

  5. Polarization-Modulated Second Harmonic Generation Microscopy in Collagen

    SciTech Connect

    Stoller, P C

    2002-09-30

    Collagen is a key structural protein in the body; several pathological conditions lead to changes in collagen. Among imaging modalities that can be used in vivo, second harmonic generation (SHG) microscopy has a key advantage: it provides {approx}1 {micro}m resolution information about collagen structure as a function of depth. A new technique--polarization-modulated SHG--is presented: it permits simultaneous measurement of collagen orientation, of a lower bound on the magnitude of the second order nonlinear susceptibility tensor, and of the ratio of the two independent elements in this tensor. It is applied to characterizing SHG in collagen and to determining effects of biologically relevant changes in collagen structure. The magnitude of the second harmonic signal in two dimensional images varies with position even in structurally homogeneous tissue; this phenomenon is due to interference between second harmonic light generated by neighboring fibrils, which are randomly oriented parallel or anti-parallel to each other. Studies in which focal spot size was varied indicated that regions where fibrils are co-oriented are less than {approx}1.5 {micro}m in diameter. A quartz reference was used to determine the spot size as well as a lower limit (d{sub xxx} > 0.3 pm/V) for the magnitude of the second order nonlinear susceptibility. The ratio of the two independent tensor elements ranged between d{sub XYY}/d{sub XXX} = 0.60 and 0.75. SHG magnitude alone was not useful for identifying structural anomalies in collagenous tissue. Instead, changes in the polarization dependence of SHG were used to analyze biologically relevant perturbations in collagen structure. Changes in polarization dependence were observed in dehydrated samples, but not in highly crosslinked samples, despite significant alterations in packing structure. Complete thermal denaturation and collagenase digestion produced samples with no detectable SHG signal. Collagen orientation was measured in thin

  6. FTIR spectro-imaging of collagen scaffold formation during glioma tumor development.

    PubMed

    Noreen, Razia; Chien, Chia-Chi; Chen, Hsiang-Hsin; Bobroff, Vladimir; Moenner, Michel; Javerzat, Sophie; Hwu, Yeukuang; Petibois, Cyril

    2013-11-01

    Evidence has recently emerged that solid and diffuse tumors produce a specific extracellular matrix (ECM) for division and diffusion, also developing a specific interface with microvasculature. This ECM is mainly composed of collagens and their scaffolding appears to drive tumor growth. Although collagens are not easily analyzable by UV-fluorescence means, FTIR imaging has appeared as a valuable tool to characterize collagen contents in tissues, specially the brain, where ECM is normally devoid of collagen proteins. Here, we used FTIR imaging to characterize collagen content changes in growing glioma tumors. We could determine that C6-derived solid tumors presented high content of triple helix after 8-11 days of growth (typical of collagen fibrils formation; 8/8 tumor samples; 91 % of total variance), and further turned to larger α-helix (days 12-15; 9/10 of tumors; 94 % of variance) and β-turns (day 18-21; 7/8 tumors; 97 % of variance) contents, which suggest the incorporation of non-fibrillar collagen types in ECM, a sign of more and more organized collagen scaffold along tumor progression. The growth of tumors was also associated to the level of collagen produced (P < 0.05). This study thus confirms that collagen scaffolding is a major event accompanying the angiogenic shift and faster tumor growth in solid glioma phenotypes. PMID:24068168

  7. Recombinant microbial systems for the production of human collagen and gelatin.

    PubMed

    Báez, Julio; Olsen, David; Polarek, James W

    2005-12-01

    The use of genetically engineered microorganisms is a cost-effective, scalable technology for the production of recombinant human collagen (rhC) and recombinant gelatin (rG). This review will discuss the use of yeast (Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha) and of bacteria (Escherichia coli, Bacillus brevis) genetically engineered for the production of rhC and rG. P. pastoris is the preferred production system for rhC and rG. Recombinant strains of P. pastoris accumulate properly hydroxylated triple helical rhC intracellularly at levels up to 1.5 g/l. Coexpression of recombinant collagen with recombinant prolyl hydroxylase results in the synthesis of hydroxylated collagen with thermal stability similar to native collagens. The purified hydroxylated rhC forms fibrils that are structurally similar to fibrils assembled from native collagen. These qualities make rhC attractive for use in many medical applications. P. pastoris can also be engineered to secrete high levels (3 to 14 g/l ) of collagen fragments with defined length, composition, and physiochemical properties that serve as substitutes for animal-derived gelatins. The replacement of animal-derived collagen and gelatin with rhC and rG will result in products with improved safety, traceability, reproducibility, and quality. In addition, the rhC and rG can be engineered to improve the performance of products containing these biomaterials.

  8. Nano-imaging collagen by atomic force, near-field and nonlinear microscope

    NASA Astrophysics Data System (ADS)

    Lim, Ken Choong; Tang, Jinkai; Li, Hao; Ng, Boon Ping; Kok, Shaw Wei; Wang, Qijie; Zhang, Ying

    2015-03-01

    As the most abundant protein in the human body, collagen has a very important role in vast numbers of bio-medical applications. The unique second order nonlinear properties of fibrillar collagen make it a very important index in nonlinear optical imaging based disease diagnosis of the brain, skin, liver, colon, kidney, bone, heart and other organs in the human body. The second-order nonlinear susceptibility of collagen has been explored at the macroscopic level and was explained as a volume-averaged molecular hyperpolarizability. However, details about the origin of optical second harmonic signals from collagen fibrils at the molecular level are still not clear. Such information is necessary for accurate interpolation of bio-information from nonlinear optical imaging techniques. The later has shown great potential in collagen based disease diagnosis methodologies. In this paper, we report our work using an atomic force microscope (AFM), near field (SNOM) and nonlinear laser scanning microscope (NLSM) to study the structure of collagen fibrils and other pro-collagen structures.

  9. Oligomers Modulate Interfibril Branching and Mass Transport Properties of Collagen Matrices

    PubMed Central

    Whittington, Catherine F.; Brandner, Eric; Teo, Ka Yaw; Han, Bumsoo; Nauman, Eric; Voytik-Harbin, Sherry L.

    2013-01-01

    Mass transport within collagen-based matrices is critical to tissue development, repair, and pathogenesis as well as the design of next generation tissue engineering strategies. This work shows how collagen precursors, specified by intermolecular cross-link composition, provide independent control of collagen matrix mechanical and transport properties. Collagen matrices were prepared from tissue-extracted monomers or oligomers. Viscoelastic behavior was measured in oscillatory shear and unconfined compression. Matrix permeability and diffusivity were measured using gravity-driven permeametry and integrated optical imaging, respectively. Both collagen types showed an increase in stiffness and permeability hindrance with increasing collagen concentration (fibril density); however, different physical property-concentration relationships were noted. Diffusivity wasn’t affected by concentration for either collagen type over the range tested. In general, oligomer matrices exhibited a substantial increase in stiffness and only a modest decrease in transport properties when compared to monomer matrices prepared at the same concentration. The observed differences in viscoelastic and transport properties were largely attributed to increased levels of interfibril branching within oligomer matrices. The ability to relate physical properties to relevant microstructure parameters, including fibril density and interfibril branching, is expected to advance the understanding of cell-matrix signaling as well as facilitate model-based prediction and design of matrix-based therapeutic strategies. PMID:23842082

  10. Second-harmonic generation scattering directionality predicts tumor cell motility in collagen gels.

    PubMed

    Burke, Kathleen A; Dawes, Ryan P; Cheema, Mehar K; Van Hove, Amy; Benoit, Danielle S W; Perry, Seth W; Brown, Edward

    2015-05-01

    Second-harmonic generation (SHG) allows for the analysis of tumor collagen structural changes throughout metastatic progression. SHG directionality, measured through the ratio of the forward-propagating to backward-propagating signal (F/B ratio), is affected by collagen fibril diameter, spacing, and disorder of fibril packing within a fiber. As tumors progress, these parameters evolve, producing concurrent changes in F/B. It has been recently shown that the F/B of highly metastatic invasive ductal carcinoma (IDC) breast tumors is significantly different from less metastatic tumors. This suggests a possible relationship between the microstructure of collagen, as measured by the F/B, and the ability of tumor cells to locomote through that collagen. Utilizing in vitro collagen gels of different F/B ratios, we explored the relationship between collagen microstructure and motility of tumor cells in a “clean” environment, free of the myriad cells, and signals found in in vivo. We found a significant relationship between F/B and the total distance traveled by the tumor cell, as well as both the average and maximum velocities of the cells. Consequently, one possible mechanism underlying the observed relationship between tumor F/B and metastatic output in IDC patient samples is a direct influence of collagen structure on tumor cell motility. PMID:25625899

  11. Second-harmonic generation scattering directionality predicts tumor cell motility in collagen gels

    NASA Astrophysics Data System (ADS)

    Burke, Kathleen A.; Dawes, Ryan P.; Cheema, Mehar K.; Van Hove, Amy; Benoit, Danielle S. W.; Perry, Seth W.; Brown, Edward

    2015-05-01

    Second-harmonic generation (SHG) allows for the analysis of tumor collagen structural changes throughout metastatic progression. SHG directionality, measured through the ratio of the forward-propagating to backward-propagating signal (F/B ratio), is affected by collagen fibril diameter, spacing, and disorder of fibril packing within a fiber. As tumors progress, these parameters evolve, producing concurrent changes in F/B. It has been recently shown that the F/B of highly metastatic invasive ductal carcinoma (IDC) breast tumors is significantly different from less metastatic tumors. This suggests a possible relationship between the microstructure of collagen, as measured by the F/B, and the ability of tumor cells to locomote through that collagen. Utilizing in vitro collagen gels of different F/B ratios, we explored the relationship between collagen microstructure and motility of tumor cells in a "clean" environment, free of the myriad cells, and signals found in in vivo. We found a significant relationship between F/B and the total distance traveled by the tumor cell, as well as both the average and maximum velocities of the cells. Consequently, one possible mechanism underlying the observed relationship between tumor F/B and metastatic output in IDC patient samples is a direct influence of collagen structure on tumor cell motility.

  12. Existence of Different Structural Intermediates on the Fibrillation Pathway of Human Serum Albumin

    PubMed Central

    Juárez, Josué; Taboada, Pablo; Mosquera, Víctor

    2009-01-01

    can be formed by an antiparallel arrangement of β-strands forming the β-sheet structure of the HSA fibrils as the most probable configuration. Very long incubation times lead to a more complex morphological variability of amyloid mature fibrils (i.e., long straight fibrils, flat-ribbon structures, laterally connected fibers, etc.). We also observed that mature straight fibrils can also grow by protein oligomers tending to align within the immediate vicinity of the fibers. This filament + monomers/oligomers scenario is an alternative pathway to the otherwise dominant filament + filament manner of the protein fibril's lateral growth. Conformational preferences for a certain pathway to become active may exist, and the influence of environmental conditions such as pH, temperature, and salt must be considered. PMID:19289061

  13. Dynamic mechanical behavior of human dentin and collagen: Methods and properties

    NASA Astrophysics Data System (ADS)

    Ryou, Heonjune

    Experimental evaluations of human coronal dentin and its collagen fibrils were performed by Dynamic Mechanical Analysis (DMA) using nanoindentation and Atomic Force Microscopy (AFM). The primary objectives were to quantify the changes in mechanical behavior of intertubular and peritubular dentin with age, and to evaluate the nanostructure and mechanical behavior of the collagen fibrils. Specimens of coronal dentin were evaluated by nanoDMA using single indents and in scanning mode via scanning probe microscopy. Collagen fibrils from coronal dentin were evaluated using Pulse-Force Mode (PFM) AFM (Peakforce QNM). Nanoindentation results showed that there were no significant differences in the storage modulus or complex modulus between the two age groups (18-25 versus 54-83 yrs) for either the intertubular or peritubular dentin. However, there were significant differences in the dampening behavior between the young and old tissues, as represented in the loss modulus and tanϕ responses. For both the intertubular and peritubular components, the capacity for dampening was significantly lower in the old group. Scanning based nanoDMA showed that the tubules of old dentin exhibit a gradient in elastic behavior, with decrease in elastic modulus from the cuff to the center of tubules filled with newly deposited mineral. AFM results showed that the stiffness of the old dentin fibrils in the peak and trough regions were greater than the young dentin fibrils. In addition, there were significant differences in the dampening behavior between the young and old dentin fibrils, as represented in the energy dissipation, phase angle and loss modulus responses. For both the peak and trough regions, the dissipative capacity was significantly lower in the old dentin fibrils.

  14. Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis

    PubMed Central

    McKleroy, William; Lee, Ting-Hein

    2013-01-01

    Pulmonary fibrosis is a vexing clinical problem with no proven therapeutic options. In the normal lung there is continuous collagen synthesis and collagen degradation, and these two processes are precisely balanced to maintain normal tissue architecture. With lung injury there is an increase in the rate of both collagen production and collagen degradation. The increase in collagen degradation is critical in preventing the formation of permanent scar tissue each time the lung is exposed to injury. In pulmonary fibrosis, collagen degradation does not keep pace with collagen production, resulting in extracellular accumulation of fibrillar collagen. Collagen degradation occurs through both extracellular and intracellular pathways. The extracellular pathway involves cleavage of collagen fibrils by proteolytic enzyme including the metalloproteinases. The less-well-described intracellular pathway involves binding and uptake of collagen fragments by fibroblasts and macrophages for lysosomal degradation. The relationship between these two pathways and their relevance to the development of fibrosis is complex. Fibrosis in the lung, liver, and skin has been associated with an impaired degradative environment. Much of the current scientific effort in fibrosis is focused on understanding the pathways that regulate increased collagen production. However, recent reports suggest an important role for collagen turnover and degradation in regulating the severity of tissue fibrosis. The objective of this review is to evaluate the roles of the extracellular and intracellular collagen degradation pathways in the development of fibrosis and to examine whether pulmonary fibrosis can be viewed as a disease of impaired matrix degradation rather than a disease of increased matrix production. PMID:23564511

  15. Collagenous gastritis: Review

    PubMed Central

    Kamimura, Kenya; Kobayashi, Masaaki; Sato, Yuichi; Aoyagi, Yutaka; Terai, Shuji

    2015-01-01

    Collagenous gastritis is a rare disease characterized by the subepithelial deposition of collagen bands thicker than 10 μm and the infiltration of inflammatory mononuclear cells in the lamina propria. Collagenous colitis and collagenous sprue have similar histological characteristics to collagenous gastritis and are thought to be part of the same disease entity. However, while collagenous colitis has become more common in the field of gastroenterology, presenting with clinical symptoms of chronic diarrhea in older patients, collagenous gastritis is rare. Since the disease was first reported in 1989, only 60 cases have been documented in the English literature. No safe and effective treatments have been identified from randomized, controlled trials. Therefore, better understanding of the disease and the reporting of more cases will help to establish diagnostic criteria and to develop therapeutic strategies. Therefore, here we review the clinical characteristics, endoscopic and histological findings, treatment, and clinical outcomes from case reports and case series published to date, and provide a summary of the latest information on the disease. This information will contribute to improved knowledge of collagenous gastritis so physicians can recognize and correctly diagnose the disease, and will help to develop a standard therapeutic strategy for future clinical trials. PMID:25789098

  16. Collagenous gastritis: Review.

    PubMed

    Kamimura, Kenya; Kobayashi, Masaaki; Sato, Yuichi; Aoyagi, Yutaka; Terai, Shuji

    2015-03-16

    Collagenous gastritis is a rare disease characterized by the subepithelial deposition of collagen bands thicker than 10 μm and the infiltration of inflammatory mononuclear cells in the lamina propria. Collagenous colitis and collagenous sprue have similar histological characteristics to collagenous gastritis and are thought to be part of the same disease entity. However, while collagenous colitis has become more common in the field of gastroenterology, presenting with clinical symptoms of chronic diarrhea in older patients, collagenous gastritis is rare. Since the disease was first reported in 1989, only 60 cases have been documented in the English literature. No safe and effective treatments have been identified from randomized, controlled trials. Therefore, better understanding of the disease and the reporting of more cases will help to establish diagnostic criteria and to develop therapeutic strategies. Therefore, here we review the clinical characteristics, endoscopic and histological findings, treatment, and clinical outcomes from case reports and case series published to date, and provide a summary of the latest information on the disease. This information will contribute to improved knowledge of collagenous gastritis so physicians can recognize and correctly diagnose the disease, and will help to develop a standard therapeutic strategy for future clinical trials. PMID:25789098

  17. ALIGNING JIG

    DOEpatents

    Culver, J.S.; Tunnell, W.C.

    1958-08-01

    A jig or device is described for setting or aligning an opening in one member relative to another member or structure, with a predetermined offset, or it may be used for measuring the amount of offset with which the parts have previously been sct. This jig comprises two blocks rabbeted to each other, with means for securing thc upper block to the lower block. The upper block has fingers for contacting one of the members to be a1igmed, the lower block is designed to ride in grooves within the reference member, and calibration marks are provided to determine the amount of offset. This jig is specially designed to align the collimating slits of a mass spectrometer.

  18. Interactions between collagen IX and biglycan measured by atomic force microscopy

    SciTech Connect

    Chen, C.-H.; Yeh, M.-L.; Geyer, Mark; Wang, Gwo-Jaw; Huang, M.-H.; Heggeness, Michael H.; Hoeoek, Magnus; Luo, Z.-P. . E-mail: luo@bcm.tmc.edu

    2006-01-06

    The stability of the lattice-like type II collagen architecture of articular cartilage is paramount to its optimal function. Such stability not only depends on the rigidity of collagen fibrils themselves, but more importantly, on their interconnections. One known interconnection is through type IX and biglycan molecules. However, the mechanical properties of this interaction and its role in the overall stability remain unrevealed. Using atomic force microscopy, this study directly measured the mechanical strength (or the rupture force) of a single bond between collagen IX and biglycan. The results demonstrated that the rupture force of this single bond was 15 pN, which was significantly smaller than those of other known molecule interactions to date. This result suggested that type IX collagen and biglycan interaction may be the weak link in the cartilage collagen architecture, vulnerable to abnormal joint force and associated with disorders such as osteoarthritis.

  19. Backbone dynamics in collagen

    NASA Astrophysics Data System (ADS)

    Aliev, Abil E.

    2004-11-01

    Peptide backbone motions of collagen have been extensively studied in the past. The experimental results were interpreted using a model of a collagen rod librating about its helix axis. Considering the size of the collagen molecule and the presence of cross-linked molecules, motional amplitudes derived for the helix axis libration were unusually high. Using solid-state NMR 13C chemical shift anisotropy and 2H quadrupolar lineshape analysis for five different isotope labelled collagens we show that motional averaging of the NMR interactions occurs primarily via small-angle librations about internal bond directions. This type of dynamics is compatible with both the presence of cross-links in collagen and the X-ray data, as well as dynamic models used for other proteins.

  20. Image alignment

    SciTech Connect

    Dowell, Larry Jonathan

    2014-04-22

    Disclosed is a method and device for aligning at least two digital images. An embodiment may use frequency-domain transforms of small tiles created from each image to identify substantially similar, "distinguishing" features within each of the images, and then align the images together based on the location of the distinguishing features. To accomplish this, an embodiment may create equal sized tile sub-images for each image. A "key" for each tile may be created by performing a frequency-domain transform calculation on each tile. A information-distance difference between each possible pair of tiles on each image may be calculated to identify distinguishing features. From analysis of the information-distance differences of the pairs of tiles, a subset of tiles with high discrimination metrics in relation to other tiles may be located for each image. The subset of distinguishing tiles for each image may then be compared to locate tiles with substantially similar keys and/or information-distance metrics to other tiles of other images. Once similar tiles are located for each image, the images may be aligned in relation to the identified similar tiles.

  1. Riboflavin/UVA Collagen Cross-Linking-Induced Changes in Normal and Keratoconus Corneal Stroma

    PubMed Central

    Hayes, Sally; Boote, Craig; Kamma-Lorger, Christina S.; Rajan, Madhavan S.; Harris, Jonathan; Dooley, Erin; Hawksworth, Nicholas; Hiller, Jennifer; Terill, Nick J.; Hafezi, Farhad; Brahma, Arun K.; Quantock, Andrew J.; Meek, Keith M.

    2011-01-01

    Purpose To determine the effect of Ultraviolet-A collagen cross-linking with hypo-osmolar and iso-osmolar riboflavin solutions on stromal collagen ultrastructure in normal and keratoconus ex vivo human corneas. Methods Using small-angle X-ray scattering, measurements of collagen D-periodicity, fibril diameter and interfibrillar spacing were made at 1 mm intervals across six normal post-mortem corneas (two above physiological hydration (swollen) and four below (unswollen)) and two post-transplant keratoconus corneal buttons (one swollen; one unswollen), before and after hypo-osmolar cross-linking. The same parameters were measured in three other unswollen normal corneas before and after iso-osmolar cross-linking and in three pairs of swollen normal corneas, in which only the left was cross-linked (with iso-osmolar riboflavin). Results Hypo-osmolar cross-linking resulted in an increase in corneal hydration in all corneas. In the keratoconus corneas and unswollen normal corneas, this was accompanied by an increase in collagen interfibrillar spacing (p<0.001); an increase in fibril diameter was also seen in two out of four unswollen normal corneas and one unswollen keratoconus cornea (p<0.001). Iso-osmolar cross-linking resulted in a decrease in tissue hydration in the swollen normal corneas only. Although there was no consistent treatment-induced change in hydration in the unswollen normal samples, iso-osmolar cross-linking of these corneas did result in a compaction of collagen fibrils and a reduced fibril diameter (p<0.001); these changes were not seen in the swollen normal corneas. Collagen D-periodicity was not affected by either treatment. Conclusion The observed structural changes following Ultraviolet-A cross-linking with hypo-osmolar or iso-osmolar riboflavin solutions are more likely a consequence of treatment-induced changes in tissue hydration rather than cross-linking. PMID:21850225

  2. Osmotically driven tensile stress in collagen-based mineralized tissues.

    PubMed

    Bertinetti, Luca; Masic, Admir; Schuetz, Roman; Barbetta, Aurelio; Seidt, Britta; Wagermaier, Wolfgang; Fratzl, Peter

    2015-12-01

    Collagen is the most abundant protein in mammals and its primary role is to serve as mechanical support in many extracellular matrices such as those of bones, tendons, skin or blood vessels. Water is an integral part of the collagen structure, but its role is still poorly understood, though it is well-known that the mechanical properties of collagen depend on hydration. Recently, it was shown that the conformation of the collagen triple helix changes upon water removal, leading to a contraction of the molecule with considerable forces. Here we investigate the influence of mineralization on this effect by studying bone and turkey leg tendon (TLT) as model systems. Indeed, TLT partially mineralizes so that well-aligned collagen with various mineral contents can be found in the same tendon. We show that water removal leads to collagen contraction in all cases generating tensile stresses up to 80MPa. Moreover, this contraction of collagen puts mineral particles under compression leading to strains of around 1%, which implies localized compressive loads in mineral of up to 800MPa. This suggests that collagen dehydration upon mineralization is at the origin of the compressive pre-strains commonly observed in bone mineral.

  3. Hyperuricemia and Atrial Fibrillation.

    PubMed

    Maharani, Nani; Kuwabara, Masanari; Hisatome, Ichiro

    2016-07-27

    The importance of atrial fibrillation (AF) as a cause of mortality and morbidity has prompted research on its pathogenesis and treatment. Recognition of AF risk factors is essential to prevent it and reduce the risk of death. Hyperuricemia has been widely accepted to be associated with the incidence of paroxysmal or persistent AF, as well as to the risk of AF in post cardiovascular surgery patients. The possible explanations for this association have been based on their relation with either oxidative stress or inflammation. To investigate the link between hyperuricemia and AF, it is necessary to refer to hyperuricemia-induced atrial remodeling. So far, both ionic channel and structural remodeling caused by hyperuricemia might be plausible explanations for the occurrence of AF. Inhibition of xanthine oxidase and nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, or the use of antioxidants, along with serum uric acid (SUA) level reduction to prevent inflammation, might be useful. Uric acid transporters (UATs) play a key role in the regulation of intracellular uric acid concentration. Intracellular rather than serum uric acid level is considered more important for the pathogenesis of AF. Identification of UATs expressed in cells is thus important, and targeting UATs might become a potential strategy to reduce the risk of hyperuricemia-induced atrial fibrillation. PMID:27396561

  4. Controlling the nano-bio interface to build collagen-silica self-assembled networks

    NASA Astrophysics Data System (ADS)

    Aimé, Carole; Mosser, Gervaise; Pembouong, Gaëlle; Bouteiller, Laurent; Coradin, Thibaud

    2012-10-01

    Bio-hybrid networks are designed based on the self-assembly of surface-engineered collagen-silica nanoparticles. Collagen triple helices can be confined on the surface of sulfonate-modified silica particles in a controlled manner. This gives rise to hybrid building blocks with well-defined diameters and surface potentials. Taking advantage of the self-assembling properties of collagen, collagen-silica networks are further built-up in solution. The structural and specific recognition properties of the collagen fibrils are well-preserved within the hybrid assembly. A combination of calorimetry, dynamic light scattering, zetametry and microscopy studies indicates that network formation occurs via a surface-mediated mechanism where pre-organization of the protein chains on the particle surface favors the fibrillogenesis process. These results enlighten the importance of the nano-bio interface on the formation and properties of self-assembled bionanocomposites.Bio-hybrid networks are designed based on the self-assembly of surface-engineered collagen-silica nanoparticles. Collagen triple helices can be confined on the surface of sulfonate-modified silica particles in a controlled manner. This gives rise to hybrid building blocks with well-defined diameters and surface potentials. Taking advantage of the self-assembling properties of collagen, collagen-silica networks are further built-up in solution. The structural and specific recognition properties of the collagen fibrils are well-preserved within the hybrid assembly. A combination of calorimetry, dynamic light scattering, zetametry and microscopy studies indicates that network formation occurs via a surface-mediated mechanism where pre-organization of the protein chains on the particle surface favors the fibrillogenesis process. These results enlighten the importance of the nano-bio interface on the formation and properties of self-assembled bionanocomposites. Electronic supplementary information (ESI) available: XPS

  5. Newly identified interfibrillar collagen crosslinking suppresses cell proliferation and remodelling.

    PubMed

    Marelli, Benedetto; Le Nihouannen, Damien; Hacking, S Adam; Tran, Simon; Li, Jingjing; Murshed, Monzur; Doillon, Charles J; Ghezzi, Chiara E; Zhang, Yu Ling; Nazhat, Showan N; Barralet, Jake E

    2015-06-01

    Copper is becoming recognised as a key cation in a variety of biological processes. Copper chelation has been studied as a potential anti-angiogenic strategy for arresting tumour growth. Conversely the delivery of copper ions and complexes in vivo can elicit a pro-angiogenic effect. Previously we unexpectedly found that copper-stimulated intraperitoneal angiogenesis was accompanied by collagen deposition. Here, in hard tissue, not only was healing accelerated by copper, but again enhanced deposition of collagen was detected at 2 weeks. Experiments with reconstituted collagen showed that addition of copper ions post-fibrillogenesis rendered plastically-compressed gels resistant to collagenases, enhanced their mechanical properties and increased the denaturation temperature of the protein. Unexpectedly, this apparently interfibrillar crosslinking was not affected by addition of glucose or ascorbic acid, which are required for crosslinking by advanced glycation end products (AGEs). Fibroblasts cultured on copper-crosslinked gels did not proliferate, whereas those cultured with an equivalent quantity of copper on either tissue culture plastic or collagen showed no effect compared with controls. Although non-proliferative, fibroblasts grown on copper-cross-linked collagen could migrate, remained metabolically active for at least 14 days and displayed a 6-fold increase in Mmps 1 and 3 mRNA expression compared with copper-free controls. The ability of copper ions to crosslink collagen fibrils during densification and independently of AGEs or Fenton type reactions is previously unreported. The effect on MMP susceptibility of collagen and the dramatic change in cell behaviour on this crosslinked ECM may contribute to shedding some light on unexplained phenomena as the apparent benefit of copper complexation in fibrotic disorders or the enhanced collagen deposition in response to localised copper delivery. PMID:25907046

  6. Newly identified interfibrillar collagen crosslinking suppresses cell proliferation and remodelling.

    PubMed

    Marelli, Benedetto; Le Nihouannen, Damien; Hacking, S Adam; Tran, Simon; Li, Jingjing; Murshed, Monzur; Doillon, Charles J; Ghezzi, Chiara E; Zhang, Yu Ling; Nazhat, Showan N; Barralet, Jake E

    2015-06-01

    Copper is becoming recognised as a key cation in a variety of biological processes. Copper chelation has been studied as a potential anti-angiogenic strategy for arresting tumour growth. Conversely the delivery of copper ions and complexes in vivo can elicit a pro-angiogenic effect. Previously we unexpectedly found that copper-stimulated intraperitoneal angiogenesis was accompanied by collagen deposition. Here, in hard tissue, not only was healing accelerated by copper, but again enhanced deposition of collagen was detected at 2 weeks. Experiments with reconstituted collagen showed that addition of copper ions post-fibrillogenesis rendered plastically-compressed gels resistant to collagenases, enhanced their mechanical properties and increased the denaturation temperature of the protein. Unexpectedly, this apparently interfibrillar crosslinking was not affected by addition of glucose or ascorbic acid, which are required for crosslinking by advanced glycation end products (AGEs). Fibroblasts cultured on copper-crosslinked gels did not proliferate, whereas those cultured with an equivalent quantity of copper on either tissue culture plastic or collagen showed no effect compared with controls. Although non-proliferative, fibroblasts grown on copper-cross-linked collagen could migrate, remained metabolically active for at least 14 days and displayed a 6-fold increase in Mmps 1 and 3 mRNA expression compared with copper-free controls. The ability of copper ions to crosslink collagen fibrils during densification and independently of AGEs or Fenton type reactions is previously unreported. The effect on MMP susceptibility of collagen and the dramatic change in cell behaviour on this crosslinked ECM may contribute to shedding some light on unexplained phenomena as the apparent benefit of copper complexation in fibrotic disorders or the enhanced collagen deposition in response to localised copper delivery.

  7. Complexation of amyloid fibrils with charged conjugated polymers.

    PubMed

    Ghosh, Dhiman; Dutta, Paulami; Chakraborty, Chanchal; Singh, Pradeep K; Anoop, A; Jha, Narendra Nath; Jacob, Reeba S; Mondal, Mrityunjoy; Mankar, Shruti; Das, Subhadeep; Malik, Sudip; Maji, Samir K

    2014-04-01

    It has been suggested that conjugated charged polymers are amyloid imaging agents and promising therapeutic candidates for neurological disorders. However, very less is known about their efficacy in modulating the amyloid aggregation pathway. Here, we studied the modulation of Parkinson's disease associated α-synuclein (AS) amyloid assembly kinetics using conjugated polyfluorene polymers (PF, cationic; PFS, anionic). We also explored the complexation of these charged polymers with the various AS aggregated species including amyloid fibrils and oligomers using multidisciplinary biophysical techniques. Our data suggests that both polymers irrespective of their different charges in the side chains increase the fibrilization kinetics of AS and also remarkably change the morphology of the resultant amyloid fibrils. Both polymers were incorporated/aligned onto the AS amyloid fibrils as evident from electron microscopy (EM) and atomic force microscopy (AFM), and the resultant complexes were structurally distinct from their pristine form of both polymers and AS supported by FTIR study. Additionally, we observed that the mechanism of interactions between the polymers with different species of AS aggregates were markedly different.

  8. Type VII Collagen Expression in the Human Vitreoretinal Interface, Corpora Amylacea and Inner Retinal Layers.

    PubMed

    Wullink, Bart; Pas, Hendri H; Van der Worp, Roelofje J; Kuijer, Roel; Los, Leonoor I

    2015-01-01

    Type VII collagen, as a major component of anchoring fibrils found at basement membrane zones, is crucial in anchoring epithelial tissue layers to their underlying stroma. Recently, type VII collagen was discovered in the inner human retina by means of immunohistochemistry, while proteomic investigations demonstrated type VII collagen at the vitreoretinal interface of chicken. Because of its potential anchoring function at the vitreoretinal interface, we further assessed the presence of type VII collagen at this site. We evaluated the vitreoretinal interface of human donor eyes by means of immunohistochemistry, confocal microscopy, immunoelectron microscopy, and Western blotting. Firstly, type VII collagen was detected alongside vitreous fibers6 at the vitreoretinal interface. Because of its known anchoring function, it is likely that type VII collagen is involved in vitreoretinal attachment. Secondly, type VII collagen was found within cytoplasmic vesicles of inner retinal cells. These cells resided most frequently in the ganglion cell layer and inner plexiform layer. Thirdly, type VII collagen was found in astrocytic cytoplasmic inclusions, known as corpora amylacea. The intraretinal presence of type VII collagen was confirmed by Western blotting of homogenized retinal preparations. These data add to the understanding of vitreoretinal attachment, which is important for a better comprehension of common vitreoretinal attachment pathologies.

  9. Effect of curcumin caged silver nanoparticle on collagen stabilization for biomedical applications.

    PubMed

    Srivatsan, Kunnavakkam Vinjimur; Duraipandy, N; Begum, Shajitha; Lakra, Rachita; Ramamurthy, Usha; Korrapati, Purna Sai; Kiran, Manikantan Syamala

    2015-04-01

    The current study aims at understanding the influence of curcumin caged silver nanoparticle (CCSNP) on stability of collagen. The results indicated that curcumin caged silver nanoparticles efficiently stabilize collagen, indicated by enhanced tensile strength, fibril formation and viscosity. The tensile strength of curcumin caged silver nanoparticle cross-linked collagen and elongation at break was also found to be higher than glutaraldehyde cross-linked collagen. The physicochemical characteristics of curcumin caged nanoparticle cross-linked collagen exhibited enhanced strength. The thermal properties were also good with both thermal degradation temperature and hydrothermal stability higher than native collagen. CD analysis showed no structural disparity in spite of superior physicochemical properties suggesting the significance of curcumin caged nanoparticle mediated cross-linking. The additional enhancement in the stabilization of collagen could be attributed to multiple sites for interaction with collagen molecule provided by curcumin caged silver nanoparticles. The results of cell proliferation and anti-microbial activity assays indicated that curcumin caged silver nanoparticles promoted cell proliferation and inhibited microbial growth making it an excellent biomaterial for wound dressing application. The study opens scope for nano-biotechnological strategies for the development of alternate non-toxic cross-linking agents facilitating multiple site interaction thereby improving therapeutic values to the collagen for biomedical application. PMID:25661876

  10. [Perioperative management of atrial fibrillation].

    PubMed

    Arguis, M J; Navarro, R; Regueiro, A; Arbelo, E; Sierra, P; Sabaté, S; Galán, J; Ruiz, A; Matute, P; Roux, C; Gomar, C; Rovira, I; Mont, L; Fita, G

    2014-05-01

    Atrial fibrillation is a frequent complication in the perioperative period. When it appears there is an increased risk of perioperative morbidity due to stroke, thromboembolism, cardiac arrest, myocardial infarction, anticoagulation haemorrhage, and hospital readmissions. The current article focuses on the recommendations for the management of perioperative atrial fibrillation based on the latest Clinical Practice Guidelines on atrial fibrillation by the European Society of Cardiology and the Spanish Society of Cardiology. This article pays special attention to the preoperative management, as well as to the acute perioperative episode. For this reason, the latest recommendations for the control of cardiac frequency, antiarrhythmic treatment and anticoagulation are included.

  11. Electroactive biomimetic collagen-silver nanowire composite scaffolds.

    PubMed

    Wickham, Abeni; Vagin, Mikhail; Khalaf, Hazem; Bertazzo, Sergio; Hodder, Peter; Dånmark, Staffan; Bengtsson, Torbjörn; Altimiras, Jordi; Aili, Daniel

    2016-08-01

    Electroactive biomaterials are widely explored as bioelectrodes and as scaffolds for neural and cardiac regeneration. Most electrodes and conductive scaffolds for tissue regeneration are based on synthetic materials that have limited biocompatibility and often display large discrepancies in mechanical properties with the surrounding tissue causing problems during tissue integration and regeneration. This work shows the development of a biomimetic nanocomposite material prepared from self-assembled collagen fibrils and silver nanowires (AgNW). Despite consisting of mostly type I collagen fibrils, the homogeneously embedded AgNWs provide these materials with a charge storage capacity of about 2.3 mC cm(-2) and a charge injection capacity of 0.3 mC cm(-2), which is on par with bioelectrodes used in the clinic. The mechanical properties of the materials are similar to soft tissues with a dynamic elastic modulus within the lower kPa range. The nanocomposites also support proliferation of embryonic cardiomyocytes while inhibiting the growth of both Gram-negative Escherichia coli and Gram-positive Staphylococcus epidermidis. The developed collagen/AgNW composites thus represent a highly attractive bioelectrode and scaffold material for a wide range of biomedical applications. PMID:27385421

  12. Mechanical properties of single electrospun collagen type I fibers.

    PubMed

    Yang, Lanti; Fitié, Carel F C; van der Werf, Kees O; Bennink, Martin L; Dijkstra, Pieter J; Feijen, Jan

    2008-03-01

    The mechanical properties of single electrospun collagen fibers were investigated using scanning mode bending tests performed with an AFM. Electrospun collagen fibers with diameters ranging from 100 to 600 nm were successfully produced by electrospinning of an 8% w/v solution of acid soluble collagen in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP). Circular dichroism (CD) spectroscopy showed that 45% of the triple helical structure of collagen molecules was denatured in the electrospun fibers. The electrospun fibers were water soluble and became insoluble after cross-linking with glutaraldehyde vapor for 24h. The bending moduli and shear moduli of both non- and cross-linked single electrospun collagen fibers were determined by scanning mode bending tests after depositing the fibers on glass substrates containing micro-channels. The bending moduli of the electrospun fibers ranged from 1.3 to 7.8 GPa at ambient conditions and ranged from 0.07 to 0.26 MPa when immersed in PBS buffer. As the diameter of the fibrils increased, a decrease in bending modulus was measured clearly indicating mechanical anisotropy of the fiber. Cross-linking of the electrospun fibers with glutaraldehyde vapor increased the shear modulus of the fiber from approximately 30 to approximately 50 MPa at ambient conditions. PMID:18082253

  13. Changes in corneal collagen induced by holmium:YAG laser irradiation

    NASA Astrophysics Data System (ADS)

    Timberlake, George T.; Reinke, Martin H.; Miller, Alvin

    1996-05-01

    Holmium:YAG laser thermokeratoplasty corrects hyperopia (farsightedness) by producing small areas of corneal collagen shrinkage that cause the central cornea to bulge outward, increasing optical power. Collagen shrinkage is probably caused by laser-heated corneal water, but details of the shrinkage mechanism are not known. We investigated the shrinkage mechanism by measuring changes in corneal ultrastructure, surface shrinkage, water content, and strength following Ho:YAG laser exposures. Morphological changes in collagen were documented by measurements from electron micrographs. Corneal adhesive strength was determined by measuring tearing force in a plane parallel to the corneal surface. Laser-induced water loss was measured by weighing corneal samples before and after exposure. Corneal surface shrinkage was assessed by photographing the movement of particles on the cornea. Lasered collagen fibrils increased in diameter, lost their orderly arrangement, and appeared `frayed.' The corneal surface contracted toward lasered areas with a maximal shift of approximately 190 micrometers , more than could be explained by a model based on collagen fibril changes. Water loss plays a minor role in corneal shrinkage since corneal samples lost about only about 1.4% of their weight after massive laser exposure. Despite marked changes in collagen structure, corneal adhesive force was unchanged.

  14. In vitro osteogenic differentiation of HOS cells on two types of collagen gels.

    PubMed

    Takitoh, Takako; Kato, Yoichi; Nakasu, Asako; Tadokoro, Mika; Bessho, Masahiko; Hirose, Motohiro; Ohgushi, Hajime; Mori, Hideki; Hara, Masayuki

    2010-10-01

    HOS cell is a model strain of human osteoblasts derived from human osteosarcoma. We cultured the HOS cells on both the conventional collagen gel (neutral gel), and the gamma-crosslinked collagen gel without collagen fibrils (acidic gel). The shape of HOS cells on the neutral gel was similar to that on the culture dish. However, HOS cells on acidic gel had an elongated shape and attached each other to form a mesh-like pattern. The cells attached to the surface of both gels but scarcely penetrated their depths. We measured the biochemical markers for osteogenic differentiation in the HOS cells cultured on both the neutral gel and the acidic gel. The expressions of alkaline phosphatase and osteocalcin were detected in the HOS cells on both types of collagen gel. Deposition of the calcium also occurred on both gels although it was higher in the neutral gel than the acidic one. These results indicate the importance of collagen for the differentiation of HOS cells, but it is not dependent on the molecular structure (fibril formation) of collagen.

  15. Immobilization of a phosphonated analog of matrix phosphoproteins within cross-linked collagen as a templating mechanism for biomimetic mineralization

    PubMed Central

    Gu, Li-sha; Kim, Young Kyung; Liu, Yan; Takahashi, Kei; Arun, Senthil; Wimmer, Courtney E.; Osorio, Raquel; Ling, Jun-qi; Looney, Stephen W.; Pashley, David H.; Tay, Franklin R.

    2010-01-01

    Immobilization of phosphoproteins on a collagen matrix is important for induction of intrafibrillar apatite mineralization. Unlike phosphate esters, polyphosphonic acid has no reactive sites for covalent binding to collagen amine groups. Binding of polyvinylphosphonic acid (PVPA), a biomimetic templating analog of matrix phosphoproteins, to collagen was found to be electrostatic in nature. Thus, an alternative retention mechanism was designed for immobilization of PVPA to collagen by cross-linking the latter with carbodiimide (EDC). This mechanism is based on the principle of size exclusion entrapment of PVPA molecules within the internal water compartments of collagen. By cross-linking collagen with EDC, a zero-length cross-linking agent, the sieving property of collagen is increased, enabling the PVPA to be immobilized within the collagen. Absence of covalent cross-linking between PVPA and collagen was confirmed by FT-IR spectroscopy. Based on these results, a concentration range for immobilized PVPA to template intrafibrillar apatite deposition was established and validated using a single-layer reconstituted type I collagen mineralization model. In the presence of a polyacrylic acid-containing mineralization medium, optimal intrafibrillar mineralization of the EDC-cross-linked collagen was achieved using 500 and 1,000 μg/mL PVPA. The mineralized fibrils exhibited a hierarchical order of intrafibrillar mineral infiltration, as manifested by the appearance of electron-dense periodicity within unstained fibrils. Understanding the basic processes in intrafibrillar mineralization of reconstituted collagen creates opportunities for the design of tissue engineering materials for hard tissue repair and regeneration. PMID:20688200

  16. Auxiliary proteins that facilitate formation of collagen-rich deposits in the posterior knee capsule in a rabbit-based joint contracture model.

    PubMed

    Steplewski, Andrzej; Fertala, Jolanta; Beredjiklian, Pedro K; Abboud, Joseph A; Wang, Mark L Y; Namdari, Surena; Barlow, Jonathan; Rivlin, Michael; Arnold, William V; Kostas, James; Hou, Cheryl; Fertala, Andrzej

    2016-03-01

    Post-traumatic joint contracture is a debilitating consequence of trauma or surgical procedures. It is associated with fibrosis that develops regardless of the nature of initial trauma and results from complex biological processes associated with inflammation and cell activation. These processes accelerate production of structural elements of the extracellular matrix, particularly collagen fibrils. Although the increased production of collagenous proteins has been demonstrated in tissues of contracted joints, researchers have not yet determined the complex protein machinery needed for the biosynthesis of collagen molecules and for their assembly into fibrils. Consequently, the purpose of our study was to investigate key enzymes and protein chaperones needed to produce collagen-rich deposits. Using a rabbit model of joint contracture, our biochemical and histological assays indicated changes in the expression patterns of heat shock protein 47 and the α-subunit of prolyl 4-hydroxylase, key proteins in processing nascent collagen chains. Moreover, our study shows that the abnormal organization of collagen fibrils in the posterior capsules of injured knees, rather than excessive formation of fibril-stabilizing cross-links, may be a key reason for observed changes in the mechanical characteristics of injured joints. This result sheds new light on pathomechanisms of joint contraction, and identifies potentially attractive anti-fibrotic targets.

  17. Collagen I self-assembly: revealing the developing structures that generate turbidity.

    PubMed

    Zhu, Jieling; Kaufman, Laura J

    2014-04-15

    Type I collagen gels are routinely used in biophysical studies and bioengineering applications. The structural and mechanical properties of these fibrillar matrices depend on the conditions under which collagen fibrillogenesis proceeds, and developing a fuller understanding of this process will enhance control over gel properties. Turbidity measurements have long been the method of choice for monitoring developing gels, whereas imaging methods are regularly used to visualize fully developed gels. In this study, turbidity and confocal reflectance microscopy (CRM) were simultaneously employed to track collagen fibrillogenesis and reconcile the information reported by the two techniques, with confocal fluorescence microscopy (CFM) used to supplement information about early events in fibrillogenesis. Time-lapse images of 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml acid-solubilized collagen I gels forming at 27°C, 32°C, and 37°C were collected. It was found that in situ turbidity measured in a scanning transmittance configuration was interchangeable with traditional turbidity measurements using a spectrophotometer. CRM and CFM were employed to reveal the structures responsible for the turbidity that develops during collagen self-assembly. Information from CRM and transmittance images was collapsed into straightforward single variables; total intensity in CRM images tracked turbidity development closely for all collagen gels investigated, and the two techniques were similarly sensitive to fibril number and dimension. Complementary CRM, CFM, and in situ turbidity measurements revealed that fibril and network formation occurred before substantial turbidity was present, and the majority of increasing turbidity during collagen self-assembly was due to increasing fibril thickness.

  18. THE DIFFERENTIAL REGULATION OF CELL MOTILE ACTIVITY THROUGH MATRIX STIFFNESS AND POROSITY IN THREE DIMENSIONAL COLLAGEN MATRICES

    PubMed Central

    Miron-Mendoza, Miguel; Seemann, Joachim; Grinnell, Frederick

    2010-01-01

    In three dimensional collagen matrices, cell motile activity results in collagen translocation, cell spreading and cell migration. Cells can penetrate into the matrix as well as spread and migrate along its surface. In the current studies, we quantitatively characterize collagen translocation, cell spreading and cell migration in relationship to collagen matrix stiffness and porosity. Collagen matrices prepared with 1 to 4 mg/ml collagen exhibited matrix stiffness (storage modulus measured by oscillating rheometry) increasing from 4 to 60 Pa and matrix porosity (measured by scanning electron microscopy) decreasing from 4 to 1 μm2. Over this collagen concentration range, the consequences of cell motile activity changed markedly. As collagen concentration increased, cells no longer were able to cause translocation of collagen fibrils. Cell migration increased and cell spreading changed from dendritic to more flattened and polarized morphology depending on location of cells within or on the surface of the matrix. Collagen translocation appeared to depend primarily on matrix stiffness, whereas cell spreading and migration were less dependent on matrix stiffness and more dependent on collagen matrix porosity. PMID:20537378

  19. Collagenous and other organizations in mature annelid cuticle and epidermis.

    PubMed

    Humphreys, S; Porter, K R

    1976-05-01

    The mature annelid cuticle contains orthogonally oriented collagen in a matrix capped superficially by a dense epicuticle with external corpuscles. The underlying epidermis is a simple columnar epithelium with two major cell types, mucous-secreting cells which secrete through channels in the cuticle to the exterior of the worm, and "supportive" cells which presumably produce and increase the cuticle by secreting into it. The structures of supportive cells, previously interpreted as specialized for establishing interfibrillar collagen order, are revealed by glutaraldehyde fixation as common cellular components without the qualities deemed useful to align collagen. Cell processes which penetrate and sometimes pass completely through the cuticle are not stable, not in geometric order, and lack cilia-like structure. Cilia, unlike the ubiquitous cellular processes, are highly restricted to regions of the epidermis with specialized functions. Cellular control, or other control, of collagen fibrillogenesis remains unestablished.

  20. Complications of collagen fillers.

    PubMed

    Lucey, Patricia; Goldberg, David J

    2014-12-01

    As the skin ages, a deficiency in collagen occurs, thus injectable collagen products have become a sensible and popular option for dermal filling and volume enhancement. Several types of collagen have been developed over the years, including animal sources such as bovine and porcine collagen, as well as human-based sources derived from pieces of the patient's own skin, cadaver skin, and later cultured from human dermal fibroblasts. While collagen overall has a relatively safe, side effect profile, there are several complications, both early and late onset, that practitioners and patients should be aware of. Early complications, occurring within days of the procedure, can be divided into non-hypersensitivity and hypersensitivity reactions. The non-hypersensitive reactions include injection site reactions, discoloration, maldistribution, infection, skin necrosis, and the very rare but dreaded risk of vision loss, whereas the hypersensitivity reactions present usually as delayed type IV reactions, but can also rarely present as an immediate type I reaction. Late complications, occurring within weeks to even years after injection, include granuloma formation, foreign body reactions, and infection secondary to atypical mycobacteria or biofilms. This review will give a detailed overview of the complications secondary to cutaneous collagen injections.

  1. Nanomechanics of collagen microfibrils

    PubMed Central

    Vesentini, Simone; Redaelli, Alberto; Gautieri, Alfonso

    2013-01-01

    Summary Collagen constitutes one third of the human proteome, providing mechanical stability, elasticity and strength to organisms and is thus the prime construction material in biology. Collagen is also the dominating material in the extracellular matrix where its stiffness controls cell differentiation, growth and pathology. We use atomistic-based hierarchical multiscale modeling to describe this complex biological material from the bottom up. This includes the use and development of large-scale computational modeling tools to investigate several aspects related to collagen-based tissues, including source of visco-elasticity and deformation mechanisms at the nanoscale level. The key innovation of this research is that until now, collagen materials have primarily been described at macroscopic scales, without explicitly understanding the mechanical contributions at the molecular and fibrillar levels. The major impact of this research will be the development of fundamental models of collagenous tissues, important to the design of new scaffolding biomaterials for regenerative medicine as well as for the understanding of collagen-related diseases. PMID:23885342

  2. Rhythm control in atrial fibrillation.

    PubMed

    Piccini, Jonathan P; Fauchier, Laurent

    2016-08-20

    Many patients with atrial fibrillation have substantial symptoms despite ventricular rate control and require restoration of sinus rhythm to improve their quality of life. Acute restoration (ie, cardioversion) and maintenance of sinus rhythm in patients with atrial fibrillation are referred to as rhythm control. The decision to pursue rhythm control is based on symptoms, the type of atrial fibrillation (paroxysmal, persistent, or long-standing persistent), patient comorbidities, general health status, and anticoagulation status. Many patients have recurrent atrial fibrillation and require further intervention to maintain long term sinus rhythm. Antiarrhythmic drug therapy is generally recommended as a first-line therapy and drug selection is on the basis of the presence or absence of structural heart disease or heart failure, electrocardiographical variables, renal function, and other comorbidities. In patients who continue to have recurrent atrial fibrillation despite medical therapy, catheter ablation has been shown to substantially reduce recurrent atrial fibrillation, decrease symptoms, and improve quality of life, although recurrence is common despite continued advancement in ablation techniques. PMID:27560278

  3. Rate control in atrial fibrillation.

    PubMed

    Van Gelder, Isabelle C; Rienstra, Michiel; Crijns, Harry J G M; Olshansky, Brian

    2016-08-20

    Control of the heart rate (rate control) is central to atrial fibrillation management, even for patients who ultimately require control of the rhythm. We review heart rate control in patients with atrial fibrillation, including the rationale for the intervention, patient selection, and the treatments available. The choice of rate control depends on the symptoms and clinical characteristics of the patient, but for all patients with atrial fibrillation, rate control is part of the management. Choice of drugs is patient-dependent. β blockers, alone or in combination with digoxin, or non-dihydropyridine calcium-channel blockers (not in heart failure) effectively lower the heart rate. Digoxin is least effective, but a reasonable choice for physically inactive patients aged 80 years or older, in whom other treatments are ineffective or are contraindicated, and as an additional drug to other rate-controlling drugs, especially in heart failure when instituted cautiously. Atrioventricular node ablation with pacemaker insertion for rate control should be used as an approach of last resort but is also an option early in the management of patients with atrial fibrillation treated with cardiac resynchronisation therapy. However, catheter ablation of atrial fibrillation should be considered before atrioventricular node ablation. Although rate control is a top priority and one of the first management issues for all patients with atrial fibrillation, many issues remain. PMID:27560277

  4. Rhythm control in atrial fibrillation.

    PubMed

    Piccini, Jonathan P; Fauchier, Laurent

    2016-08-20

    Many patients with atrial fibrillation have substantial symptoms despite ventricular rate control and require restoration of sinus rhythm to improve their quality of life. Acute restoration (ie, cardioversion) and maintenance of sinus rhythm in patients with atrial fibrillation are referred to as rhythm control. The decision to pursue rhythm control is based on symptoms, the type of atrial fibrillation (paroxysmal, persistent, or long-standing persistent), patient comorbidities, general health status, and anticoagulation status. Many patients have recurrent atrial fibrillation and require further intervention to maintain long term sinus rhythm. Antiarrhythmic drug therapy is generally recommended as a first-line therapy and drug selection is on the basis of the presence or absence of structural heart disease or heart failure, electrocardiographical variables, renal function, and other comorbidities. In patients who continue to have recurrent atrial fibrillation despite medical therapy, catheter ablation has been shown to substantially reduce recurrent atrial fibrillation, decrease symptoms, and improve quality of life, although recurrence is common despite continued advancement in ablation techniques.

  5. Stroke prevention in atrial fibrillation.

    PubMed

    Freedman, Ben; Potpara, Tatjana S; Lip, Gregory Y H

    2016-08-20

    Atrial fibrillation is found in a third of all ischaemic strokes, even more after post-stroke atrial fibrillation monitoring. Data from stroke registries show that both unknown and untreated or under treated atrial fibrillation is responsible for most of these strokes, which are often fatal or debilitating. Most could be prevented if efforts were directed towards detection of atrial fibrillation before stroke occurs, through screening or case finding, and treatment of all patients with atrial fibrillation at increased risk of stroke with well-controlled vitamin K antagonists or non-vitamin K antagonist anticoagulants. The default strategy should be to offer anticoagulant thromboprophylaxis to all patients with atrial fibrillation unless defined as truly low risk by simple validated risk scores, such as CHA2DS2-VASc. Assessment of bleeding risk using the HAS-BLED score should focus attention on reversible bleeding risk factors. Finally, patients need support from physicians and various other sources to start anticoagulant treatment and to ensure adherence to and persistence with treatment in the long term. PMID:27560276

  6. In vivo multiphoton imaging of the cornea: polarization-resolved second harmonic generation from stromal collagen

    NASA Astrophysics Data System (ADS)

    Latour, G.; Gusachenko, I.; Kowalczuk, L.; Lamarre, I.; Schanne-Klein, M.-C.

    2012-03-01

    Multiphoton microscopy provides specific and contrasted images of unstained collagenous tissues such as tendons or corneas. Polarization-resolved second harmonic generation (SHG) measurements have been implemented in a laserscanning multiphoton microscope. Distortion of the polarimetric response due to birefringence and diattenuation during propagation of the laser excitation has been shown in rat-tail tendons. A model has been developed to account for these effects and correct polarization-resolved SHG images in thick tissues. This new modality is then used in unstained human corneas to access two quantitative parameters: the fibrils orientation within the collagen lamellae and the ratio of the main second-order nonlinear tensorial components. Orientation maps obtained from polarization resolution of the trans-detected SHG images are in good agreement with the striated features observed in the raw images. Most importantly, polarization analysis of the epi-detected SHG images also enables to map the fibrils orientation within the collagen lamellae while epi-detected SHG images of corneal stroma are spatially homogenous and do not enable direct visualization of the fibrils orientation. Depth profiles of the polarimetric SHG response are also measured and compared to models accounting for orientation changes of the collagen lamellae within the focal volume. Finally, in vivo polarization-resolved SHG is performed in rat corneas and structural organization of corneal stroma is determined using epi-detected signals.

  7. The mRNAs for the three chains of human collagen type XI are widely distributed but not necessarily co-expressed: implications for homotrimeric, heterotrimeric and heterotypic collagen molecules.

    PubMed Central

    Lui, V C; Kong, R Y; Nicholls, J; Cheung, A N; Cheah, K S

    1995-01-01

    In cartilage collagen type XI exists as heterotrimeric molecules composed of alpha 1(XI), alpha 2(XI) and alpha 3(XI) subunits. Messenger RNAs for some of the alpha chains of collagen type XI have also been found in non-chondrogenic tissues but the chain composition of the molecule in these sites is not known. Some non-chondrogenic tissues also contain heterotrimers containing collagen alpha 2(V) and alpha 1(XI) chains. We have explored the possibility that collagen type XI could exist in differing trimeric forms in non-chondrogenic tissues and aimed to predict the subunit composition of this collagen in those tissues. The distribution and relative levels of expression of collagen alpha 1(XI), alpha 2(XI) and alpha 3(XI)/alpha 1(II) mRNAs in different human fetal tissues were studied. Expression of mRNAs for all three genes of collagen type XI is not restricted to cartilage but is widespread. However, in some non-chondrogenic tissues, the mRNAs for all three alpha chains of collagen type XI were not co-expressed, but collagen alpha 1(XI) and alpha 2(XI) mRNAs were found either singly or without collagen alpha 3(XI) transcripts. Collagen type XI may therefore exist as homotrimers and/or heterotrimers composed of two collagen alpha(XI) chains in some tissues. The distribution of mRNAs for collagen alpha 2(V) and alpha 1(I) were also studied. Co-expression of collagen type XI, alpha 2(V) and alpha 1(I) mRNAs was found for many tissues. These findings have implications for the possibility of additional chain associations for collagen types XI and V in cross-type heterotrimers within heterotypic fibrils. Images Figure 1 Figure 2 Figure 3 PMID:7487888

  8. Small-Angle X-ray Study of the Three-Dimensional Collagen/Mineral Superstructure in Intramuscular Fish Bone

    SciTech Connect

    Zhou,H.; Burger, C.; Sics, I.; Hsiao, B.; Chu, B.; Graham, L.; Glimcher, M.

    2007-01-01

    Synchrotron small-angle X-ray scattering (SAXS) was conducted on native intramuscular shad/herring bone samples. Two-dimensional SAXS patterns were quantitatively analyzed with special consideration for preferred orientation effects, leading to new insights into the three-dimensional superstructure of mineralized collagen fibrils in shad/herring bone.

  9. Global alignment: Finding rearrangements during alignment

    SciTech Connect

    Brudno, Michael; Malde, Sanket; Poliakov, Alexander; Do, Chuong B.; Couronne, Olivier; Dubchak, Inna; Batzoglou, Serafim

    2003-01-06

    Motivation: To compare entire genomes from different species, biologists increasingly need alignment methods that are efficient enough to handle long sequences, and accurate enough to correctly align the conserved biological features between distant species. The two main classes of pairwise alignments are global alignment, where one string is transformed into the other, and local alignment, where all locations of similarity between the two strings are returned. Global alignments are less prone to demonstrating false homology as each letter of one sequence is constrained to being aligned to only one letter of the other. Local alignments, on the other hand, can cope with rearrangements between non-syntenic, orthologous sequences by identifying similar regions in sequences; this, however, comes at the expense of a higher false positive rate due to the inability of local aligners to take into account overall conservation maps.

  10. PAK1 is involved in sensing the orientation of collagen stiffness gradients in mouse fibroblasts.

    PubMed

    Pinto, V I; Mohammadi, H; Lee, W S; Cheung, A H; McCulloch, C A

    2015-10-01

    Migrating cells sense variations of stiffness in connective tissue matrices but how cells detect and respond to stiffness orientation is not defined. We examined cell extension formation on collagen with underlying support (vertical stiffness gradient) or on collagen laterally supported by nylon (lateral stiffness gradient). At 6 h after plating, cells plated on laterally-supported collagen exhibited >2-fold more abundant and ~2-fold longer cell extensions than cells plated on collagen with underlying support. We examined whether p21-activated kinase 1 (PAK1) influences extension formation that is dependent on the orientation of support. At 6 h after plating on collagen with underlying support, wild-type cell extensions were 40% shorter than PAK1 knockdown cells. In contrast, on laterally-supported collagen, wild-type cell extensions were 2-fold longer than PAK1 knockdown cells. In cells plated on laterally-supported collagen, there were ~2-fold reductions of collagen fiber alignment and compaction in PAK1 knockdown cells compared with wild-type cells. PAK1 knockdown did not affect collagen fiber alignment or compaction by cells plated on collagen with underlying support. Wild-type cells with lateral support of collagen exhibited 3-fold increases of phospho-myosin staining at 6h, which was 2-fold lower in PAK1 knockdown cells. In contrast, cells on collagen with underlying support showed no increase of phospho-myosin staining at any times. PAK1 knockdown did not affect α2 or β1 integrin expression or function. We conclude that PAK1 is involved in the ability of cells to sense the orientation of stiffness in collagen substrates and generate contractile forces that affect cell extension formation.

  11. Ventricular fibrillation and atrial fibrillation are two different beasts

    NASA Astrophysics Data System (ADS)

    Gray, R. A.; Jalife, J.

    1998-03-01

    Although the mechanisms of fibrillation are no doubt multi-faceted, the geometry of the heart may play a major role in the dynamics of wave propagation during fibrillation [A. T. Winfree, Science 266, 1003-1006 (1994)]. The ventricles are thick chambers made up of sheets of parallel muscle fibers with the direction of fibers rotating across the ventricular walls (rotational anisotropy). The thick walls of the ventricles allow reentry to develop transmurally, provided the wavelength is sufficiently small. Depending on the kinetics of heart cells, the dynamics of rotating waves in three dimensions may be fundamentally different than in two dimensions, leading to destabilization of reentry and ventricular fibrillation (VF) in thick ventricles. The atria have an intricate geometry comprised of a thin sheet of cardiac tissue attached to a very co