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Sample records for enhances collagen fibril

  1. 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

  2. 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.

  3. 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

  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. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  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. 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

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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).

  15. 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

  16. 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.

  17. 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.

  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. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. Fibril Fragmentation Enhances Amyloid Cytotoxicity*♦

    PubMed Central

    Xue, Wei-Feng; Hellewell, Andrew L.; Gosal, Walraj S.; Homans, Steve W.; Hewitt, Eric W.; Radford, Sheena E.

    2009-01-01

    Fibrils associated with amyloid disease are molecular assemblies of key biological importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chemical composition or molecular properties of the amyloid fibrils, but their physical attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biological properties of amyloid fibrils. In addition to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged molecular architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biological properties such as amyloid cytotoxicity. PMID:19808677

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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."

  12. 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

  13. 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.

  14. 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

  15. 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%).

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. Enhanced cell colonization of collagen scaffold by ultraviolet/ozone surface processing.

    PubMed

    Liu, Chaozong; McKenna, Fiona-Mairead; Liang, He; Johnstone, Alan; Abel, Eric W

    2010-12-01

    Both physical and chemical crosslinking methods have been shown to be effective in improving the biological stability and mechanical properties of porous collagen scaffolds. However, the wetting of the collagen fibril surface by a culture medium is reduced and it is difficult for the medium to diffuse into the 3D structure of a porous collagen scaffold. This article reports a strategy for the surface processing of crosslinked collagen scaffolds by an integrated ultraviolet/ozone perfuse processing technique. Ultraviolet/ozone perfuse processing improved surface wettability for both the exterior and interior surfaces of the porous 3D collagen scaffold. This leads to a significant improvement in the scaffolds ability to take up water without compromising the bulk biological stability and mechanical properties. In vitro evaluation using mesenchymal stem cell demonstrated that surface processing enhanced cell colonization of the scaffolds, cells could migrate deep into the structure of the scaffolds, and significantly higher levels of cell proliferation were achieved. In contrast, the cells were unable to migrate deep into the scaffolds, and most of the cells that survived were observed only in the top seeding layer resulting in a low level of cell activity in the unprocessed scaffolds.

  12. Enhanced second-harmonic-generation detection of collagen by means of optical wavefront shaping

    NASA Astrophysics Data System (ADS)

    Thompson, Jonathan V.; Throckmorton, Graham A.; Hokr, Brett H.; Yakovlev, Vladislav V.

    2016-03-01

    Second-harmonic generation (SHG) has proven to be an effective method to both image and detect structural variations in fibrillar collagen. The ability to detect these differences is especially useful in studying diseases like cancer and fibrosis.1 SHG techniques have historically been limited by their ability to penetrate and image through strongly scattering tissues. Recently, optical wavefront shaping has enabled light to be focused through highly scattering media such as biological tissue.2-4 This technology also enables us to examine the dependence of second harmonic generation on the spatial phase of the pump laser. Here, we demonstrate that wavefront shaping can be used to enhance the generation of second harmonic light from collagen fibrils even when scattering is low or non-existent.

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. Alfacalcidol enhances collagen quality in ovariectomized rat bones.

    PubMed

    Nagaoka, Hideaki; Terajima, Masahiko; Yamada, Shizuka; Azuma, Yoshiaki; Chida, Takayuki; Yamauchi, Mitsuo

    2014-08-01

    The aim of this study was to investigate the effects of alfacalcidol (1α(OH)D3 : ALF) on bone collagen employing an ovariectomized rat model. Thirty-five 16-week-old female Sprague-Dawley rats were divided into five groups: SHAM (sham-operated + vehicle), OVX (ovariectomy + vehicle), and three ALF-treated groups, that is, ovariectomy + 0.022 µg/kg/day ALF, ovariectomy + 0.067 µg/kg/day ALF, and ovariectomy + 0.2 µg/kg/day ALF. After 12 weeks of treatment, tibiae were subjected to histological, biochemical and immunohistochemical analyses. Collagen matrices in OVX bone appeared as immature and poorly organized; however, with the ALF treatment, it was improved in a dose-dependent manner. Contents of collagen and pyridinoline cross-link were decreased in OVX compared with SHAM, but they increased to the level comparable to SHAM in ALF-treated groups. The total aldehyde, that is, a sum of free and those involved cross-links, in the highest dose of ALF was significantly higher than the rest of the groups (p < 0.05). In addition, the expression of lysyl oxidase was increased in the all ALF-treated groups compared with OVX (p < 0.05). In conclusion, ALF increases not only the amount of collagen but also enhances the maturation of collagen in ovariectomy-induced osteoporotic bones, which likely contributes to the improvement of bone quality.

  6. Effects of fibrillation on the wood fibers' enzymatic hydrolysis enhanced by mechanical refining.

    PubMed

    Liu, Wei; Wang, Bing; Hou, Qingxi; Chen, Wei; Wu, Ming

    2016-04-01

    The hardwood bleached kraft pulp (HBKP) fibers were pretreated by PFI mill to obtain the substrates, the effects of fibrillation on HBKP fibers' enzymatic hydrolysis was studied. The results showed that the enzymatic hydrolysis efficiency was enhanced obviously by mechanical refining. The mechanical refining alterated the fibers' characteristics such as fibrillation degree, specific surface area, swelling ability, crystallinity, fiber length and fines content. All these factors correlating to the enzymatic hydrolysis were evaluated through mathematical analysis. Among these factors, the fibrillation degree has the profoundest impact on the enzymatic hydrolysis of wood fibers. Consequently, the mechanical refining aiming for a high fibrillation degree was feasible to enhance the enzymatic hydrolysis of lignocellulosic biomass. PMID:26851576

  7. ADS-J1 inhibits semen-derived amyloid fibril formation and blocks fibril-mediated enhancement of HIV-1 infection.

    PubMed

    Xun, Tianrong; Li, Wenjuan; Chen, Jinquan; Yu, Fei; Xu, Wei; Wang, Qian; Yu, Ruizhe; Li, Xiaojuan; Zhou, Xuefeng; Lu, Lu; Jiang, Shibo; Li, Lin; Tan, Suiyi; Liu, Shuwen

    2015-09-01

    Semen-derived enhancer of viral infection (SEVI) is composed of amyloid fibrils that can greatly enhance HIV-1 infectivity. By its cationic property, SEVI promotes viral sexual transmission by facilitating the attachment and internalization of HIV-1 to target cells. Therefore, semen-derived amyloid fibrils are potential targets for microbicide design. ADS-J1 is an anionic HIV-1 entry inhibitor. In this study, we explored an additional function of ADS-J1: inhibition of SEVI fibril formation and blockage of SEVI-mediated enhancement of viral infection. We found that ADS-J1 bound to an amyloidogenic peptide fragment (PAP248-286, comprising amino acids 248 to 286 of the enzyme prostatic acid phosphatase), thereby inhibiting peptide assembly into amyloid fibrils. In addition, ADS-J1 binds to mature amyloid fibrils and antagonizes fibril-mediated enhancement of viral infection. Unlike cellulose sulfate, a polyanion that failed in clinical trial to prevent HIV-1 sexual transmission, ADS-J1 shows no ability to facilitate fibril formation. More importantly, the combination of ADS-J1 with several antiretroviral drugs exhibited synergistic effects against HIV-1 infection in semen, with little cytotoxicity to vaginal epithelial cells. Our results suggest that ADS-J1 or a derivative may be incorporated into a combination microbicide for prevention of the sexual transmission of HIV-1.

  8. Colloidal graphite/graphene nanostructures using collagen showing enhanced thermal conductivity

    PubMed Central

    Bhattacharya, Soumya; Dhar, Purbarun; Das, Sarit K; Ganguly, Ranjan; Webster, Thomas J; Nayar, Suprabha

    2014-01-01

    In the present study, the exfoliation of natural graphite (GR) directly to colloidal GR/graphene (G) nanostructures using collagen (CL) was studied as a safe and scalable process, akin to numerous natural processes and hence can be termed “biomimetic”. Although the exfoliation and functionalization takes place in just 1 day, it takes about 7 days for the nano GR/G flakes to stabilize. The predominantly aromatic residues of the triple helical CL forms its own special micro and nanoarchitecture in acetic acid dispersions. This, with the help of hydrophobic and electrostatic forces, interacts with GR and breaks it down to nanostructures, forming a stable colloidal dispersion. Surface enhanced Raman spectroscopy, X-ray diffraction, photoluminescence, fluorescence, and X-ray photoelectron spectroscopy of the colloid show the interaction between GR and CL on day 1 and 7. Differential interference contrast images in the liquid state clearly reveal how the GR flakes are entrapped in the CL fibrils, with a corresponding fluorescence image showing the intercalation of CL within GR. Atomic force microscopy of graphene-collagen coated on glass substrates shows an average flake size of 350 nm, and the hexagonal diffraction pattern and thickness contours of the G flakes from transmission electron microscopy confirm ≤ five layers of G. Thermal conductivity of the colloid shows an approximate 17% enhancement for a volume fraction of less than approximately 0.00005 of G. Thus, through the use of CL, this new material and process may improve the use of G in terms of biocompatibility for numerous medical applications that currently employ G, such as internally controlled drug-delivery assisted thermal ablation of carcinoma cells. PMID:24648728

  9. Colloidal graphite/graphene nanostructures using collagen showing enhanced thermal conductivity.

    PubMed

    Bhattacharya, Soumya; Dhar, Purbarun; Das, Sarit K; Ganguly, Ranjan; Webster, Thomas J; Nayar, Suprabha

    2014-01-01

    In the present study, the exfoliation of natural graphite (GR) directly to colloidal GR/graphene (G) nanostructures using collagen (CL) was studied as a safe and scalable process, akin to numerous natural processes and hence can be termed "biomimetic". Although the exfoliation and functionalization takes place in just 1 day, it takes about 7 days for the nano GR/G flakes to stabilize. The predominantly aromatic residues of the triple helical CL forms its own special micro and nanoarchitecture in acetic acid dispersions. This, with the help of hydrophobic and electrostatic forces, interacts with GR and breaks it down to nanostructures, forming a stable colloidal dispersion. Surface enhanced Raman spectroscopy, X-ray diffraction, photoluminescence, fluorescence, and X-ray photoelectron spectroscopy of the colloid show the interaction between GR and CL on day 1 and 7. Differential interference contrast images in the liquid state clearly reveal how the GR flakes are entrapped in the CL fibrils, with a corresponding fluorescence image showing the intercalation of CL within GR. Atomic force microscopy of graphene-collagen coated on glass substrates shows an average flake size of 350 nm, and the hexagonal diffraction pattern and thickness contours of the G flakes from transmission electron microscopy confirm ≤ five layers of G. Thermal conductivity of the colloid shows an approximate 17% enhancement for a volume fraction of less than approximately 0.00005 of G. Thus, through the use of CL, this new material and process may improve the use of G in terms of biocompatibility for numerous medical applications that currently employ G, such as internally controlled drug-delivery assisted thermal ablation of carcinoma cells.

  10. 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.

  11. Gallic Acid Is an Antagonist of Semen Amyloid Fibrils That Enhance HIV-1 Infection.

    PubMed

    LoRicco, Josephine G; Xu, Changmingzi Sherry; Neidleman, Jason; Bergkvist, Magnus; Greene, Warner C; Roan, Nadia R; Makhatadze, George I

    2016-07-01

    Recent in vitro studies have demonstrated that amyloid fibrils found in semen from healthy and HIV-infected men, as well as semen itself, can markedly enhance HIV infection rates. Semen fibrils are made up of multiple naturally occurring peptide fragments derived from semen. The best characterized of these fibrils are SEVI (semen-derived enhancer of viral infection), made up of residues 248-286 of prostatic acidic phosphatase, and the SEM1 fibrils, made up of residues 86-107 of semenogelin 1. A small molecule screen for antagonists of semen fibrils identified four compounds that lowered semen-mediated enhancement of HIV-1 infectivity. One of the four, gallic acid, was previously reported to antagonize other amyloids and to exert anti-inflammatory effects. To better understand the mechanism by which gallic acid modifies the properties of semen amyloids, we performed biophysical measurements (atomic force microscopy, electron microscopy, confocal microscopy, thioflavin T and Congo Red fluorescence assays, zeta potential measurements) and quantitative assays on the effects of gallic acid on semen-mediated enhancement of HIV infection and inflammation. Our results demonstrate that gallic acid binds to both SEVI and SEM1 fibrils and modifies their surface electrostatics to render them less cationic. In addition, gallic acid decreased semen-mediated enhancement of HIV infection but did not decrease the inflammatory response induced by semen. Together, these observations identify gallic acid as a non-polyanionic compound that inhibits semen-mediated enhancement of HIV infection and suggest the potential utility of incorporating gallic acid into a multicomponent microbicide targeting both the HIV virus and host components that promote viral infection.

  12. 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.

  13. Enhanced osteoblast proliferation and collagen gene expression by estradiol

    SciTech Connect

    Ernest, M.; Schmid, Ch.; Froesch, E.R. )

    1988-04-01

    Estrogens play a crucial role in the development of postmenopausal osteoporosis. However, the mechanism by which estrogens exert their effects on bone is unknown. To examine possible direct effects of 17{beta}-estradiol on bone-forming cells, the authors used pure rat osteoblast-like cells in vitro as a model. Osteoblast-like cells prepared from calvaria of newborn rats were cultured serum-free in methylcellulose-containing medium for 21 days. Osteoblast-like cells proliferate selectively into clonally derived cell clusters of spherical morphorlogy. 17{beta}-Estradiol at concentrations of 0.1 nM and 1 nM enhanced osteoblast-like cell proliferation by 41% and 68% above vehicle-treated controls. The biologically inactive stereoisomer 17{alpha}-estradiol (same concentrations) had no effect. Moreover, the antiestrogen tamoxifen abolished the stimulation of osteoblast-like cell proliferation by 17{beta}-estradiol. After 21 days of culture, RNA was prepared and analyzed in a dot-hybridization assay for the abundance of pro{alpha}1(I) collagen mRNA. Steady-state mRNA levels were increased in cultures treated with 17{beta}-estradiol in a dose-dependent manner with maximal stimulation at 1 nM and 10 nM. At the same concentrations, the percentage of synthesized protein (labeled by ({sup 3}H)proline pulse) that was digestible by collagenase was increased, indicating that 17{beta}-estradiol acts as pretranslational levels to enhance synthesis of bone collagen. These data show that the osteoblast is a direct target for 17{beta}-estradiol.

  14. 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.

  15. 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

  16. Platelet-collagen adhesion enhances platelet aggregation induced by binding of VWF to platelets

    SciTech Connect

    Laduca, F.M.; Bell, W.R.; Bettigole, R.E. State Univ. of New York, Buffalo )

    1987-11-01

    Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of ({sup 3}H)serotonin, or alter the dose-responsive binding of {sup 125}I-labeled VWF to platelets, which occurred with increasing ristocetin concentrations. However, ASA inhibition of the platelet release reaction prevented collagen-enhanced RIPA. These results demonstrate that platelet-collagen adhesion altered the platelet-release reaction induced by the binding of VWF to platelets causing a platelet-release reaction at a level of VWF-platelet binding not normally initiating a secondary aggregation. These findings suggest that platelet-collagen adhesion enhances platelet function mediated by VWF.

  17. Structural Characterization of Semen Coagulum-Derived SEM1(86–107) Amyloid Fibrils That Enhance HIV-1 Infection

    PubMed Central

    2015-01-01

    SEM1(86–107) is a 22-residue peptide corresponding to residues 86–107 in the semenogelin I protein. SEM1(86–107) is an abundant component of freshly liquefied semen and forms amyloid fibrils capable of enhancing HIV infection. To probe the factors affecting fibril formation and gain a better understanding of how differences in pH between semen and vaginal fluid affect fibril stability, this study determined the effect of pH on SEM1(86–107) fibril formation and dissociation. The SEM1(86–107) fibril structure (i.e., residues that comprise the fibrillar core) was also probed using hydrogen–deuterium exchange mass spectrometry (HDXMS) and hydroxyl radical-mediated protein modification. The average percent exposure to hydroxyl radical-mediated modification in the SEM1(86–107) fibrils was determined without requiring tandem mass spectrometry spectral acquisition or complete separation of modified peptides. It was found that the residue exposures calculated from HDXMS and hydroxyl radical-mediated modification were similar. These techniques demonstrated that three regions of SEM1(86–107) comprise the amyloid fibril core and that positively charged residues are exposed, suggesting that electrostatic interactions between SEM1(86–107) and HIV or the cell surface may be responsible for mediating HIV infection enhancement by the SEM1(86–107) fibrils. PMID:24811874

  18. 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.

  19. 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

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

    SciTech Connect

    Chen, Chih-Hao; Kuo, Shyh Ming; Liu, Guei-Sheung; Chen, Wan-Nan U.; Chuang, Chin-Wen; Liu, Li-Feng

    2012-11-09

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

  1. 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

  2. Nanoscale Heterogeneity of the Molecular Structure of Individual hIAPP Amyloid Fibrils Revealed with Tip-Enhanced Raman Spectroscopy.

    PubMed

    vandenAkker, Corianne C; Deckert-Gaudig, Tanja; Schleeger, Michael; Velikov, Krassimir P; Deckert, Volker; Bonn, Mischa; Koenderink, Gijsje H

    2015-09-01

    Type 2 diabetes mellitus is characterized by the pathological deposition of fibrillized protein, known as amyloids. It is thought that oligomers and/or amyloid fibrils formed from human islet amyloid polypeptide (hIAPP or amylin) cause cell death by membrane damage. The molecular structure of hIAPP amyloid fibrils is dominated by β-sheet structure, as probed with conventional infrared and Raman vibrational spectroscopy. However, with these techniques it is not possible to distinguish between the core and the surface structure of the fibrils. Since the fibril surface crucially affects amyloid toxicity, it is essential to know its structure. Here the surface molecular structure and amino acid residue composition of hIAPP fibrils are specifically probed with nanoscale resolution using tip-enhanced Raman spectroscopy (TERS). The fibril surface mainly contains unordered or α-helical structures, in contrast to the β-sheet-rich core. This experimentally validates recent models of hIAPP amyloids based on NMR measurements. Spatial mapping of the surface structure reveals a highly heterogeneous surface structure. Finally, TERS can probe fibrils formed on a lipid interface, which is more representative of amyloids in vivo.

  3. 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

  4. Collagen VI enhances cartilage tissue generation by stimulating chondrocyte proliferation.

    PubMed

    Smeriglio, Piera; Dhulipala, Lakshmi; Lai, Janice H; Goodman, Stuart B; Dragoo, Jason L; Smith, Robert L; Maloney, William J; Yang, Fan; Bhutani, Nidhi

    2015-02-01

    Regeneration of human cartilage is inherently inefficient. Current cell-based approaches for cartilage repair, including autologous chondrocytes, are limited by the paucity of cells, associated donor site morbidity, and generation of functionally inferior fibrocartilage rather than articular cartilage. Upon investigating the role of collagen VI (Col VI), a major component of the chondrocyte pericellular matrix (PCM), we observe that soluble Col VI stimulates chondrocyte proliferation. Interestingly, both adult and osteoarthritis chondrocytes respond to soluble Col VI in a similar manner. The proliferative effect is, however, strictly due to the soluble Col VI as no proliferation is observed upon exposure of chondrocytes to immobilized Col VI. Upon short Col VI treatment in 2D monolayer culture, chondrocytes maintain high expression of characteristic chondrocyte markers like Col2a1, agc, and Sox9 whereas the expression of the fibrocartilage marker Collagen I (Col I) and of the hypertrophy marker Collagen X (Col X) is minimal. Additionally, Col VI-expanded chondrocytes show a similar potential to untreated chondrocytes in engineering cartilage in 3D biomimetic hydrogel constructs. Our study has, therefore, identified soluble Col VI as a biologic that can be useful for the expansion and utilization of scarce sources of chondrocytes, potentially for autologous chondrocyte implantation. Additionally, our results underscore the importance of further investigating the changes in chondrocyte PCM with age and disease and the subsequent effects on chondrocyte growth and function.

  5. 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.

  6. 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.

  7. 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.

  8. Enhanced regeneration of corneal tissue via a bioengineered collagen construct implanted by a nondisruptive surgical technique.

    PubMed

    Koulikovska, Marina; Rafat, Mehrdad; Petrovski, Goran; Veréb, Zoltán; Akhtar, Saeed; Fagerholm, Per; Lagali, Neil

    2015-03-01

    Severe shortage of donor corneas for transplantation, particularly in developing countries, has prompted the advancement of bioengineered tissue alternatives. Bioengineered corneas that can withstand transplantation while maintaining transparency and compatibility with host cells, and that are additionally amenable to standardized low-cost mass production are sought. In this study, a bioengineered porcine construct (BPC) was developed to function as a biodegradable scaffold to promote corneal stromal regeneration by host cells. Using high-purity medical-grade type I collagen, high 18% collagen content and optimized EDC-NHS cross-linker ratio, BPCs were fabricated into hydrogel corneal implants with over 90% transparency and four-fold increase in strength and stiffness compared with previous versions. Remarkably, optical transparency was achieved despite the absence of collagen fibril organization at the nanoscale. In vitro testing indicated that BPC supported confluent human epithelial and stromal-derived mesenchymal stem cell populations. With a novel femtosecond laser-assisted corneal surgical model in rabbits, cell-free BPCs were implanted in vivo in the corneal stroma of 10 rabbits over an 8-week period. In vivo, transparency of implanted corneas was maintained throughout the postoperative period, while healing occurred rapidly without inflammation and without the use of postoperative steroids. BPC implants had a 100% retention rate at 8 weeks, when host stromal cells began to migrate into implants. Direct histochemical evidence of stromal tissue regeneration was observed by means of migrated host cells producing new collagen from within the implants. This study indicates that a cost-effective BPC extracellular matrix equivalent can incorporate cells passively to initiate regenerative healing of the corneal stroma, and is compatible with human stem or organ-specific cells for future therapeutic applications as a stromal replacement for treating blinding

  9. The exercise-induced biochemical milieu enhances collagen content and tensile strength of engineered ligaments.

    PubMed

    West, Daniel W D; Lee-Barthel, Ann; McIntyre, Todd; Shamim, Baubak; Lee, Cassandra A; Baar, Keith

    2015-10-15

    Exercise stimulates a dramatic change in the concentration of circulating hormones, such as growth hormone (GH), but the biological functions of this response are unclear. Pharmacological GH administration stimulates collagen synthesis; however, whether the post-exercise systemic milieu has a similar action is unknown. We aimed to determine whether the collagen content and tensile strength of tissue-engineered ligaments is enhanced by serum obtained post-exercise. Primary cells from a human anterior cruciate ligament (ACL) were used to engineer ligament constructs in vitro. Blood obtained from 12 healthy young men 15 min after resistance exercise contained GH concentrations that were ∼7-fold greater than resting serum (P < 0.001), whereas IGF-1 was not elevated at this time point (P = 0.21 vs. rest). Ligament constructs were treated for 7 days with medium supplemented with serum obtained at rest (RestTx) or 15 min post-exercise (ExTx), before tensile testing and collagen content analysis. Compared with RestTx, ExTx enhanced collagen content (+19%; 181 ± 33 vs. 215 ± 40 μg per construct P = 0.001) and ligament mechanical properties - maximal tensile load (+17%, P = 0.03 vs. RestTx) and ultimate tensile strength (+10%, P = 0.15 vs. RestTx). In a separate set of engineered ligaments, recombinant IGF-1, but not GH, enhanced collagen content and mechanics. Bioassays in 2D culture revealed that acute treatment with post-exercise serum activated mTORC1 and ERK1/2. In conclusion, the post-exercise biochemical milieu, but not recombinant GH, enhances collagen content and tensile strength of engineered ligaments, in association with mTORC1 and ERK1/2 activation.

  10. Lysophosphatidic acid enhances collagen deposition and matrix thickening in engineered tissue.

    PubMed

    Chabaud, Stéphane; Marcoux, Thomas-Louis; Deschênes-Rompré, Marie-Pier; Rousseau, Alexandre; Morissette, Amélie; Bouhout, Sara; Bernard, Geneviève; Bolduc, Stéphane

    2015-11-01

    The time needed to produce engineered tissue is critical. A self-assembly approach provided excellent results regarding biological functions and cell differentiation because it closely respected the microenvironment of cells. Nevertheless, the technique was time consuming for producing tissue equivalents with enough extracellular matrix to allow manipulations. Unlike L-arginine supplementation that only increased accumulation of collagen in cell culture supernatant in our model, addition of lysophosphatidic acid, a natural bioactive lipid, did not modify the amount of accumulated collagen in the cell culture supernatant; however, it enhanced the matrix deposition rate without inducing fibroblast hyperproliferation and tissue fibrosis.

  11. 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.

  12. Enhanced osteoprogenitor elongated collagen fiber matrix formation by bioactive glass ionic silicon dependent on Sp7 (osterix) transcription.

    PubMed

    Varanasi, Venu G; Odatsu, Tetsurou; Bishop, Timothy; Chang, Joyce; Owyoung, Jeremy; Loomer, Peter M

    2016-10-01

    Bioactive glasses release ions, those enhance osteoblast collagen matrix synthesis and osteogenic marker expression during bone healing. Collagen matrix density and osteogenic marker expression depend on osteogenic transcription factors, (e.g., Osterix (OSX)). We hypothesize that enhanced expression and formation of collagen by Si(4+) depends on enhanced expression of OSX transcription. Experimental bioactive glass (6P53-b) and commercial Bioglass(TM) (45S5) were dissolved in basal medium to make glass conditioned medium (GCM). ICP-MS analysis was used to measure bioactive glass ion release rates. MC3T3-E1 cells were cultured for 20 days, and gene expression and extracellular matrix collagen formation was analyzed. In a separate study, siRNA was used to determine the effect of OSX knockdown on impacting the effect of Si(4+) on osteogenic markers and matrix collagen formation. Each bioactive glass exhibited similar ion release rates for all ions, except Mg(2+) released by 6P53-b. Gene expression results showed that GCM markedly enhanced many osteogenic markers, and 45S5 GCM showed higher levels of expression and collagen matrix fiber bundle density than 6P53-b GCM. Upon knockdown of OSX transcription, collagen type 5, alkaline phosphatase, and matrix density were not enhanced as compared to wild type cells. This study illustrates that the enhancement of elongated collagen fiber matrix formation by Si(±) depends on OSX transcription. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2604-2615, 2016.

  13. Improvement of Interfacial Adhesion in Bamboo Polymer Composite Enhanced with Micro-Fibrillated Cellulose

    NASA Astrophysics Data System (ADS)

    Okubo, Kazuya; Fujii, Toru; Yamashita, Naoya

    Current study presents one of effective techniques to improve mechanical properties of PLA (Poly-Lactic Acid)-based bamboo fiber composite. Commercially available Micro-Fibrillated Cellulose (MFC) obtained from wood pulp was applied as an enhancer to the composite. The bamboo fibers were extracted by steam explosion method and they were also rubbed in water to remove xylem (soft-wall cells). The liquid-based MFC, PLA and the bamboo fiber were mixed in water for several minutes and they were filtrated under vacuum pressure. To fabricate the composite, remained sheets were then hot pressed after dry. Three-point bending strength and Mode I fracture toughness of the composite were significantly improved, even when 10% of the MFC was added into the PLA/BF composite in weight. If small amount of MFC added into the bamboo fiber composite, tangled MFC fibers prevented the growth of micro crack along the interface between bamboo fiber and matrix.

  14. A modified collagen gel enhances healing outcome in a preclinical swine model of excisional wounds.

    PubMed

    Elgharably, Haytham; Roy, Sashwati; Khanna, Savita; Abas, Motaz; Dasghatak, Piya; Das, Amitava; Mohammed, Kareem; Sen, Chandan K

    2013-01-01

    Collagen-based dressings are of great interest in wound care. However, evidence supporting their mechanism of action is scanty. This work provides first results from a preclinical swine model of excisional wounds, elucidating the mechanism of action of a modified collagen gel (MCG) dressing. Following wounding, wound-edge tissue was collected at specific time intervals (3, 7, 14, and 21 days postwounding). On day 7, histological analysis showed significant increase in the length of rete ridges, suggesting improved biomechanical properties of the healing wound tissue. Rapid and transient mounting of inflammation is necessary for efficient healing. MCG significantly accelerated neutrophil and macrophage recruitment to the wound site on day 3 and day 7 with successful resolution of inflammation on day 21. MCG induced monocyte chemotactic protein-1 expression in neutrophil-like human promyelocytic leukemia-60 cells in vitro. In vivo, MCG-treated wound tissue displayed elevated vascular endothelial growth factor expression. Consistently, MCG-treated wounds displayed significantly higher abundance of endothelial cells with increased blood flow to the wound area indicating improved vascularization. This observation was explained by the finding that MCG enhanced proliferation of wound-site endothelial cells. In MCG-treated wound tissue, Masson's trichrome and picrosirius red staining showed higher abundance of collagen and increased collagen type I:III ratio. This work presents first evidence from a preclinical setting explaining how a collagen-based dressing may improve wound closure by targeting multiple key mechanisms. The current findings warrant additional studies to determine whether the responses to the MCG are different from other collagen-based products used in clinical setting.

  15. Jararhagin disruption of endothelial cell anchorage is enhanced in collagen enriched matrices.

    PubMed

    Baldo, C; Lopes, D S; Faquim-Mauro, E L; Jacysyn, J F; Niland, S; Eble, J A; Clissa, P B; Moura-da-Silva, A M

    2015-12-15

    Hemorrhage is one of the most striking effects of bites by viper snakes resulting in fast bleeding and ischemia in affected tissues. Snake venom metalloproteinases (SVMPs) are responsible for hemorrhagic activity, but the mechanisms involved in SVMP-induced hemorrhage are not entirely understood and the study of such mechanisms greatly depends on in vivo experiments. In vivo, hemorrhagic SVMPs accumulate on basement membrane (BM) of venules and capillary vessels allowing the hydrolysis of collagen IV with consequent weakness and rupture of capillary walls. These effects are not reproducible in vitro with conventional endothelial cell cultures. In this study we used two-dimension (2D) or three-dimension (3D) cultures of HUVECs on matrigel and observed the same characteristics as in ex vivo experiments: only the hemorrhagic toxin was able to localize on surfaces or internalize endothelial cells in 2D cultures or in the surface of tubules formed on 3D cultures. The contribution of matrigel, fibronectin and collagen matrices in jararhagin-induced endothelial cell damage was then analyzed. Collagen and matrigel substrates enhanced the endothelial cell damage induced by jararhagin allowing toxin binding to focal adhesions, disruption of stress fibers, detachment and apoptosis. The higher affinity of jararhagin to collagen than to fibronectin explains the localization of the toxin within BM. Moreover, once located in BM, interactions of jararhagin with α2β1 integrin would favor its localization on focal adhesions, as observed in our study. The accumulation of toxin in focal adhesions, observed only in cells grown in collagen matrices, would explain the enhancement of cell damage in these matrices and reflects the actual interaction among toxin, endothelial cells and BM components that occurs in vivo and results in the hemorrhagic lesions induced by viper venoms.

  16. Mesenchymal stem cell-coated sutures enhance collagen depositions in sutured tissues.

    PubMed

    Casado, Javier G; Blazquez, Rebeca; Jorge, Inmaculada; Alvarez, Veronica; Gomez-Mauricio, Guadalupe; Ortega-Muñoz, Mariano; Vazquez, Jesus; Sanchez-Margallo, Francisco M

    2014-01-01

    Sutures are commonly used for surgical procedures and new sutures are being developed to improve wound healing. In the past decade, it has been extensively shown that mesenchymal stem cells (MSCs) have a wound healing potential. To benefit the overall wound healing process, we aimed to analyze the usage of pretreated sutures for improving the implantation of MSCs in the tissues. Our results firstly showed that suture pretreatments with gelatin, poly-L-lysine, and NaOH improved the adhesive strength of MSCs to sutures. These cells remained surrounding the sutured tissue and no significant phenotypic changes were found in those cells cultured onto pretreated sutures. In vivo experiments showed that the implantation of MSCs by suturing increases the collagen content in the sutured tissue. Moreover, proteomics analysis of secreted proteins showed that collagen alpha-1(I) chain was the most abundant collagen found. To our knowledge, this is the first report that aimed to improve the implantation of MSCs in tissue by suture pretreatments. Moreover, in vivo experiments suggest that MSC-coated sutures may enhance wound healing and tissue remodeling through the release of different collagen types being applicable for those patients that tend to have difficulty healing.

  17. 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

  18. 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

  19. Surface modification of PVDF using non-mammalian sources of collagen for enhancement of endothelial cell functionality.

    PubMed

    Wang, Jun Kit; Xiong, Gordon Minru; Luo, Baiwen; Choo, Chee Chong; Yuan, Shaojun; Tan, Nguan Soon; Choong, Cleo

    2016-03-01

    Although polyvinylidene fluoride (PVDF) is non-toxic and stable in vivo, its hydrophobic surface has limited its bio-applications due to poor cell-material interaction and thrombus formation when used in blood contacting devices. In this study, surface modification of PVDF using naturally derived non-mammalian collagen was accomplished via direct surface-initiated atom transfer radical polymerisation (SI-ATRP) to enhance its cytocompatibility and hemocompatibility. Results showed that Type I collagen was successfully extracted from fish scales and bullfrog skin. The covalent immobilisation of fish scale-derived collagen (FSCOL) and bullfrog skin-derived collagen (BFCOL) onto the PVDF surface improves the attachment and proliferation of human umbilical vein endothelial cells (HUVECs). Furthermore, both FSCOL and BFCOL had comparable anti-thrombogenic profiles to that of commercially available bovine collagen (BVCOL). Also, cell surface expression of the leukocyte adhesion molecule was lower on HUVECs cultured on non-mammalian collagen surfaces than on BVCOL, which is an indication of lower pro-inflammatory response. Overall, results from this study demonstrated that non-mammalian sources of collagen could be used to confer bioactivity to PVDF, with comparable cell-material interactions and hemocompatibility to BVCOL. Additionally, higher expression levels of Type IV collagen in HUVECs cultured on FSCOL and BFCOL were observed as compared to BVCOL, which is an indication that the non-mammalian sources of collagen led to a better pro-angiogenic properties, thus making them suitable for blood contacting applications.

  20. 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.

  1. 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

  2. RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils

    PubMed Central

    Zaarur, Nava; Xu, Xiaobin; Lestienne, Patrick; Meriin, Anatoli B; McComb, Mark; Costello, Catherine E; Newnam, Gary P; Ganti, Rakhee; Romanova, Nina V; Shanmugasundaram, Maruda; Silva, Sara TN; Bandeiras, Tiago M; Matias, Pedro M; Lobachev, Kirill S; Lednev, Igor K; Chernoff, Yury O; Sherman, Michael Y

    2015-01-01

    The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome-like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin-1 interacted directly with the RuvbL1 barrel-like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation. PMID:26303906

  3. Strain-enhanced stress relaxation impacts nonlinear elasticity in collagen gels.

    PubMed

    Nam, Sungmin; Hu, Kenneth H; Butte, Manish J; Chaudhuri, Ovijit

    2016-05-17

    The extracellular matrix (ECM) is a complex assembly of structural proteins that provides physical support and biochemical signaling to cells in tissues. The mechanical properties of the ECM have been found to play a key role in regulating cell behaviors such as differentiation and malignancy. Gels formed from ECM protein biopolymers such as collagen or fibrin are commonly used for 3D cell culture models of tissue. One of the most striking features of these gels is that they exhibit nonlinear elasticity, undergoing strain stiffening. However, these gels are also viscoelastic and exhibit stress relaxation, with the resistance of the gel to a deformation relaxing over time. Recent studies have suggested that cells sense and respond to both nonlinear elasticity and viscoelasticity of ECM, yet little is known about the connection between nonlinear elasticity and viscoelasticity. Here, we report that, as strain is increased, not only do biopolymer gels stiffen but they also exhibit faster stress relaxation, reducing the timescale over which elastic energy is dissipated. This effect is not universal to all biological gels and is mediated through weak cross-links. Mechanistically, computational modeling and atomic force microscopy (AFM) indicate that strain-enhanced stress relaxation of collagen gels arises from force-dependent unbinding of weak bonds between collagen fibers. The broader effect of strain-enhanced stress relaxation is to rapidly diminish strain stiffening over time. These results reveal the interplay between nonlinear elasticity and viscoelasticity in collagen gels, and highlight the complexity of the ECM mechanics that are likely sensed through cellular mechanotransduction. PMID:27140623

  4. Hypoxia-induced collagen crosslinking as a mechanism for enhancing mechanical properties of engineered articular cartilage

    PubMed Central

    Makris, E.A.; Hu, J.C.; Athanasiou, K.A.

    2013-01-01

    Objective The focus of tissue engineering of neocartilage has traditionally been on enhancing extracellular matrix and thus biomechanical properties. Emphasis has been placed on the enhancement of collagen type and quantity, and, concomitantly, tensile properties. The objective of this study was to improve crosslinking of the collagen network by testing the hypothesis that hypoxia could promote pyridinoline (PYR) crosslinks and, thus, improve neocartilage’s tensile properties. Methods Chondrocyte expression of lysyl oxidase (LOX), an enzyme responsible for the formation of collagen PYR crosslinks, was first assessed pre- and post- hypoxia application. Then, the mechanical properties of self-assembled neocartilage constructs were measured, after 4 weeks of culture, for groups exposed to 4% O2 at different initiation times and durations, i.e., during the 1st and 3rd weeks, 3rd and 4th weeks, 4th week only, continuously after cell seeding, or never. Results Results showed that LOX gene expression was upregulated ~20-fold in chondrocytes in response to hypoxia. Hypoxia applied during the 3rd and 4th weeks significantly increased PYR crosslinks without affecting collagen content. Excitingly, neocartilage tensile properties were increased ~2-fold. It should be noted that these properties exhibited a distinct temporal dependence to hypoxia exposure, since upregulation of these properties was due to hypoxia applied only during the 3rd and 4th weeks. Conclusion These data elucidate the role of hypoxia-mediated upregulation of LOX and subsequent increases in PYR crosslinks in engineered cartilage. These results hold promise toward applying hypoxia at precise time points to promote tensile integrity and direct construct maturation. PMID:23353112

  5. Enhanced osteoprogenitor elongated collagen fiber matrix formation by bioactive glass ionic silicon dependent on Sp7 (osterix) transcription.

    PubMed

    Varanasi, Venu G; Odatsu, Tetsurou; Bishop, Timothy; Chang, Joyce; Owyoung, Jeremy; Loomer, Peter M

    2016-10-01

    Bioactive glasses release ions, those enhance osteoblast collagen matrix synthesis and osteogenic marker expression during bone healing. Collagen matrix density and osteogenic marker expression depend on osteogenic transcription factors, (e.g., Osterix (OSX)). We hypothesize that enhanced expression and formation of collagen by Si(4+) depends on enhanced expression of OSX transcription. Experimental bioactive glass (6P53-b) and commercial Bioglass(TM) (45S5) were dissolved in basal medium to make glass conditioned medium (GCM). ICP-MS analysis was used to measure bioactive glass ion release rates. MC3T3-E1 cells were cultured for 20 days, and gene expression and extracellular matrix collagen formation was analyzed. In a separate study, siRNA was used to determine the effect of OSX knockdown on impacting the effect of Si(4+) on osteogenic markers and matrix collagen formation. Each bioactive glass exhibited similar ion release rates for all ions, except Mg(2+) released by 6P53-b. Gene expression results showed that GCM markedly enhanced many osteogenic markers, and 45S5 GCM showed higher levels of expression and collagen matrix fiber bundle density than 6P53-b GCM. Upon knockdown of OSX transcription, collagen type 5, alkaline phosphatase, and matrix density were not enhanced as compared to wild type cells. This study illustrates that the enhancement of elongated collagen fiber matrix formation by Si(±) depends on OSX transcription. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2604-2615, 2016. PMID:27279631

  6. 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

  7. Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates

    SciTech Connect

    Kato, Shigemi; Ben, T.L.; De Luca, L.M. )

    1988-11-01

    The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10{sup {minus}9} and 10{sup {minus}7} M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.

  8. L-arginine mediated renaturation enhances yield of human, α6 Type IV collagen non-collagenous domain from bacterial inclusion bodies.

    PubMed

    Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Sudhakar, Yakkanti Akul

    2012-10-01

    The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.

  9. Polyanionic Candidate Microbicides Accelerate the Formation of Semen-Derived Amyloid Fibrils to Enhance HIV-1 Infection

    PubMed Central

    Tan, Suiyi; Lu, Lu; Li, Lin; Liu, Jixiang; Oksov, Yelena; Lu, Hong; Jiang, Shibo; Liu, Shuwen

    2013-01-01

    Polyanionic candidate microbicides, including cellulose sulfate, carrageenan, PRO 2000, were proven ineffective in preventing HIV-1 transmission and even cellulose sulfate showed increased risk of HIV acquisition in the Phase III efficacy trials. Semen plays critical roles in HIV-1 sexual transmission. Specifically, amyloid fibrils formed by fragments of prostatic acidic phosphatase (PAP) in semen termed semen-derived enhancer of virus infection (SEVI) could drastically enhance HIV-1 infection. Here we investigated the interaction between polyanions and PAP248-286, a prototype peptide of SEVI, to understand the possible cause of polyanionic candidate microbicides to fail in clinical trials. We found anionic polymers could efficiently promote SEVI fibril formation, most likely mediated by the natural electrostatic interaction between polyanions and PAP248-286, as revealed by acid native PAGE and Western blot. The overall anti-HIV-1 activity of polyanions in the presence or absence of PAP248-286 or semen was evaluated. In the viral infection assay, the supernatants of polyanions/PAP248-286 or polyanions/semen mixtures containing the free, unbound polyanionic molecules showed a general reduction in antiviral efficacy, while the pellets containing amyloid fibrils formed by the polyanion-bound PAP248-286 showed aggravated enhancement of viral infection. Collectively, from the point of drug-host protein interaction, our study revealed that polyanions facilitate SEVI fibril formation to promote HIV-1 infection, thus highlighting a molecular mechanism underlying the failure of polyanions in clinical trials and the importance of drug-semen interaction in evaluating the anti-HIV-1 efficacy of candidate microbicides. PMID:23544097

  10. Hypoxia-mimicking bioactive glass/collagen glycosaminoglycan composite scaffolds to enhance angiogenesis and bone repair.

    PubMed

    Quinlan, Elaine; Partap, Sonia; Azevedo, Maria M; Jell, Gavin; Stevens, Molly M; O'Brien, Fergal J

    2015-06-01

    One of the biggest challenges in regenerative medicine is promoting sufficient vascularisation of tissue-engineered constructs. One approach to overcome this challenge is to target the cellular hypoxia inducible factor (HIF-1α) pathway, which responds to low oxygen concentration (hypoxia) and results in the activation of numerous pro-angiogenic genes including vascular endothelial growth factor (VEGF). Cobalt ions are known to mimic hypoxia by artificially stabilising the HIF-1α transcription factor. Here, resorbable bioactive glass particles (38 μm and 100 μm) with cobalt ions incorporated into the glass network were used to create bioactive glass/collagen-glycosaminoglycan scaffolds optimised for bone tissue engineering. Inclusion of the bioactive glass improved the compressive modulus of the resulting composite scaffolds while maintaining high degrees of porosity (>97%). Moreover, in vitro analysis demonstrated that the incorporation of cobalt bioactive glass with a mean particle size of 100 μm significantly enhanced the production and expression of VEGF in endothelial cells, and cobalt bioactive glass/collagen-glycosaminoglycan scaffold conditioned media also promoted enhanced tubule formation. Furthermore, our results prove the ability of these scaffolds to support osteoblast cell proliferation and osteogenesis in all bioactive glass/collagen-glycosaminoglycan scaffolds irrespective of the particle size. In summary, we have developed a hypoxia-mimicking tissue-engineered scaffold with pro-angiogenic and pro-osteogenic capabilities that may encourage bone tissue regeneration and overcome the problem of inadequate vascularisation of grafts commonly seen in the field of tissue engineering.

  11. Adiponectin exacerbates collagen-induced arthritis via enhancing Th17 response and prompting RANKL expression.

    PubMed

    Sun, Xiaoxuan; Feng, Xiaoke; Tan, Wenfeng; Lin, Na; Hua, Minhui; Wei, Yu; Wang, Fang; Li, Ningli; Zhang, Miaojia

    2015-01-01

    We previously reported adiponectin (AD) is highly expressed in the inflamed synovial joint tissue and correlates closely with progressive bone erosion in Rheumatoid arthritis (RA) patients. Here, we investigate the role of adiponectin in regulating Th17 response and the expression of receptor activator of nuclear factor-κB ligand (RANKL) in mice with CIA mice by intraarticularly injection of adiponectin into knee joints on day 17, day 20 and day 23 post first collagen immunization. The increased adiponectin expression was found in inflamed joint tissue of collagen-induced arthritis (CIA) mice. Adiponectin injection resulted in an earlier onset of arthritis, an aggravated arthritic progression, more severe synovial hyperplasia, bone erosion and osteoporosis in CIA mice. CD4(+)IL-17(+) Th17 cells, IL-17 mRNA and RANKL mRNA expression were markedly increased in the joint tissue of adiponectin treated CIA mice. Moreover, adiponectin treatment markedly enhanced Th17 cell generation from naive CD4(+) T cells in vitro, which accompanied by the high expression of Th17 transcription factor ROR-γt, and Th17 cytokine genes included IL-22 and IL-23. This study reveals a novel effect of adiponectin in exacerbating CIA progression by enhancing Th17 cell response and RANKL expression. PMID:26063682

  12. Electron microscopy of Staphylococcus epidermidis fibril and biofilm formation using image-enhancing ionic liquid.

    PubMed

    Takahashi, Chisato; Kalita, Golap; Ogawa, Noriko; Moriguchi, Keiichi; Tanemura, Masaki; Kawashima, Yoshiaki; Yamamoto, Hiromitsu

    2015-02-01

    We established an optimized biofilm observation method using a hydrophilic ionic liquid (IL), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]). In the present study, a biofilm was formed by Staphylococcus epidermidis. Using field emission (FE) scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the colonization of assemblages formed by microbial cells was observed as a function of the cultivation time. FE-TEM analysis revealed that the fibril comprises three types of protein. In addition, the ultrastructure of each protein monomer was visualized. It was expected that the curly-structured protein plays an important role in extension during fibril formation. Compared to the conventional sample preparation method for electron microscopy, a fine structure was easily obtained by the present method using IL. This observation technique can provide valuable information to characterize the ultrastructure of the fibril and biofilm that has not been revealed till date. Furthermore, these findings of the molecular architecture of the fibril and the colonization behavior of microbial cells during biofilm formation are useful for the development of antibacterial drugs and microbial utilization.

  13. Electron microscopy of Staphylococcus epidermidis fibril and biofilm formation using image-enhancing ionic liquid.

    PubMed

    Takahashi, Chisato; Kalita, Golap; Ogawa, Noriko; Moriguchi, Keiichi; Tanemura, Masaki; Kawashima, Yoshiaki; Yamamoto, Hiromitsu

    2015-02-01

    We established an optimized biofilm observation method using a hydrophilic ionic liquid (IL), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]). In the present study, a biofilm was formed by Staphylococcus epidermidis. Using field emission (FE) scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the colonization of assemblages formed by microbial cells was observed as a function of the cultivation time. FE-TEM analysis revealed that the fibril comprises three types of protein. In addition, the ultrastructure of each protein monomer was visualized. It was expected that the curly-structured protein plays an important role in extension during fibril formation. Compared to the conventional sample preparation method for electron microscopy, a fine structure was easily obtained by the present method using IL. This observation technique can provide valuable information to characterize the ultrastructure of the fibril and biofilm that has not been revealed till date. Furthermore, these findings of the molecular architecture of the fibril and the colonization behavior of microbial cells during biofilm formation are useful for the development of antibacterial drugs and microbial utilization. PMID:25542577

  14. Collagen esterification enhances the function and survival of pancreatic β cells in 2D and 3D culture systems

    SciTech Connect

    Ko, Jae Hyung; Kim, Yang Hee; Jeong, Seong Hee; Lee, Song; Park, Si-Nae; Shim, In Kyong; Kim, Song Cheol

    2015-08-07

    Collagen, one of the most important components of the extracellular matrix (ECM), may play a role in the survival of pancreatic islet cells. In addition, chemical modifications that change the collagen charge profile to a net positive charge by esterification have been shown to increase the adhesion and proliferation of various cell types. The purpose of this study was to characterize and compare the effects of native collagen (NC) and esterified collagen (EC) on β cell function and survival. After isolation by the collagenase digestion technique, rat islets were cultured with NC and EC in 2 dimensional (2D) and 3 dimensional (3D) environments for a long-term duration in vitro. The cells were assessed for islet adhesion, morphology, viability, glucose-induced insulin secretion, and mRNA expression of glucose metabolism-related genes, and visualized by scanning electron microscopy (SEM). Islet cells attached tightly in the NC group, but islet cell viability was similar in both the NC and EC groups. Glucose-stimulated insulin secretion was higher in the EC group than in the NC group in both 2D and 3D culture. Furthermore, the mRNA expression levels of glucokinase in the EC group were higher than those in the NC group and were associated with glucose metabolism and insulin secretion. Finally, SEM observation confirmed that islets had more intact component cells on EC sponges than on NC sponges. These results indicate that modification of collagen may offer opportunities to improve function and viability of islet cells. - Highlights: • We changed the collagen charge profile to a net positive charge by esterification. • Islets cultured on esterified collagen improved survival in both 2D and 3D culture. • Islets cultured on esterified collagen enhanced glucose-stimulated insulin release. • High levels of glucokinase mRNA may be associated with increased insulin release.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. Suboptimal Activation of Protease-activated Receptors Enhances α2β1 Integrin-mediated Platelet Adhesion to Collagen*

    PubMed Central

    Marjoram, Robin J.; Voss, Bryan; Pan, Yumei; Dickeson, S. Kent; Zutter, Mary M.; Hamm, Heidi E.; Santoro, Samuel A.

    2009-01-01

    Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the α2β1 integrin (α2β1) and the glycoprotein VI (GPVI)/FcRγ chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (α2-CRP) containing the α2β1-specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to α2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was α2β1-dependent and GPVI/FcRγ-independent as revealed in experiments with α2β1- or FcRγ-deficient mouse platelets. We further show that suboptimal activation of other platelet Gq-linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to α2-CRP. The enhanced α2β1-mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of αIIbβ3 integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet Gq-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced α2β1 binding to collagens containing GFOGER sites. PMID:19815553

  20. 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

  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. PMID:27524008

  2. 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.

  3. Water absorbing and antibacterial properties of N-isopropyl acrylamide grafted and collagen/chitosan immobilized polypropylene nonwoven fabric and its application on wound healing enhancement.

    PubMed

    Wang, Chyung-Chyung; Su, Ching-Hua; Chen, Cheng-Chi

    2008-03-15

    A durable sandwich wound dressing system with high liquid absorbing, biocompatibility, and antibacterial properties was designed. Various solution weight ratios of collagen to chitosan were used to immobilize on the polypropylene nonwoven fabric, which were pregrafted with acrylic acid (AA) or N-isopropyl acrylamide (NIPAAm) to construct a durable sandwich wound dressing membrane with high water absorbing, easy removal, and antibacterial activity. Swelling properties and antibacterial activity of the membranes were measured, and wound healing enhancement by skin full-thickness excision on animal model was examined. The results indicated that NIPAAm-grafted and collagen/chitosan-immobilized polypropylene nonwoven fabric (PP-NIPAAm-collagen-chitosan) showed a better healing effect than AA-grafted and collagen/chitosan-immobilized polypropylene nonwoven fabric (PP-AA-collagen-chitosan). The wound treated with PP-NIPAAm-collagen-chitosan demonstrated the excellent remodeling effect in histological examination with respect to the construction of vein, epidermis, and dermis at 21 days after skin injury. The values of water uptake and water diffusion coefficient for PP-NIPAAm-collagen-chitosan were higher than that for PP-AA-collagen-chitosan under a given solution weight ratio of collagen/chitosan. Both PP-NIPAAm-collagen-chitosan and PP-AA-collagen-chitosan demonstrated antibacterial activity.

  4. 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.

  5. Disaggregation-induced fluorescence enhancement of NIAD-4 for the optical imaging of amyloid-β fibrils.

    PubMed

    Peccati, Francesca; Hernando, Jordi; Blancafort, Lluís; Solans-Monfort, Xavier; Sodupe, Mariona

    2015-08-14

    The main hallmark of Alzheimer's disease is the deposition of amyloid-β (Aβ) aggregates in the brain. An early diagnosis of the disease requires a fast and accurate detection of such aggregates in vivo. NIAD-4 is one of the most promising in vivo markers developed due to its high emission at λ > 600 nm and its ability to rapidly cross the blood-brain barrier (BBB) and target Aβ deposits. Furthermore, it shows a dramatic fluorescence enhancement upon binding to amyloid fibrils, which is essential for attaining good imaging contrast. Aiming at establishing novel design concepts for the preparation of optimized optical probes, the current work rationalizes the excellent performance of NIAD-4 by using a pool of computational (TD-DFT and CASPT2 calculations, ab initio molecular dynamics and protein energy landscape exploration) and spectroscopic techniques. Unlike other markers operating as molecular rotors or polarity-sensitive dyes, we uncover herein that the high fluorescence imaging contrast observed upon NIAD-4 binding to amyloid fibrils results from reversible aggregation. NIAD-4 forms non-emissive assemblies in aqueous solution already at very low concentrations, which convert into the highly fluorescent monomeric species by diffusion into the hydrophobic voids of Aβ deposits. This result paves the way to exploit aggregation-induced processes as a new strategy towards advanced fluorescence markers for amyloid detection. PMID:26123993

  6. Disaggregation-induced fluorescence enhancement of NIAD-4 for the optical imaging of amyloid-β fibrils.

    PubMed

    Peccati, Francesca; Hernando, Jordi; Blancafort, Lluís; Solans-Monfort, Xavier; Sodupe, Mariona

    2015-08-14

    The main hallmark of Alzheimer's disease is the deposition of amyloid-β (Aβ) aggregates in the brain. An early diagnosis of the disease requires a fast and accurate detection of such aggregates in vivo. NIAD-4 is one of the most promising in vivo markers developed due to its high emission at λ > 600 nm and its ability to rapidly cross the blood-brain barrier (BBB) and target Aβ deposits. Furthermore, it shows a dramatic fluorescence enhancement upon binding to amyloid fibrils, which is essential for attaining good imaging contrast. Aiming at establishing novel design concepts for the preparation of optimized optical probes, the current work rationalizes the excellent performance of NIAD-4 by using a pool of computational (TD-DFT and CASPT2 calculations, ab initio molecular dynamics and protein energy landscape exploration) and spectroscopic techniques. Unlike other markers operating as molecular rotors or polarity-sensitive dyes, we uncover herein that the high fluorescence imaging contrast observed upon NIAD-4 binding to amyloid fibrils results from reversible aggregation. NIAD-4 forms non-emissive assemblies in aqueous solution already at very low concentrations, which convert into the highly fluorescent monomeric species by diffusion into the hydrophobic voids of Aβ deposits. This result paves the way to exploit aggregation-induced processes as a new strategy towards advanced fluorescence markers for amyloid detection.

  7. Myofibroblast expression in skin wounds is enhanced by collagen III suppression.

    PubMed

    Al-Qattan, Mohammed M; Abd-Elwahed, Mervat M; Hawary, Khalid; Arafah, Maha M; Shier, Medhat K

    2015-01-01

    Generally speaking, the excessive expression of myofibroblasts is associated with excessive collagen production. One exception is seen in patients and animal models of Ehlers-Danlos syndrome type IV in which the COL3A1 gene mutation results in reduced collagen III but with concurrent increased myofibroblast expression. This paradox has not been examined with the use of external drugs/modalities to prevent hypertrophic scars. In this paper, we injected the rabbit ear wound model of hypertrophic scarring with two doses of a protein called nAG, which is known to reduce collagen expression and to suppress hypertrophic scarring in that animal model. The higher nAG dose was associated with significantly less collagen III expression and concurrent higher degree of myofibroblast expression. We concluded that collagen III content of the extracellular matrix may have a direct or an indirect effect on myofibroblast differentiation. However, further research is required to investigate the pathogenesis of this paradoxical phenomenon. PMID:25789326

  8. A Modified Collagen Gel Enhances Healing Outcome in a Pre-Clinical Swine Model of Excisional Wounds

    PubMed Central

    Elgharably, Haytham; Roy, Sashwati; Khanna, Savita; Abas, Motaz; DasGhatak, Piya; Das, Amitava; Mohammed, Kareem; Sen, Chandan K.

    2013-01-01

    Collagen-based dressings are of great interest in wound care. However, evidence supporting their mechanism of action in a wound setting in vivo is scanty. This work providesfirst results from a pre-clinical swine model of excisional wounds elucidating the mechanism of action of a modified collagen gel (MCG) dressing. Following wounding, wound-edge tissue was collected at specific time intervals (3, 7, 14, and 21 days post-wounding). On day 7, histological analysis showed significant increase in the length of rete ridges suggesting improved biomechanical properties of the healing wound tissue. Rapid and transient mounting of inflammation is necessary for efficient healing. MCG significantly accelerated neutrophil and macrophages recruitment to the wound site on day 3 and day 7 with successful resolution of inflammation on day 21. MCG induced MCP-1 expression in neutrophil-like HL-60 cells in vitro. In vivo, MCG treated wound tissue displayed elevated VEGF expression. Consistently, MCG-treated wounds displayed significantly higher abundance of endothelial cells with increased blood flow to the wound area indicating improved vascularization. This observation was explained by the finding that MCG enhanced proliferation of wound-site endothelial cells. In MCG-treated wound tissue, Masson’s Trichrome and Picrosirius red staining showed higher abundance of collagen and increased collagen type I:III ratio. This work presents first evidence from a pre-clinical experimental setting explaining how a collagen-based dressing may improve wound closure by targeting multiple key mechanisms as compared to standard of care i.e., Tegadem treated wounds. The current findings warrant additional studies to determine whether the responses to the MCG are different from other modified or unmodified collagen based products used in clinical setting. PMID:23607796

  9. A biomaterial composed of collagen and solubilized elastin enhances angiogenesis and elastic fiber formation without calcification.

    PubMed

    Daamen, Willeke F; Nillesen, Suzan T M; Wismans, Ronnie G; Reinhardt, Dieter P; Hafmans, Theo; Veerkamp, Jacques H; van Kuppevelt, Toin H

    2008-03-01

    Elastin is the prime protein in elastic tissues that contributes to elasticity of, for example, lung, aorta, and skin. Upon injury, elastic fibers are not readily replaced, which hampers tissue regeneration. Incorporation of solubilized elastin (hydrolyzed insoluble elastin fibers or elastin peptides) in biomaterials may improve regeneration, because solubilized elastin is able to promote proliferation as well as elastin synthesis. Porous biomaterials composed of highly purified collagen without and without elastin fibers or solubilized elastin were prepared by freezing and lyophilization. Solubilized elastin formed spherical structures that were incorporated in the collagenous part of the scaffolds and that persisted after chemical crosslinking of the scaffolds. Crosslinked scaffolds were subcutaneously implanted in young Sprague Dawley rats. Collagen-solubilized elastin and collagen scaffolds showed no calcification in this sensitive calcification model, in contrast to scaffolds containing elastin fibers. Collagen-solubilized elastin scaffolds also induced angiogenesis, as revealed by type IV collagen staining, and promoted elastic fiber synthesis, as shown with antibodies against rat elastin and fibrillin-1. It is concluded that scaffolds produced from collagen and solubilized elastin present a non-calcifying biomaterial with a capacity for soft-tissue regeneration, especially in relation to elastic fiber synthesis.

  10. Determining collagen distribution in articular cartilage using contrast-enhanced micro-computed tomography

    PubMed Central

    Nieminen, H.J.; Ylitalo, T.; Karhula, S.; Suuronen, J.-P.; Kauppinen, S.; Serimaa, R.; Hæggström, E.; Pritzker, K.P.H.; Valkealahti, M.; Lehenkari, P.; Finnilä, M.; Saarakkala, S.

    2015-01-01

    Summary Objective Collagen distribution within articular cartilage (AC) is typically evaluated from histological sections, e.g., using collagen staining and light microscopy (LM). Unfortunately, all techniques based on histological sections are time-consuming, destructive, and without extraordinary effort, limited to two dimensions. This study investigates whether phosphotungstic acid (PTA) and phosphomolybdic acid (PMA), two collagen-specific markers and X-ray absorbers, could (1) produce contrast for AC X-ray imaging or (2) be used to detect collagen distribution within AC. Method We labeled equine AC samples with PTA or PMA and imaged them with micro-computed tomography (micro-CT) at pre-defined time points 0, 18, 36, 54, 72, 90, 180, 270 h during staining. The micro-CT image intensity was compared with collagen distributions obtained with a reference technique, i.e., Fourier-transform infrared imaging (FTIRI). The labeling time and contrast agent producing highest association (Pearson correlation, Bland–Altman analysis) between FTIRI collagen distribution and micro-CT -determined PTA distribution was selected for human AC. Results Both, PTA and PMA labeling permitted visualization of AC features using micro-CT in non-calcified cartilage. After labeling the samples for 36 h in PTA, the spatial distribution of X-ray attenuation correlated highly with the collagen distribution determined by FTIRI in both equine (mean ± S.D. of the Pearson correlation coefficients, r = 0.96 ± 0.03, n = 12) and human AC (r = 0.82 ± 0.15, n = 4). Conclusions PTA-induced X-ray attenuation is a potential marker for non-destructive detection of AC collagen distributions in 3D. This approach opens new possibilities in development of non-destructive 3D histopathological techniques for characterization of OA. PMID:26003951

  11. Type I collagen-mediated synthesis of noble metallic nanoparticles networks and the applications in Surface-Enhanced Raman Scattering and electrochemistry.

    PubMed

    Sun, Yujing; Sun, Lanlan; Zhang, Baohua; Xu, Fugang; Liu, Zhelin; Guo, Cunlan; Zhang, Yue; Li, Zhuang

    2009-08-15

    In this paper, we demonstrated an effective environmentally friendly synthesis route to prepare noble metallic (Au, Ag, Pt and Pd) nanoparticles (NPs) networks mediated by type I collagen in the absence of any seeds or surfactants. In the reactions, type I collagen served as stabilizing agent and assembly template for the synthesized metallic NPs. The hydrophobic interaction between collagen and mica interface as well as the hydrogen bonds between inter- and intra-collagen molecules play important roles in the formation of collagen-metallic NPs networks. The noble metallic NPs networks have many advantages in the applications of Surface-Enhanced Raman Scattering (SERS) and electrochemistry detection. Typically, the as-prepared Ag NPs networks reveal great Raman enhancement activity for 4-ATP, and can even be used to detect low concentration of DNA base, adenine, without any label step. Furthermore, the cyclic voltammograms showed Pt NPs networks have good electrocatalytic ability for the reduction of O(2).

  12. A copper sulfate and hydroxylysine treatment regimen for enhancing collagen cross-linking and biomechanical properties in engineered neocartilage

    PubMed Central

    Makris, Eleftherios A.; MacBarb, Regina F.; Responte, Donald J.; Hu, Jerry C.; Athanasiou, Kyriacos A.

    2013-01-01

    The objective of this study was to improve the biomechanical properties of engineered neotissues through promoting the development of collagen cross-links. It was hypothesized that supplementing medium with copper sulfate and the amino acid hydroxylysine would enhance the activity of lysyl oxidase enzyme to form collagen cross-links, increasing the strength and integrity of the neotissue. Neocartilage constructs were generated using a scaffoldless, self-assembling process and treated with copper sulfate and hydroxylysine, either alone or in combination, following a 2-factor, full-factorial study design. Following a 6-wk culture period, the biomechanical and biochemical properties of the constructs were measured. Results found copper sulfate to significantly increase pyridinoline (PYR) cross-links in all copper sulfate-containing groups over controls. When copper sulfate and hydroxylysine were combined, the result was synergistic, with a 10-fold increase in PYR content over controls. This increase in PYR cross-links manifested in a 3.3-fold significant increase in the tensile properties of the copper sulfate + hydroxylysine group. In addition, an 123% increase over control values was detected in the copper sulfate group in terms of the aggregate modulus. These data elucidate the role of copper sulfate and hydroxylysine toward improving the biomechanical properties of neotissues through collagen cross-linking enhancement.—Makris, E. A., MacBarb, R. F., Responte, D. J., Hu, J. C., Athanasiou, K. A. A copper sulfate and hydroxylysine treatment regimen for enhancing collagen cross-linking and biomechanical properties in engineered neocartilage. PMID:23457219

  13. Electrical and Neurotrophin Enhancement of Neurite Outgrowth within a 3D Collagen Scaffold

    PubMed Central

    Adams, Robert D.; Rendell, Sara R.; Counts, Lauren R.; Papke, Jason B.; Willits, Rebecca K.; Harkins, Amy B.

    2016-01-01

    Electrical and chemical stimulation have been studied as potent mechanisms of enhancing nerve regeneration and wound healing. However, it remains unclear how electrical stimuli affect nerve growth, particularly in the presence of neurotrophic factors. The objective of this study was to explore (1) the effect of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) supplementation to support neurite outgrowth in a 3D scaffold, and (2) the effect of brief, low voltage, electrical stimulation (ES) on neurite outgrowth prior to neurotrophin supplementation. Dissociated E11 chick dorsal root ganglia (DRG) were seeded within a 1.5 mg/mL type-I collagen scaffold. For neurotrophin treatments, scaffolds were incubated for 24 hrs in culture media containing nerve growth factor (NGF, 10 ng/mL) or BDNF (200 ng/mL), or both. For ES groups, scaffolds containing neurons were stimulated for 10 min at 8–10 V/m DC, then incubated for 24 hrs with neurotrophin. Fixed and labeled neurons were imaged to measure neurite growth and directionality. BDNF supplementation was not as effective as NGF at supporting DRG neurite outgrowth. ES prior to NGF supplementation improved DRG neurite outgrowth compared to NGF alone. This combination of brief ES with NGF treatment was the most effective treatment compared to NGF or BDNF alone. Brief ES had no impact on neurite directionality in the 3D scaffolds. These results demonstrate that ES improves neurite outgrowth in the presence of neurotrophins, and could provide a potential therapeutic approach to improve nerve regeneration when coupled with neurotrophin treatment. PMID:24710795

  14. Enhanced biomineralization in osteoblasts on a novel electrospun biocomposite nanofibrous substrate of hydroxyapatite/collagen/chitosan.

    PubMed

    Zhang, Yanzhong; Reddy, Venugopal Jayarama; Wong, Siew Yee; Li, Xu; Su, Bo; Ramakrishna, Seeram; Lim, Chwee Teck

    2010-06-01

    Electrospun chitosan (CTS)-based hydroxyapatite (HAp)/CTS biocomposite nanofibers for bone tissue engineering could afford a close biomimicry to the fibrous nanostructure and constituents of the hierarchically organized natural bone, but their biological performance is somewhat deficient compared with the HAp/collagen (Col) biocomposite system. This necessitates doping the electrospun HAp/CTS hybrid with the bioactive component of Col. We show herein that Col-doped HAp/CTS biocomposite (i.e., HAp/Col/CTS) containing 27.8 wt% HAp nanoparticles, 7.2 wt% Col, and 57.8 wt% CTS can be successfully electrospun into nanofibrous form through using small amount (7.2 wt%) of ultrahigh-molecular-weight poly(ethylene oxide) as the fiber-forming additive. Morphology, structure, composition, and mechanical properties of the electrospun HAp/Col/CTS scaffolds were examined by field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy, and tensile tests, respectively. Human fetal osteoblasts on the nanofibrous HAp/Col/CTS scaffolds were cultured for up to 15 days to assess the cell-scaffold interaction and biomineralization effect. In comparison with different controls, significant increments in osteoblast proliferation, alkaline phosphatase expression, and mineral deposition were observed. Results obtained thus highlight that introduction of Col can significantly enhance the biological performance of osteoblasts on the CTS-based nanofibrous substrates and suggest that current electrospun HAp/Col/CTS biocomposite, as a highly biomimetic and bioactive nanofibrous structure, may be one of the most attractive candidates for various osteoregeneration-related applications. PMID:20088700

  15. Calcium alginate enhances wound healing by up-regulating the ratio of collagen types I/III in diabetic rats.

    PubMed

    Wang, Tao; Gu, Qisheng; Zhao, Jun; Mei, Jiacai; Shao, Mingzhe; Pan, Ye; Zhang, Jian; Wu, Haisheng; Zhang, Zhen; Liu, Fang

    2015-01-01

    Calcium alginate has been proved to favor the skin ulcer healing and collagen synthesis was a critical factor for the wound closure. The present study was to elucidate the mechanism of calcium alginate on the diabetes skin ulceration. Calcium alginate dressing was applied daily on the full-thickness exercising wound created on the back of diabetic rat model as Alg-group (n=6), and the vaseline dressing was used as control (n=6). Rats were respectively sacrificed and the wound tissues were removed and used for the evaluation of various biochemical analysis contained collagen (type I and III) by Western blotting and hydroxyproline level changes by ELISA assay at 3 d, 7 d and 14 d after wounding. The expression of skin collagen I in Alg-group was enhanced from day 3 (0.66 ± 0.25 vs. 0.42 ± 0.09, P<0.05) to day 14 (1.09 ± 0.14 vs. 0.78 ± 0.16, P<0.05). However, no significant difference of collagen III expression was found between two groups during wound healing (P>0.05). And the ratio of collagen I/III in Alg-group was greater than that of Vas-group at day 7 (1.07 ± 0.31 vs. 0.77 ± 0.11, P<0.05) and 14 (1.18 ± 0.30 vs. 0.83 ± 0.14, P<0.05). The hydroxyproline level in skin homogenate of Alg-group was higher than that of Vas-group from day 3 (30.29 ± 0.92 ng/ml vs. 27.52 ± 0.83 ng/ml, P<0.05) to day 14 (89.58 ± 4.97 ng/ml vs. 79.30 ± 4.42 ng/ml, P<0.05). Calcium alginate accelerates the process of wound healing through improving type I collagen synthesis and increasing ratio of collagen I/III in diabetic rats.

  16. Calcium alginate enhances wound healing by up-regulating the ratio of collagen types I/III in diabetic rats

    PubMed Central

    Wang, Tao; Gu, Qisheng; Zhao, Jun; Mei, Jiacai; Shao, Mingzhe; Pan, Ye; Zhang, Jian; Wu, Haisheng; Zhang, Zhen; Liu, Fang

    2015-01-01

    Calcium alginate has been proved to favor the skin ulcer healing and collagen synthesis was a critical factor for the wound closure. The present study was to elucidate the mechanism of calcium alginate on the diabetes skin ulceration. Calcium alginate dressing was applied daily on the full-thickness exercising wound created on the back of diabetic rat model as Alg-group (n=6), and the vaseline dressing was used as control (n=6). Rats were respectively sacrificed and the wound tissues were removed and used for the evaluation of various biochemical analysis contained collagen (type I and III) by Western blotting and hydroxyproline level changes by ELISA assay at 3 d, 7 d and 14 d after wounding. The expression of skin collagen I in Alg-group was enhanced from day 3 (0.66±0.25 vs. 0.42±0.09, P<0.05) to day 14 (1.09±0.14 vs. 0.78±0.16, P<0.05). However, no significant difference of collagen III expression was found between two groups during wound healing (P>0.05). And the ratio of collagen I/III in Alg-group was greater than that of Vas-group at day 7 (1.07±0.31 vs. 0.77±0.11, P<0.05) and 14 (1.18±0.30 vs. 0.83±0.14, P<0.05). The hydroxyproline level in skin homogenate of Alg-group was higher than that of Vas-group from day 3 (30.29±0.92 ng/ml vs. 27.52±0.83 ng/ml, P<0.05) to day 14 (89.58±4.97 ng/ml vs. 79.30±4.42 ng/ml, P<0.05). Calcium alginate accelerates the process of wound healing through improving type I collagen synthesis and increasing ratio of collagen I/III in diabetic rats. PMID:26261545

  17. Polymorphism of amyloid fibrils formed by a peptide from the yeast prion protein Sup35: AFM and Tip-Enhanced Raman Scattering studies.

    PubMed

    Krasnoslobodtsev, Alexey V; Deckert-Gaudig, Tanja; Zhang, Yuliang; Deckert, Volker; Lyubchenko, Yuri L

    2016-06-01

    Aggregation of prion proteins is the cause of various prion related diseases. The infectious form of prions, amyloid aggregates, exist as multiple strains. The strains are thought to represent structurally different prion protein molecules packed into amyloid aggregates, but the knowledge on the structure of different types of aggregates is limited. Here we report on the use of AFM (Atomic Force Microscopy) and TERS (Tip-Enhanced Raman Scattering) to study morphological heterogeneity and access underlying conformational features of individual amyloid aggregates. Using AFM we identified the morphology of amyloid fibrils formed by the peptide (CGNNQQNY) from the yeast prion protein Sup35 that is critically involved in the aggregation of the full protein. TERS results demonstrate that morphologically different amyloid fibrils are composed of a distinct set of conformations. Fibrils formed at pH 5.6 are composed of a mixture of peptide conformations (β-sheets, random coil and α-helix) while fibrils formed in pH~2 solution primarily have β-sheets. Additionally, peak positions in the amide III region of the TERS spectra suggested that peptides have parallel arrangement of β-sheets for pH~2 fibrils and antiparallel arrangement for fibrils formed at pH 5.6. We also developed a methodology for detailed analysis of the peptide secondary structure by correlating intensity changes of Raman bands in different regions of TERS spectra. Such correlation established that structural composition of peptides is highly localized with large contribution of unordered secondary structures on a fibrillar surface. PMID:27060278

  18. 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

  19. 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.

  20. Administration of adipose-derived stem cells enhances vascularity, induces collagen deposition, and dermal adipogenesis in burn wounds.

    PubMed

    Bliley, Jacqueline M; Argenta, Anne; Satish, Latha; McLaughlin, Meghan M; Dees, Aaron; Tompkins-Rhoades, Casey; Marra, Kacey G; Rubin, J Peter

    2016-09-01

    Current treatment options for severe burn wounds are often insufficient in reconstructing skin and soft tissue defects. Adipose-derived stem cells (ASCs), a readily available source of multipotent stem cells, represent a promising therapy for the treatment of full-thickness burn wounds. Full-thickness burn wounds were created on the paraspinal region of athymic mice. A one-time, sub-eschar injection of 6.8×10(6) ASCs in PBS or PBS alone was administered at 24-h postoperatively. Time to healing was quantified using Image J analysis. At days 4, 7, 14, and 21, mice were sacrificed and tissues were excised for molecular and histological analysis. ASCs were able to survive in burn wounds as determined by the presence of PKH labeling and human PPARγ expression within the wounds. CD-31 staining demonstrated increased vascularity in ASC-treated wounds at POD 4 (p<0.05). Molecular studies showed enhanced adipogenesis, as well as type III and type I collagen deposition in the ASC treated group (p<0.05). An increase in the mRNA expression ratio of type III to type I collagen was also observed following ASC treatment (p<0.05). By enhancing vascularity, collagen deposition, and adipogenesis, ASCs show promise as an adjunctive therapy for the current treatment of full thickness burn wounds.

  1. Enhanced bioavailability of subcutaneously injected insulin coadministered with collagen in rats and humans

    SciTech Connect

    Hori, R.; Komada, F.; Iwakawa, S.; Seino, Y.; Okumura, K. )

    1989-09-01

    The present study was undertaken to develop an agent that stabilizes insulin injected subcutaneously. {sup 125}I-Porcine insulin with 0.2 U/kg unlabeled porcine insulin was subcutaneously injected with or without collagen in the rat under the depilated skin of the back. At various times, the radioactivity in subcutaneous tissue was assayed for insulin and its metabolites by gel filtration. The degradation and absorption rate constants of insulin at the subcutaneous injection site were estimated according to a one-compartment model. The degradation rate constant of insulin in the presence of collagen at the injection site was less than half of the control rate. The inhibition was confirmed by increases in the immunoreactive insulin plasma levels and the hypoglycemic effect in rats and healthy volunteers. We postulate that collagen prevents insulin from being degraded by inhibiting proteolytic enzymes, mainly collagenase-like peptidase, in subcutaneous tissue.

  2. 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.

  3. 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.

  4. 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

  5. Traumatic Acid Reduces Oxidative Stress and Enhances Collagen Biosynthesis in Cultured Human Skin Fibroblasts.

    PubMed

    Jabłońska-Trypuć, Agata; Pankiewicz, Walentyn; Czerpak, Romuald

    2016-09-01

    Traumatic acid (TA) is a plant hormone (cytokinin) that in terms of chemical structure belongs to the group of fatty acids derivatives. It was isolated from Phaseolus vulgaris. TA activity and its influence on human cells and organism has not previously been the subject of research. The aim of this study was to examine the effects of TA on collagen content and basic oxidative stress parameters, such as antioxidative enzyme activity, reduced glutathione, thiol group content, and lipid peroxidation in physiological conditions. The results show a stimulatory effect of TA on tested parameters. TA caused a decrease in membrane phospholipid peroxidation and exhibited protective properties against ROS production. It also increases protein and collagen biosynthesis and its secretion into the culture medium. The present findings reveal that TA exhibits multiple and complex activity in fibroblast cells in vitro. TA, with its activity similar to unsaturated fatty acids, shows antioxidant and stimulatory effects on collagen biosynthesis. It is a potentially powerful agent with applications in the treatment of many skin diseases connected with oxidative stress and collagen biosynthesis disorders. PMID:27423205

  6. Contrast enhancement in second harmonic imaging: discriminating between muscle and collagen

    NASA Astrophysics Data System (ADS)

    Psilodimitrakopoulos, Sotiris; Artigas, David; Soria, Guadalupe; Amat-Roldan, Ivan; Torre, Iratxe; Gratacos, Eduard; Planas, Anna M.; Loza-Alvarez, Pablo

    2009-07-01

    In this study, polarization second harmonic generation (SHG) imaging is used and data analysis is developed to gain contrast and to discriminate with pixel resolution, in the same image, SHG source architectures. We use mammalian tissue in which both skeletal muscle and fibrilar collagen can be found. The images are fitted point by point using an algorithm based on a biophysical model, where the coefficient of determination is utilized as a filtering mechanism. For the whole image we retrieve for every pixel, the effective orientation, θe , of the SHG active structures. As a result a new image is formed which its contrast depends on the values of θe . Collagen presented in the forward direction for a predefined region of interest (ROI), peak distribution of angles θe centered in the region of ~45°, while muscle in the region of ~65°. Consequently, collagen and muscle are represented in different colors in the same image. Thus, here we show that it is possible to gain contrast and to discriminate between collagen and muscle without the use of any exogenous labeling or any co-localization with fluorescence imaging.

  7. Vegetable peptones increase production of type I collagen in human fibroblasts by inducing the RSK-CCAAT/enhancer binding protein-β phosphorylation pathway.

    PubMed

    Jung, Eunsun; Cho, Jae Youl; Park, Deokhoon; Kim, Min Hee; Park, Beomseok; Lee, Sang Yeol; Lee, Jongsung

    2015-02-01

    Skin aging appears to be principally attributed to a decrease in type I collagen level and the regeneration ability of dermal fibroblasts. We hypothesized that vegetable peptones promote cell proliferation and production of type I collagen in human dermal fibroblasts. Therefore, we investigated the effects of vegetable peptones on cell proliferation and type I collagen production and their possible mechanisms in human dermal fibroblasts. Vegetable peptones significantly promoted cell proliferation in a concentration-dependent manner. In addition, the human luciferase type I collagen α2 promoter and type I procollagen synthesis assays showed that the vegetable peptones induced type I procollagen production by activating the type I collagen α2 promoter. Moreover, the vegetable peptones activated p90 ribosomal s6 kinase, which was mediated by activating the Raf-p44/42 mitogen-activated protein kinase signaling pathway. Furthermore, the vegetable peptone-induced increase in cell proliferation and type I collagen production decreased upon treatment with the ERK inhibitor PD98059. Taken together, these findings suggest that increased proliferation of human dermal fibroblasts and enhanced production of type I collagen by vegetable peptones occur primarily by inducing the p90 ribosomal s6 kinase-CCAAT/enhancer binding protein β phosphorylation pathway, which is mediated by activating Raf-ERK signaling.

  8. 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 ...

  9. 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 ...

  10. Transdermal nicotine patch enhances type I collagen synthesis in abstinent smokers.

    PubMed

    Sørensen, Lars T; Jorgensen, Lars N; Zillmer, Rikke; Vange, Jakob; Hemmingsen, Ulla; Gottrup, Finn

    2006-01-01

    Cigarette smokers deposit less collagen, expressed as hydroxyproline, in granulation tissue than nonsmokers. We studied the effect of abstinence from smoking and transdermal nicotine patches on deposition of hydroxyproline, proline, type I procollagen, and total proteins. Fifty-four healthy smokers were studied during 10 days of smoking and again from days 10 to 20 following smoking cessation. After the first 10 days of abstinence they were randomized to double-blind treatment with transdermal nicotine patches of 25 mg/day or placebo for a period of 10 days. During this period and during smoking, an expanded polytetrafluoroethylene tube was implanted into the subcutis. Following removal of the implant, total amino acids and peptides were extracted. Hydroxyproline and proline were analyzed by high-pressure liquid chromatography, type I procollagen was analyzed by enzyme-linked immunoassay, and total proteins were determined colorimetrically. In the 39 subjects who complied with the study protocol, abstinence from smoking did not affect the deposition of hydroxyproline, proline, type I procollagen, or total protein in the implants. During abstinence, the type I procollagen level increased by 18% in the transdermal nicotine patches group and decreased by 10% in the placebo group (p<0.05). We conclude that 20 days of abstinence from smoking does not affect collagen deposition in granulation tissue. However, in abstinent smokers, transdermal nicotine patches appears to increase type I collagen synthesis.

  11. SEVI, the semen enhancer of HIV infection along with fragments from its central region, form amyloid fibrils that are toxic to neuronal cells.

    PubMed

    Elias, Abigail K; Scanlon, Denis; Musgrave, Ian F; Carver, John A

    2014-09-01

    Semen-derived enhancer of viral infection (SEVI) is the term given to the amyloid fibrils formed by a 39-amino acid fragment (PAP248-286) of prostatic acidic phosphatase (PAP) found in human semen. SEVI enhances human immunodeficiency virus (HIV) infectivity by four to five orders of magnitude (Münch et al., 2007). Here, we show by various biophysical techniques including Thioflavin T fluorescence, circular dichroism spectroscopy and transmission electron microscopy that fragments encompassing the central region of SEVI, i.e. PAP248-271 and PAP257-267, form fibrils of similar morphology to SEVI. Our results show that the central region, residues PAP267-271, is crucially important in promoting SEVI fibril formation. Furthermore, SEVI and fibrillar forms of these peptide fragments are toxic to neuronal pheochromocytoma 12 cells but not to epithelial colon carcinoma cells. These findings imply that although SEVI assists in the attachment of HIV-1 to immune cells, it may not facilitate HIV entry by damaging the epithelial cell layer that presents a barrier to the HIV.

  12. Development and characterization of a rapid polymerizing collagen for soft tissue augmentation

    PubMed Central

    Devore, Dale; Zhu, Jiaxun; Brooks, Robert; McCrate, Rebecca Rone; Grant, David A.

    2015-01-01

    Abstract A liquid collagen has been developed that fibrilizes upon injection. Rapid polymerizing collagen (RPC) is a type I porcine collagen that undergoes fibrillization upon interaction with ionic solutions, such as physiological solutions. The ability to inject liquid collagen would be beneficial for many soft tissue augmentation applications. In this study, RPC was synthesized and characterized as a possible dermal filler. Transmission electron microscopy, ion induced RPC fibrillogenesis tests, collagenase resistance assay, and injection force studies were performed to assess RPC's physicochemical properties. An in vivo study was performed which consisted of a 1‐, 3‐, and 6‐month study where RPC was injected into the ears of miniature swine. The results demonstrated that the liquid RPC requires low injection force (<7 N); fibrillogenesis and banding of collagen occurs when RPC is injected into ionic solutions, and RPC has enhanced resistance to collagenase breakdown. The in vivo study demonstrated long‐term biocompatibility with low irritation scores. In conclusion RPC possesses many of the desirable properties of a soft tissue augmentation material. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 758–767, 2016. PMID:26488368

  13. Enhancing cell migration in shape-memory alginate-collagen composite scaffolds: In vitro and ex vivo assessment for intervertebral disc repair.

    PubMed

    Guillaume, Olivier; Naqvi, Syeda Masooma; Lennon, Kerri; Buckley, Conor Timothy

    2015-04-01

    weeks. Taken together, these findings illustrate the advantages of incorporating collagen as a means to enhance cell migration and proliferation in porous scaffolds which could be used to augment tissue repair strategies.

  14. Prognostic Implications of Left Ventricular Scar Determined by Late Gadolinium Enhanced Cardiac Magnetic Resonance in Patients With Atrial Fibrillation.

    PubMed

    Suksaranjit, Promporn; McGann, Christopher J; Akoum, Nazem; Biskupiak, Joseph; Stoddard, Gregory J; Kholmovski, Eugene G; Navaravong, Leenhapong; Rassa, Allen; Bieging, Erik; Chang, Lowell; Haider, Imran; Marrouche, Nassir F; Wilson, Brent D

    2016-10-01

    Left ventricular (LV) scar identified by late gadolinium enhanced (LGE) cardiac magnetic resonance (CMR) is associated with adverse outcomes in coronary artery disease and cardiomyopathies. We sought to determine the prognostic significance of LV-LGE in atrial fibrillation (AF). We studied 778 consecutive patients referred for radiofrequency ablation of AF who underwent CMR. Patients with coronary artery disease, previous myocardial infarction, or hypertrophic or dilated cardiomyopathy were excluded. The end points of interest were major adverse cardiac and cerebrovascular events (MACCE), defined as a composite of cardiovascular death, myocardial infarction, and ischemic stroke/transient ischemic attack. Of the 754 patients who met the inclusion criteria, 60% were men with an average age of 64 years. Most (87%) had a normal LV ejection fraction of ≥55%. LV-LGE was found in 46 patients (6%). There were 32 MACCE over the mean follow-up period of 55 months. The MACCE rate was higher for patients with LV-LGE (13.0% vs 3.7%; p = 0.002). In multivariate analysis, CHA2DS2-VASc score (hazard ratio [HR] 1.36, 95% CI 1.05 to 1.76), the presence of LV-LGE (HR 3.21, 95% CI 1.31 to 7.88), and the LV-LGE extent (HR 1.43, 95% CI 1.15 to 1.78) were independent predictors of MACCE. In addition, the presence of LV-LGE was an independent predictor for ischemic stroke/transient ischemic attack (HR 3.61, 95% CI 1.18 to 11.01) after adjusting for CHA2DS2-VASc score. In conclusion, the presence and extent of LV scar identified by LGE-CMR were independent predictors of MACCE in patients with AF. PMID:27553101

  15. 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.

  16. 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.

  17. 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

  18. Royal jelly protects against ultraviolet B-induced photoaging in human skin fibroblasts via enhancing collagen production.

    PubMed

    Park, Hye Min; Hwang, Eunson; Lee, Kwang Gill; Han, Sang-Mi; Cho, Yunhi; Kim, Sun Yeou

    2011-09-01

    Royal jelly (RJ) is a honeybee product containing proteins, carbohydrates, fats, free amino acids, vitamins, and minerals. As its principal unsaturated fatty acid, RJ contains 10-hydroxy-2-decenoic acid (10-HDA), which may have antitumor and antibacterial activity and a capacity to stimulate collagen production. RJ has attracted interest in various parts of the world for its pharmacological properties. However, the effects of RJ on ultraviolet (UV)-induced photoaging of the skin have not been reported. In this study we measured the 10-HDA content of RJ by high-performance liquid chromatography and tested the effects of RJ on UVB-induced skin photoaging in normal human dermal fibroblasts. The effects of RJ and 10-HDA on UVB-induced photoaging were tested by measuring procollagen type I, transforming growth factor (TGF)-β1, and matrix metalloproteinase (MMP)-1 after UVB irradiation. The RJ contained about 0.211% 10-HDA. The UVB-irradiated human skin fibroblasts treated with RJ and 10-HDA had increased procollagen type I and TGF-β1 productions, but the level of MMP-1 was not changed. Thus RJ may potentially protect the skin from UVB-induced photoaging by enhancing collagen production.

  19. 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.

  20. Multimodal Spectroscopic Study of Amyloid Fibril Polymorphism.

    PubMed

    VandenAkker, Corianne C; Schleeger, Michael; Bruinen, Anne L; Deckert-Gaudig, Tanja; Velikov, Krassimir P; Heeren, Ron M A; Deckert, Volker; Bonn, Mischa; Koenderink, Gijsje H

    2016-09-01

    Amyloid fibrils are a large class of self-assembled protein aggregates that are formed from unstructured peptides and unfolded proteins. The fibrils are characterized by a universal β-sheet core stabilized by hydrogen bonds, but the molecular structure of the peptide subunits exposed on the fibril surface is variable. Here we show that multimodal spectroscopy using a range of bulk- and surface-sensitive techniques provides a powerful way to dissect variations in the molecular structure of polymorphic amyloid fibrils. As a model system, we use fibrils formed by the milk protein β-lactoglobulin, whose morphology can be tuned by varying the protein concentration during formation. We investigate the differences in the molecular structure and composition between long, straight fibrils versus short, wormlike fibrils. We show using mass spectrometry that the peptide composition of the two fibril types is similar. The overall molecular structure of the fibrils probed with various bulk-sensitive spectroscopic techniques shows a dominant contribution of the β-sheet core but no difference in structure between straight and wormlike fibrils. However, when probing specifically the surface of the fibrils with nanometer resolution using tip-enhanced Raman spectroscopy (TERS), we find that both fibril types exhibit a heterogeneous surface structure with mainly unordered or α-helical structures and that the surface of long, straight fibrils contains markedly more β-sheet structure than the surface of short, wormlike fibrils. This finding is consistent with previous surface-specific vibrational sum-frequency generation (VSFG) spectroscopic results ( VandenAkker et al. J. Am. Chem. Soc. , 2011 , 133 , 18030 - 18033 , DOI: 10.1021/ja206513r ). In conclusion, only advanced vibrational spectroscopic techniques sensitive to surface structure such as TERS and VSFG are able to reveal the difference in structure that underlies the distinct morphology and rigidity of different amyloid

  1. A three-dimensional hierarchical collagen scaffold fabricated by a combined solid freeform fabrication (SFF) and electrospinning process to enhance mesenchymal stem cell (MSC) proliferation

    NASA Astrophysics Data System (ADS)

    Ahn, SeungHyun; Koh, Young Ho; Kim, GeunHyung

    2010-06-01

    Collagen has the advantage of being very similar to macromolecular substances that can be recognized and metabolized in the biological environment. Although the natural material has superior property for this purpose, its use to fabricate reproducible and pore-structure-controlled 3D structures, which are designed to allow the entry of sufficient cells and the easy diffusion of nutrients, has been limited due to its low processability. Here, we propose a hybrid technology that combines a cryogenic plotting system with an electrospinning process. Using this technique, an easily pore-size-controllable hierarchical 3D scaffold consisting of micro-sized highly porous collagen strands and micro/nano-sized collagen fibers was fabricated. The pore structure of the collagen scaffold was controlled by the collagen micro/nanofibers, which were layered in the scaffold. The hierarchical scaffolds were characterized with respect to initial cell attachment and proliferation of bone marrow-derived mesenchymal stem cells within the scaffolds. The hierarchical scaffold exhibited incredibly enhanced initial cell attachment and cell compactness between pores of the plotted scaffold relative to the normally designed 3D collagen scaffold.

  2. Comparative effects of biodynes, tocotrienol-rich fraction, and tocopherol in enhancing collagen synthesis and inhibiting collagen degradation in stress-induced premature senescence model of human diploid fibroblasts.

    PubMed

    Makpol, Suzana; Jam, Faidruz Azura; Khor, Shy Cian; Ismail, Zahariah; Mohd Yusof, Yasmin Anum; Ngah, Wan Zurinah Wan

    2013-01-01

    Biodynes, tocotrienol-rich fraction (TRF), and tocopherol have shown antiaging properties. However, the combined effects of these compounds on skin aging are yet to be investigated. This study aimed to elucidate the skin aging effects of biodynes, TRF, and tocopherol on stress-induced premature senescence (SIPS) model of human diploid fibroblasts (HDFs) by determining the expression of collagen and MMPs at gene and protein levels. Primary HDFs were treated with biodynes, TRF, and tocopherol prior to hydrogen peroxide (H2O2) exposure. The expression of COL1A1, COL3A1, MMP1, MMP2, MMP3, and MMP9 genes was determined by qRT-PCR. Type I and type III procollagen proteins were measured by Western blotting while the activities of MMPs were quantified by fluorometric Sensolyte MMP Kit. Our results showed that biodynes, TRF, and tocopherol upregulated collagen genes and downregulated MMP genes (P < 0.05). Type I procollagen and type III procollagen protein levels were significantly increased in response to biodynes, TRF, and tocopherol treatment (P < 0.05) with reduction in MMP-1, MMP-2, MMP-3, and MMP-9 activities (P < 0.05). These findings indicated that biodynes, TRF, and tocopherol effectively enhanced collagen synthesis and inhibited collagen degradation and therefore may protect the skin from aging.

  3. Comparative effects of biodynes, tocotrienol-rich fraction, and tocopherol in enhancing collagen synthesis and inhibiting collagen degradation in stress-induced premature senescence model of human diploid fibroblasts.

    PubMed

    Makpol, Suzana; Jam, Faidruz Azura; Khor, Shy Cian; Ismail, Zahariah; Mohd Yusof, Yasmin Anum; Ngah, Wan Zurinah Wan

    2013-01-01

    Biodynes, tocotrienol-rich fraction (TRF), and tocopherol have shown antiaging properties. However, the combined effects of these compounds on skin aging are yet to be investigated. This study aimed to elucidate the skin aging effects of biodynes, TRF, and tocopherol on stress-induced premature senescence (SIPS) model of human diploid fibroblasts (HDFs) by determining the expression of collagen and MMPs at gene and protein levels. Primary HDFs were treated with biodynes, TRF, and tocopherol prior to hydrogen peroxide (H2O2) exposure. The expression of COL1A1, COL3A1, MMP1, MMP2, MMP3, and MMP9 genes was determined by qRT-PCR. Type I and type III procollagen proteins were measured by Western blotting while the activities of MMPs were quantified by fluorometric Sensolyte MMP Kit. Our results showed that biodynes, TRF, and tocopherol upregulated collagen genes and downregulated MMP genes (P < 0.05). Type I procollagen and type III procollagen protein levels were significantly increased in response to biodynes, TRF, and tocopherol treatment (P < 0.05) with reduction in MMP-1, MMP-2, MMP-3, and MMP-9 activities (P < 0.05). These findings indicated that biodynes, TRF, and tocopherol effectively enhanced collagen synthesis and inhibited collagen degradation and therefore may protect the skin from aging. PMID:24396567

  4. Comparative Effects of Biodynes, Tocotrienol-Rich Fraction, and Tocopherol in Enhancing Collagen Synthesis and Inhibiting Collagen Degradation in Stress-Induced Premature Senescence Model of Human Diploid Fibroblasts

    PubMed Central

    Jam, Faidruz Azura; Ismail, Zahariah; Wan Ngah, Wan Zurinah

    2013-01-01

    Biodynes, tocotrienol-rich fraction (TRF), and tocopherol have shown antiaging properties. However, the combined effects of these compounds on skin aging are yet to be investigated. This study aimed to elucidate the skin aging effects of biodynes, TRF, and tocopherol on stress-induced premature senescence (SIPS) model of human diploid fibroblasts (HDFs) by determining the expression of collagen and MMPs at gene and protein levels. Primary HDFs were treated with biodynes, TRF, and tocopherol prior to hydrogen peroxide (H2O2) exposure. The expression of COL1A1, COL3A1, MMP1, MMP2, MMP3, and MMP9 genes was determined by qRT-PCR. Type I and type III procollagen proteins were measured by Western blotting while the activities of MMPs were quantified by fluorometric Sensolyte MMP Kit. Our results showed that biodynes, TRF, and tocopherol upregulated collagen genes and downregulated MMP genes (P < 0.05). Type I procollagen and type III procollagen protein levels were significantly increased in response to biodynes, TRF, and tocopherol treatment (P < 0.05) with reduction in MMP-1, MMP-2, MMP-3, and MMP-9 activities (P < 0.05). These findings indicated that biodynes, TRF, and tocopherol effectively enhanced collagen synthesis and inhibited collagen degradation and therefore may protect the skin from aging. PMID:24396567

  5. Chitosan-collagen biomembrane embedded with calcium-aluminate enhances dentinogenic potential of pulp cells.

    PubMed

    Soares, Diana Gabriela; Rosseto, Hebert Luís; Basso, Fernanda Gonçalves; Scheffel, Débora Salles; Hebling, Josimeri; Costa, Carlos Alberto de Souza

    2016-01-01

    The development of biomaterials capable of driving dental pulp stem cell differentiation into odontoblast-like cells able to secrete reparative dentin is the goal of current conservative dentistry. In the present investigation, a biomembrane (BM) composed of a chitosan/collagen matrix embedded with calcium-aluminate microparticles was tested. The BM was produced by mixing collagen gel with a chitosan solution (2:1), and then adding bioactive calcium-aluminate cement as the mineral phase. An inert material (polystyrene) was used as the negative control. Human dental pulp cells were seeded onto the surface of certain materials, and the cytocompatibility was evaluated by cell proliferation and cell morphology, assessed after 1, 7, 14 and 28 days in culture. The odontoblastic differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, total protein production, gene expression of DMP-1/DSPP and mineralized nodule deposition. The pulp cells were able to attach onto the BM surface and spread, displaying a faster proliferative rate at initial periods than that of the control cells. The BM also acted on the cells to induce more intense ALP activity, protein production at 14 days, and higher gene expression of DSPP and DMP-1 at 28 days, leading to the deposition of about five times more mineralized matrix than the cells in the control group. Therefore, the experimental biomembrane induced the differentiation of pulp cells into odontoblast-like cells featuring a highly secretory phenotype. This innovative bioactive material can drive other protocols for dental pulp exposure treatment by inducing the regeneration of dentin tissue mediated by resident cells. PMID:27119587

  6. Maternal overnutrition enhances mRNA expression of adipogenic markers and collagen deposition in skeletal muscle of beef cattle fetuses.

    PubMed

    Duarte, M S; Gionbelli, M P; Paulino, P V R; Serão, N V L; Nascimento, C S; Botelho, M E; Martins, T S; Filho, S C V; Dodson, M V; Guimarães, S E F; Du, M

    2014-09-01

    Twenty-four pregnant Nellore cows were randomly assigned into 2 feeding level groups (control [CTL]; fed 1.0 times the maintenance requirement; n = 12; and overnourished [ON]; fed at 1.5 times the maintenance requirement; n = 12) to evaluate effects of maternal overnutrition on fetal skeletal muscle development. Cows were slaughtered at 135, 190, and 240 d of gestation and samples of fetal LM were collected for analysis of mRNA expression analysis and for histological evaluation of collagen content and number of muscle cells. There was no interaction between gestational period and maternal nutrition for the variables evaluated (P > 0.05). The mRNA expression of Cadherin-associated protein, β 1 (β-catenin) tended to be greater in fetuses from ON cows (P = 0.08), while myogenic differentiation 1 (MyoD; P = 0.56), myogenin (MyoG; P = 0.70), and the number of muscle cells (P = 0.90) were not affected by maternal overnutrition. Gestational period did not affect the mRNA expression of β-catenin (P = 0.60) and MyoG (P = 0.21). The mRNA expression of MyoD tended to increase with days of gestation (P = 0.06). The mRNA expression of zinc finger protein 423 (Zfp423; P < 0.0001), C/EBPα (P = 0.01), and PPARγ (P < 0.0001) were enhanced in ON fetuses. No effects of days of gestation were observed for mRNA expression of Zfp423 (P = 0.75) and C/EBPα (P = 0.48). The mRNA expression of PPARγ in fetuses at 190 d of gestation tended to be greater than those at 135 and 240 d of gestation (P = 0.06). The mRNA expression of transforming growth factor β (TGF-β; P < 0.0001), collagen type III, α I (COL3A1; P < 0.0001), and collagen content (P = 0.01) were increased in ON fetuses. Gestational period did not affect the mRNA expression of collagen type I, α I (COL1A1; P = 0.65). The mRNA expression of COL3A1 (P = 0.09) in fetuses at 190 d of gestation tended to be greater than fetuses at 135 and 240 d of gestation. The mRNA expression of TGF-β in fetuses at 190 d of gestation was

  7. TGF-beta enhances the integrin alpha2beta1-mediated attachment of mesenchymal stem cells to type I collagen.

    PubMed

    Warstat, Katrin; Meckbach, Diana; Weis-Klemm, Michaela; Hack, Anita; Klein, Gerd; de Zwart, Peter; Aicher, Wilhelm K

    2010-05-01

    The heterodimeric integrins are important receptors for the attachment of cells to their extracellular matrix. Here, we studied the attachment of human mesenchymal stem cells (MSCs) to type I collagen (col-1), which is part of the extracellular matrix in bone, skin, and connective tissues. Furthermore, we examined how TGF-beta influences the integrin expression and attachment of MSC. Using flow cytometry, immunoblot, and RT-PCR, we report that MSC express several integrin subunits, including the alpha(2)beta(1) integrin (VLA-2, CD49b/CD29). TGF-beta increases the expression of integrin subunits alpha(2), alpha(6), and beta(1) in MSC, thereby enhancing the attachment of MSC to col-1. The TGF-beta-mediated up-regulation of the expression of the integrin subunits alpha(2) and alpha(6) is mainly mediated in MSC by Smad2.

  8. 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.

  9. Modulating the Rigidity and Mineralization of Collagen Gels Using Poly(Lactic-Co-Glycolic Acid) Microparticles

    PubMed Central

    DeVolder, Ross J.; Kim, Il Won; Kim, Eun-Suk

    2012-01-01

    Extensive efforts have been made to prepare osteoconductive collagen gels for the regeneration of normal bone and the pathological examination of diseased bone; however, collagen gels are often plagued by limited controllability of their rigidity and mineral deposition. This study reports a simple but efficient strategy that tunes the mechanical properties of, and apatite formation in, collagen gels by incorporating hydrolyzable poly(lactic-co-glycolic acid) (PLGA) microparticles within the gels. The PLGA microparticles are associated with the collagen fibrils and increased both the gel's elasticity and rigidity while minimally influencing its permeability. As compared with pure collagen gels, the PLGA microparticle-filled collagen gels, termed PLGA-Col hydrogels, significantly enhanced the deposition of apatite-like minerals within the gels when incubated in simulated body fluid or encapsulated with mesenchymal stem cells (MSCs) undergoing osteogenic differentiation. Finally, PLGA-Col hydrogels mineralized by differentiated MSCs led to an enhanced formation of bone-like tissues within the hydrogels. Overall, the PLGA-Col hydrogel system developed in this study will serve to improve the quality of osteoconductive matrices for both fundamental and clinical studies that are relevant to bone repair, regeneration, and pathogenesis. PMID:22480235

  10. 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.

  11. 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

  12. 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

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

    PubMed

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

    2015-12-01

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

  14. 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.

  15. 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.

  16. Exacerbation of collagen induced arthritis by Fcγ receptor targeted collagen peptide due to enhanced inflammatory chemokine and cytokine production

    PubMed Central

    Szarka, Eszter; Neer, Zsuzsa; Balogh, Péter; Ádori, Monika; Angyal, Adrienn; Prechl, József; Kiss, Endre; Kövesdi, Dorottya; Sármay, Gabriella

    2012-01-01

    Antibodies specific for bovine type II collagen (CII) and Fcγ receptors play a major role in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). Our aim was to clarify the mechanism of immune complex-mediated inflammation and modulation of the disease. CII pre-immunized DBA/1 mice were intravenously boosted with extravidin coupled biotinylated monomeric CII-peptide epitope (ARGLTGRPGDA) and its complexes with biotinylated FcγRII/III specific single chain Fv (scFv) fragment. Disease scores were monitored, antibody titers and cytokines were determined by ELISA, and binding of complexes was detected by flow cytometry and immune histochemistry. Cytokine and chemokine secretion was monitored by protein profiler microarray. When intravenously administered into collagen-primed DBA/1 mice, both CII-peptide and its complex with 2.4G2 scFv significantly accelerated CIA and increased the severity of the disease, whereas the monomeric peptide and monomeric 2.4G2 scFv had no effect. FcγRII/III targeted CII-peptide complexes bound to marginal zone macrophages and dendritic cells, and significantly elevated the synthesis of peptide-specific IgG2a. Furthermore, CII-peptide containing complexes augmented the in vivo secretion of cytokines, including IL-10, IL-12, IL-17, IL-23, and chemokines (CXCL13, MIP-1, MIP-2). These data indicate that complexes formed by the CII-peptide epitope aggravate CIA by inducing the secretion of chemokines and the IL-12/23 family of pro-inflammatory cytokines. Taken together, these results suggest that the in vivo emerging immune complexes formed with autoantigen(s) may trigger the IL-12/23 dependent pathways, escalating the inflammation in RA. Thus blockade of these cytokines may be beneficial to downregulate immune complex-induced inflammation in autoimmune arthritis. PMID:22532778

  17. Exacerbation of collagen induced arthritis by Fcγ receptor targeted collagen peptide due to enhanced inflammatory chemokine and cytokine production.

    PubMed

    Szarka, Eszter; Neer, Zsuzsa; Balogh, Péter; Adori, Monika; Angyal, Adrienn; Prechl, József; Kiss, Endre; Kövesdi, Dorottya; Sármay, Gabriella

    2012-01-01

    Antibodies specific for bovine type II collagen (CII) and Fcγ receptors play a major role in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). Our aim was to clarify the mechanism of immune complex-mediated inflammation and modulation of the disease. CII pre-immunized DBA/1 mice were intravenously boosted with extravidin coupled biotinylated monomeric CII-peptide epitope (ARGLTGRPGDA) and its complexes with biotinylated FcγRII/III specific single chain Fv (scFv) fragment. Disease scores were monitored, antibody titers and cytokines were determined by ELISA, and binding of complexes was detected by flow cytometry and immune histochemistry. Cytokine and chemokine secretion was monitored by protein profiler microarray. When intravenously administered into collagen-primed DBA/1 mice, both CII-peptide and its complex with 2.4G2 scFv significantly accelerated CIA and increased the severity of the disease, whereas the monomeric peptide and monomeric 2.4G2 scFv had no effect. FcγRII/III targeted CII-peptide complexes bound to marginal zone macrophages and dendritic cells, and significantly elevated the synthesis of peptide-specific IgG2a. Furthermore, CII-peptide containing complexes augmented the in vivo secretion of cytokines, including IL-10, IL-12, IL-17, IL-23, and chemokines (CXCL13, MIP-1, MIP-2). These data indicate that complexes formed by the CII-peptide epitope aggravate CIA by inducing the secretion of chemokines and the IL-12/23 family of pro-inflammatory cytokines. Taken together, these results suggest that the in vivo emerging immune complexes formed with autoantigen(s) may trigger the IL-12/23 dependent pathways, escalating the inflammation in RA. Thus blockade of these cytokines may be beneficial to downregulate immune complex-induced inflammation in autoimmune arthritis. PMID:22532778

  18. 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

  19. 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

  20. Incorporation of the natural marine multi-mineral dietary supplement Aquamin enhances osteogenesis and improves the mechanical properties of a collagen-based bone graft substitute.

    PubMed

    Brennan, Orlaith; Stenson, Barry; Widaa, Amro; O'Gorman, Denise M; O'Brien, Fergal J

    2015-07-01

    Aquamin is a commercially-available supplement derived from the algae species Lithothamnion, which has proven osteogenic potential. By harnessing this potential and combining Aquamin with a collagen scaffold, with architecture and composition optimised for bone repair, the aim of this study was to develop a natural osteo-stimulative bone graft substitute. A fabrication process was developed to incorporate Aquamin into scaffolds to produce collagen-Aquamin (CollAqua) scaffolds at two different Aquamin concentrations, 100F or 500F (equivalent weight% of collagen or five times the weight of collagen respectively). CollAqua constructs had improved mechanical properties which were achieved without reducing the scaffold׳s permeability or porosity below the minimum level required for successful bone tissue engineering. The fabrication process produced a homogenous Aquamin distribution throughout the scaffold. Release kinetics revealed that in the first 12h, the entire Aquamin content was released from the 100F however, less than half of Aquamin in the 500F was released with the remainder released approximately 21 days later giving an initial burst release followed by a delayed release. Osteoblasts cultured on the CollAqua scaffolds showed improved osteogenesis as measured by alkaline phosphatase, osteopontin and osteocalcin expression. This was confirmed by increased mineralisation as determined by von Kossa and Alizarin red staining. In conclusion, a cell and growth factor free collagen-based bone graft substitute with enhanced mechanical properties has been developed. The addition of Aquamin to the collagen biomaterial significantly improved mineralisation by osteoblasts and results in a new product which may be capable of enhancing osteogenesis to facilitate bone repair in vivo.

  1. 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

  2. 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.

  3. 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.

  4. Enhancement of tendon-bone healing via the combination of biodegradable collagen-loaded nanofibrous membranes and a three-dimensional printed bone-anchoring bolt.

    PubMed

    Chou, Ying-Chao; Yeh, Wen-Lin; Chao, Chien-Lin; Hsu, Yung-Heng; Yu, Yi-Hsun; Chen, Jan-Kan; Liu, Shih-Jung

    2016-01-01

    A composite biodegradable polymeric model was developed to enhance tendon graft healing. This model included a biodegradable polylactide (PLA) bolt as the bone anchor and a poly(D,L-lactide-co-glycolide) (PLGA) nanofibrous membrane embedded with collagen as a biomimic patch to promote tendon-bone interface integration. Degradation rate and compressive strength of the PLA bolt were measured after immersion in a buffer solution for 3 months. In vitro biochemical characteristics and the nanofibrous matrix were assessed using a water contact angle analyzer, pH meter, and tetrazolium reduction assay. In vivo efficacies of PLGA/collagen nanofibers and PLA bolts for tendon-bone healing were investigated on a rabbit bone tunnel model with histological and tendon pullout tests. The PLGA/collagen-blended nanofibrous membrane was a hydrophilic, stable, and biocompatible scaffold. The PLA bolt was durable for tendon-bone anchoring. Histology showed adequate biocompatibility of the PLA bolt on a medial cortex with progressive bone ingrowth and without tissue overreaction. PLGA nanofibers within the bone tunnel also decreased the tunnel enlargement phenomenon and enhanced tendon-bone integration. Composite polymers of the PLA bolt and PLGA/collagen nanofibrous membrane can effectively promote outcomes of tendon reconstruction in a rabbit model. The composite biodegradable polymeric system may be useful in humans for tendon reconstruction. PMID:27601901

  5. Enhancement of tendon–bone healing via the combination of biodegradable collagen-loaded nanofibrous membranes and a three-dimensional printed bone-anchoring bolt

    PubMed Central

    Chou, Ying-Chao; Yeh, Wen-Lin; Chao, Chien-Lin; Hsu, Yung-Heng; Yu, Yi-Hsun; Chen, Jan-Kan; Liu, Shih-Jung

    2016-01-01

    A composite biodegradable polymeric model was developed to enhance tendon graft healing. This model included a biodegradable polylactide (PLA) bolt as the bone anchor and a poly(D,L-lactide-co-glycolide) (PLGA) nanofibrous membrane embedded with collagen as a biomimic patch to promote tendon–bone interface integration. Degradation rate and compressive strength of the PLA bolt were measured after immersion in a buffer solution for 3 months. In vitro biochemical characteristics and the nanofibrous matrix were assessed using a water contact angle analyzer, pH meter, and tetrazolium reduction assay. In vivo efficacies of PLGA/collagen nanofibers and PLA bolts for tendon–bone healing were investigated on a rabbit bone tunnel model with histological and tendon pullout tests. The PLGA/collagen-blended nanofibrous membrane was a hydrophilic, stable, and biocompatible scaffold. The PLA bolt was durable for tendon–bone anchoring. Histology showed adequate biocompatibility of the PLA bolt on a medial cortex with progressive bone ingrowth and without tissue overreaction. PLGA nanofibers within the bone tunnel also decreased the tunnel enlargement phenomenon and enhanced tendon–bone integration. Composite polymers of the PLA bolt and PLGA/collagen nanofibrous membrane can effectively promote outcomes of tendon reconstruction in a rabbit model. The composite biodegradable polymeric system may be useful in humans for tendon reconstruction.

  6. Enhancement of tendon–bone healing via the combination of biodegradable collagen-loaded nanofibrous membranes and a three-dimensional printed bone-anchoring bolt

    PubMed Central

    Chou, Ying-Chao; Yeh, Wen-Lin; Chao, Chien-Lin; Hsu, Yung-Heng; Yu, Yi-Hsun; Chen, Jan-Kan; Liu, Shih-Jung

    2016-01-01

    A composite biodegradable polymeric model was developed to enhance tendon graft healing. This model included a biodegradable polylactide (PLA) bolt as the bone anchor and a poly(D,L-lactide-co-glycolide) (PLGA) nanofibrous membrane embedded with collagen as a biomimic patch to promote tendon–bone interface integration. Degradation rate and compressive strength of the PLA bolt were measured after immersion in a buffer solution for 3 months. In vitro biochemical characteristics and the nanofibrous matrix were assessed using a water contact angle analyzer, pH meter, and tetrazolium reduction assay. In vivo efficacies of PLGA/collagen nanofibers and PLA bolts for tendon–bone healing were investigated on a rabbit bone tunnel model with histological and tendon pullout tests. The PLGA/collagen-blended nanofibrous membrane was a hydrophilic, stable, and biocompatible scaffold. The PLA bolt was durable for tendon–bone anchoring. Histology showed adequate biocompatibility of the PLA bolt on a medial cortex with progressive bone ingrowth and without tissue overreaction. PLGA nanofibers within the bone tunnel also decreased the tunnel enlargement phenomenon and enhanced tendon–bone integration. Composite polymers of the PLA bolt and PLGA/collagen nanofibrous membrane can effectively promote outcomes of tendon reconstruction in a rabbit model. The composite biodegradable polymeric system may be useful in humans for tendon reconstruction. PMID:27601901

  7. 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

  8. Biomimetic mineralization of woven bone-like nanocomposites: role of collagen cross-links.

    PubMed

    Li, Yuping; Thula, Taili T; Jee, Sangsoo; Perkins, Sasha L; Aparicio, Conrado; Douglas, Elliot P; Gower, Laurie B

    2012-01-01

    Ideal biomaterials for bone grafts must be biocompatible, osteoconductive, osteoinductive and have appropriate mechanical properties. For this, the development of synthetic bone substitutes mimicking natural bone is desirable, but this requires controllable mineralization of the collagen matrix. In this study, densified collagen films (up to 100 μm thick) were fabricated by a plastic compression technique and cross-linked using carbodiimide. Then, collagen-hydroxyapatite composites were prepared by using a polymer-induced liquid-precursor (PILP) mineralization process. Compared to traditional methods that produce only extrafibrillar hydroxyapatite (HA) clusters on the surface of collagen scaffolds, by using the PILP mineralization process, homogeneous intra- and extrafibrillar minerals were achieved on densified collagen films, leading to a similar nanostructure as bone, and a woven microstructure analogous to woven bone. The role of collagen cross-links on mineralization was examined and it was found that the cross-linked collagen films stimulated the mineralization reaction, which in turn enhanced the mechanical properties (hardness and modulus). The highest value of hardness and elastic modulus was 0.7 ± 0.1 and 9.1 ± 1.4 GPa in the dry state, respectively, which is comparable to that of woven bone. In the wet state, the values were much lower (177 ± 31 and 8 ± 3 MPa) due to inherent microporosity in the films, but still comparable to those of woven bone in the same conditions. Mineralization of collagen films with controllable mineral content and good mechanical properties provide a biomimetic route toward the development of bone substitutes for the next generation of biomaterials. This work also provides insight into understanding the role of collagen fibrils on mineralization.

  9. Collagen-containing scaffolds enhance attachment and proliferation of non-cultured bone marrow multipotential stromal cells.

    PubMed

    El-Jawhari, Jehan J; Sanjurjo-Rodríguez, Clara; Jones, Elena; Giannoudis, Peter V

    2016-04-01

    Large bone defects are ideally treated with autografts, which have many limitations. Therefore, osteoconductive scaffolds loaded with autologous bone marrow (BM) aspirate are increasingly used as alternatives. The purpose of this study was to compare the growth of multipotential stromal cells (MSCs) from unprocessed BM on a collagen-containing bovine bone scaffold (Orthoss(®) Collagen) with a non-collagen-containing bovine bone scaffold, Orthoss(®) . Another collagen-containing synthetic scaffold, Vitoss(®) was included in the comparison. Colonization of scaffolds by BM MSCs (n = 23 donors) was evaluated using microscopy, colony forming unit-fibroblast assay and flow-cytometry. The number of BM MSCs initially attached to Orthoss(®) Collagen and Vitoss(®) was similar but greater than Orthoss(®) (p = 0.001 and p = 0.041, respectively). Furthermore, the number of MSCs released from Orthoss(®) Collagen and Vitoss(®) after 2-week culture was also higher compared to Orthoss(®) (p = 0.010 and p = 0.023, respectively). Interestingly, collagen-containing scaffolds accommodated larger numbers of lymphocytic and myelomonocytic cells. Additionally, the proliferation of culture-expanded MSCs on Orthoss(®) collagen and Vitoss(®) was greater compared to Orthoss(®) (p = 0.047 and p = 0.004, respectively). Collectively, collagen-containing scaffolds were superior in supporting the attachment and proliferation of MSCs when they were loaded with unprocessed BM aspirates. This highlights the benefit of collagen incorporation into bone scaffolds for use with autologous bone marrow aspirates as autograft substitutes.

  10. 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.

  11. 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

  12. In vitro study of a novel nanogold-collagen composite to enhance the mesenchymal stem cell behavior for vascular regeneration.

    PubMed

    Hung, Huey-Shan; Chang, Chih-Hsuan; Chang, Chen-Jung; Tang, Cheng-Ming; Kao, Wei-Chien; Lin, Shinn-Zong; Hsieh, Hsien-Hsu; Chu, Mei-Yun; Sun, Wei-Shen; Hsu, Shan-Hui

    2014-01-01

    Novel nanocomposites based on type I collagen (Col) containing a small amount (17.4, 43.5, and 174 ppm) of gold nanoparticles (AuNPs, approximately 5 nm) were prepared in this study. The pure Col and Col-AuNP composites (Col-Au) were characterized by the UV-Vis spectroscopy (UV-Vis), surface-enhanced raman spectroscopy (SERS) and atomic force microscopy (AFM). The interaction between Col and AuNPs was confirmed by infrared (IR) spectra. The effect of AuNPs on the biocompatibility of Col, evaluated by the proliferation and reactive oxygen species (ROS) production of mesenchymal stem cells (MSCs) as well as the activation of monocytes and platelets, was investigated. Results showed that Col-Au had better biocompatibility than Col. Upon stimulation by vascular endothelial growth factor (VEGF) and stromal derived factor-1α (SDF-1α), MSCs expressed the highest levels of αvβ3 integrin/CXCR4, focal adhesion kinase (FAK), matrix metalloproteinase-2 (MMP-2), and Akt/endothelial nitric oxide synthase (eNOS) proteins when grown on the Col-Au (43.5 ppm) nanocomposite. Taken together, Col-Au nanocomposites may promote the proliferation and migration of MSCs and stimulate the endothelial cell differentiation. These results suggest that Col-Au may be used to construct tissue engineering scaffolds for vascular regeneration.

  13. In Vitro Study of a Novel Nanogold-Collagen Composite to Enhance the Mesenchymal Stem Cell Behavior for Vascular Regeneration

    PubMed Central

    Hung, Huey-Shan; Chang, Chih-Hsuan; Chang, Chen-Jung; Tang, Cheng-Ming; Kao, Wei-Chien; Lin, Shinn-Zong; Hsieh, Hsien-Hsu; Chu, Mei-Yun; Sun, Wei-Shen; Hsu, Shan-hui

    2014-01-01

    Novel nanocomposites based on type I collagen (Col) containing a small amount (17.4, 43.5, and 174 ppm) of gold nanoparticles (AuNPs, approximately 5 nm) were prepared in this study. The pure Col and Col-AuNP composites (Col-Au) were characterized by the UV-Vis spectroscopy (UV-Vis), surface-enhanced raman spectroscopy (SERS) and atomic force microscopy (AFM). The interaction between Col and AuNPs was confirmed by infrared (IR) spectra. The effect of AuNPs on the biocompatibility of Col, evaluated by the proliferation and reactive oxygen species (ROS) production of mesenchymal stem cells (MSCs) as well as the activation of monocytes and platelets, was investigated. Results showed that Col-Au had better biocompatibility than Col. Upon stimulation by vascular endothelial growth factor (VEGF) and stromal derived factor-1α (SDF-1α), MSCs expressed the highest levels of αvβ3 integrin/CXCR4, focal adhesion kinase (FAK), matrix metalloproteinase-2 (MMP-2), and Akt/endothelial nitric oxide synthase (eNOS) proteins when grown on the Col-Au (43.5 ppm) nanocomposite. Taken together, Col-Au nanocomposites may promote the proliferation and migration of MSCs and stimulate the endothelial cell differentiation. These results suggest that Col-Au may be used to construct tissue engineering scaffolds for vascular regeneration. PMID:25093502

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. High-voltage pulsed current stimulation enhances wound healing in diabetic rats by restoring the expression of collagen, α-smooth muscle actin, and TGF-β1.

    PubMed

    Kim, Tae Hoon; Cho, Hwi-Young; Lee, Suk Min

    2014-01-01

    Impaired wound healing is a common complication of diabetes mellitus and a major morbidity that leads to pain and severely diminished quality of life. Diabetic wounds are commonly associated with defective immune cell responses or abnormality of extracellular matrix. Various types of electrical stimulation interventions have been used to promote tissue healing. However, it is unclear whether high-voltage pulsed current stimulation (HVPCS) enhances diabetic wound healing. In this study, the effects of HVPCS on wound healing were investigated in diabetic rats. Three groups of rats (10 per group) were used: non-diabetic control, diabetic control, and diabetic rats that were administered HVPCS for 40 minutes daily for 1 week. Rats from control groups were administered sham interventions. Dorsal incision wounds were generated in all animals, and wound-healing rate was determined during one-week intervention. After interventions, we measured the relative expression levels of collagen type I (collagen-I), α-smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1) mRNAs in the wounded skin. Wound closure was delayed in diabetic control rats compared to the non-diabetic control rats, and the diabetic control rats showed the reduced expression levels of collagen-I, α-SMA and TGF-β1 mRNAs. Importantly, compared to diabetic control rats, rats with HVPCS showed accelerated wound closure and healing (p < 0.01) and restored expression levels of collagen-I (p = 0.02), α-SMA (p = 0.04), and TGF-β1 (p = 0.01) mRNAs. In conclusion, HVPCS may be beneficial for enhancing the healing of diabetic wounds by restoring the expression levels of TGF-β1, collagen-I, and α-SMA. PMID:25169252

  20. The collagen I mimetic peptide DGEA enhances an osteogenic phenotype in mesenchymal stem cells when presented from cell-encapsulating hydrogels.

    PubMed

    Mehta, Manav; Madl, Christopher M; Lee, Shimwoo; Duda, Georg N; Mooney, David J

    2015-11-01

    Interactions between cells and the extracellular matrix (ECM) are known to play critical roles in regulating cell phenotype. The identity of ECM ligands presented to mesenchymal stem cells (MSCs) has previously been shown to direct the cell fate commitment of these cells. To enhance osteogenic differentiation of MSCs, alginate hydrogels were prepared that present the DGEA ligand derived from collagen I. When presented from hydrogel surfaces in 2D, the DGEA ligand did not facilitate cell adhesion, while hydrogels presenting the RGD ligand derived from fibronectin did encourage cell adhesion and spreading. However, the osteogenic differentiation of MSCs encapsulated within alginate hydrogels presenting the DGEA ligand was enhanced when compared with unmodified alginate hydrogels and hydrogels presenting the RGD ligand. MSCs cultured in DGEA-presenting gels exhibited increased levels of osteocalcin production and mineral deposition. These data suggest that the presentation of the collagen I-derived DGEA ligand is a feasible approach for selectively inducing an osteogenic phenotype in encapsulated MSCs.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. Angiotensin II enhances adenylyl cyclase signaling via Ca2+/calmodulin. Gq-Gs cross-talk regulates collagen production in cardiac fibroblasts.

    PubMed

    Ostrom, Rennolds S; Naugle, Jennifer E; Hase, Miki; Gregorian, Caroline; Swaney, James S; Insel, Paul A; Brunton, Laurence L; Meszaros, J Gary

    2003-07-01

    Cardiac fibroblasts regulate formation of extracellular matrix in the heart, playing key roles in cardiac remodeling and hypertrophy. In this study, we sought to characterize cross-talk between Gq and Gs signaling pathways and its impact on modulating collagen synthesis by cardiac fibroblasts. Angiotensin II (ANG II) activates cell proliferation and collagen synthesis but also potentiates cyclic AMP (cAMP) production stimulated by beta-adrenergic receptors (beta-AR). The potentiation of beta-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of Gq. Ionomycin and thapsigargin increased intracellular Ca2+ levels and potentiated isoproterenol- and forskolin-stimulated cAMP production, whereas chelation of Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/AM inhibited such potentiation. Inhibitors of tyrosine kinases, protein kinase C, or Gbetagamma did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca2+-activated isoform, along with AC2, AC4, AC5, AC6, and AC7. Of those isoforms, only AC3 and AC5/6 proteins were detected in caveolin-rich fractions. Overexpression of AC6 increased betaAR-stimulated cAMP accumulation but did not alter the size of the ANG II potentiation, suggesting that the cross-talk is AC isoform-specific. Isoproterenol-mediated inhibition of serum-stimulated collagen synthesis increased from 31 to 48% in the presence of ANG II, indicating that betaAR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca2+-dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of cross-talk between Gq and Gs signaling pathways in cardiac fibroblasts. PMID:12711600

  10. Angiotensin II enhances adenylyl cyclase signaling via Ca2+/calmodulin. Gq-Gs cross-talk regulates collagen production in cardiac fibroblasts.

    PubMed

    Ostrom, Rennolds S; Naugle, Jennifer E; Hase, Miki; Gregorian, Caroline; Swaney, James S; Insel, Paul A; Brunton, Laurence L; Meszaros, J Gary

    2003-07-01

    Cardiac fibroblasts regulate formation of extracellular matrix in the heart, playing key roles in cardiac remodeling and hypertrophy. In this study, we sought to characterize cross-talk between Gq and Gs signaling pathways and its impact on modulating collagen synthesis by cardiac fibroblasts. Angiotensin II (ANG II) activates cell proliferation and collagen synthesis but also potentiates cyclic AMP (cAMP) production stimulated by beta-adrenergic receptors (beta-AR). The potentiation of beta-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of Gq. Ionomycin and thapsigargin increased intracellular Ca2+ levels and potentiated isoproterenol- and forskolin-stimulated cAMP production, whereas chelation of Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/AM inhibited such potentiation. Inhibitors of tyrosine kinases, protein kinase C, or Gbetagamma did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca2+-activated isoform, along with AC2, AC4, AC5, AC6, and AC7. Of those isoforms, only AC3 and AC5/6 proteins were detected in caveolin-rich fractions. Overexpression of AC6 increased betaAR-stimulated cAMP accumulation but did not alter the size of the ANG II potentiation, suggesting that the cross-talk is AC isoform-specific. Isoproterenol-mediated inhibition of serum-stimulated collagen synthesis increased from 31 to 48% in the presence of ANG II, indicating that betaAR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca2+-dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of cross-talk between Gq and Gs signaling pathways in cardiac fibroblasts.

  11. 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

  12. 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

  13. 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

  14. [Atrial fibrillation and physical activity].

    PubMed

    Apor, Péter

    2013-03-31

    Atrial fibrillation is the most frequent arrhythmia. Its "lone" form (when underlying pathology is not discovered) can be detected in a small percentage of endurance sports participants, and in growing numbers among veterans, probably as a result of some cardiac or other irregularities. Enhanced vagal tone and sudden sympathetic impulse, repetitive oxidative stress, inflammatory processes, enlarged atria, electric instabilization can explain the higher occurrence. Treatment of atrial fibrillation enables the affected persons to participate in regular medium-intensity exercise, 3-5 hours a week, which offers a protective role against cardiovascular, metabolic and mental illnesses. PMID:23524234

  15. In Vitro Mineralization of an Osteoid-Like Dense Collagen Construct for Bone Tissue Engineering

    NASA Astrophysics Data System (ADS)

    Marelli, Benedetto

    The aim of this doctoral research was to design and evaluate strategies to rapidly achieve an acellular mineralization of an osteoid-like dense collagen gel for potential applications in bone regeneration. It was hypothesized that the collagen fibrillar density (CFD) affects the microenvironment and the physical properties of the framework of collagen gels. To test this hypothesis, and as a first objective, the mineralization of collagen gel sheets, rolls and strips with increasing CFDs was investigated in vitro in simulated body fluid (SBF). Collagen gels with physiologically relevant CFDs (14.1 wt%) led to greater extent of mineralization (12 dry wt% at day 14 in SBF), when compared to highly hydrated gels. Chemical characterization confirmed this mineral phase to be CHA, which significantly increased the gel apparent modulus and ultimate tensile strength (UTS). Surprisingly, CFD also affected the electrostatic properties of collagen gel, as investigated by quantifying the extent of anionic and cationic dyes bound to collagen gels with different CFDs. It was therefore proposed that the increase in gel CFD led to a more physiological microenvironment, resulting in a higher number of fibril-to-fibril contact points and an increase in charge concentration, which facilitated the mineral formation and validated the proposed osteoid model. As a second objective, the mineralization of dense collagen (DC) gels with physiologically relevant CFD (14.1 wt%) was enhanced and accelerated by mimicking the role of anionic non collagenous proteins (NCPs) in the native osteoid, which act as CHA nucleators. Two strategies were implemented: first, the influence of collagen fibrillization pH on the extent of DC gel mineralization was investigated. Since the collagen molecule is slightly positively charged at physiological pH (isoelectric point at pH 7.8), it was hypothesized that it would be more negatively charged if formed in an alkaline environment, i.e., above its isoelectric

  16. 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

  17. Signal Transducer and Activator of Transcription 3/MicroRNA-21 Feedback Loop Contributes to Atrial Fibrillation by Promoting Atrial Fibrosis in a Rat Sterile Pericarditis Model

    PubMed Central

    Huang, Zhengrong; Chen, Xiao-jun; Qian, Cheng; Dong, Qian; Ding, Dan; Wu, Qiong-feng; Li, Jing; Wang, Hong-fei; Li, Wei-hua; Xie, Qiang; Cheng, Xiang; Liao, Yu-hua

    2016-01-01

    Background— Postoperative atrial fibrillation is a frequent complication in cardiac surgery. The aberrant activation of signal transducer and activator of transcription 3 (STAT3) contributes to the pathogenesis of atrial fibrillation. MicroRNA-21 (miR-21) promotes atrial fibrosis. Recent studies support the existence of reciprocal regulation between STAT3 and miR-21. Here, we test the hypothesis that these 2 molecules might form a feedback loop that contributes to postoperative atrial fibrillation by promoting atrial fibrosis. Methods and Results— A sterile pericarditis model was created using atrial surfaces dusted with sterile talcum powder in rats. The inflammatory cytokines interleukin (IL)-1β, IL-6, transforming growth factor-β, and tumor necrosis factor-α, along with STAT3 and miR-21, were highly upregulated in sterile pericarditis rats. The inhibition of STAT3 by S3I-201 resulted in miR-21 downregulation, which ameliorated atrial fibrosis and decreased the expression of the fibrosis-related genes, α-smooth muscle actin, collagen-1, and collagen-3; reduced the inhomogeneity of atrial conduction; and attenuated atrial fibrillation vulnerability. Meanwhile, treatment with antagomir-21 decreased STAT3 phosphorylation, alleviated atrial remodeling, abrogated sterile pericarditis–induced inhomogeneous conduction, and prevented atrial fibrillation promotion. The culturing of cardiac fibroblasts with IL-6 resulted in progressively augmented STAT3 phosphorylation and miR-21 levels. S3I-201 blocked IL-6 induced the expression of miR-21 and fibrosis-related genes in addition to cardiac fibroblast proliferation. Transfected antagomir-21 decreased the IL-6–induced cardiac fibroblast activation and STAT3 phosphorylation. The overexpression of miR-21 in cardiac fibroblasts caused the upregulation of STAT3 phosphorylation, enhanced fibrosis-related genes, and increased cell numbers. Conclusions— Our results have uncovered a novel reciprocal loop between STAT3

  18. 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.

  19. 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

  20. 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

  1. Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    PubMed Central

    Hozhabri, Neda S. T.; Benson, M. Douglas; Vu, Michael D.; Patel, Rinkesh H.; Martinez, Rebecca M.; Nakhaie, Fatemeh N.; Kim, Harry K. W.; Varanasi, Venu G.

    2015-01-01

    Inflammatory response in the dental pulp can alter the collagen matrix formation by dental pulp stem cells and lead to a delay or poor healing of the pulp. This inflammatory response is mediated by cytokines, including interleukin-1β and tumor necrosis factor-α. In this study, it is hypothesized that suppressing the actions of these inflammatory cytokines by knocking down the activity of transcription factor Nuclear Factor–κB will lead to dental pulp stem cell differentiation into odontoblasts and the production of collagen. Here, the role of Nuclear Factor–κB signaling and its reduction was examined during odontogenic behavior in the presence of these cytokines. The results showed a significant increase in Nuclear Factor–κB gene expression and p65 protein expression by interleukin-1β and tumor necrosis factor-α. Nuclear Factor–κB activation in the presence of these cytokines decreased significantly in a dose-dependent manner by a Nuclear Factor–κB inhibitor (MG132) and p65 siRNA. Down-regulation of Nuclear Factor–κB activity also enhanced the gene expression of the odontoblastic markers (dentin sialophosphoprotein, Nestin, and alkaline phosphatase) and displayed an odontoblastic cell morphology indicating the promotion of odontogenic differentiation of dental pulp stem cells. Finally, dental pulp stem cells exposed to reduced Nuclear Factor–κB activity resulted in a significant increase in collagen (I)-α1 expression in the presence of these cytokines. In conclusion, a decrease in Nuclear Factor-κB in dental pulp stem cells in the presence of inflammatory cytokines enhanced odontoblastic differentiation and collagen matrix formation. PMID:25629155

  2. Comparative vibrational spectroscopy of intracellular tau and extracellular collagen I reveals parallels of gelation and fibrillar structure.

    PubMed

    Juszczak, Laura J

    2004-02-27

    The N-terminal tau 2-19 peptide undergoes gelation, syneresis, and aggregation over a period of years. These changes may be approximated on a shorter time scale by agitation and partial dehydration. The anomalously enhanced (229 nm) ultraviolet resonance Raman (UVRR) imide II band reveals a common structural feature for gels of nondehydrated tau 2-19 and collagen I and insoluble paired helical filaments (PHFs) and collagen I of weak hydrogen bonding at proline carbonyls. Anomalous UVRR enhancement of amide bands at 229 nm results from gel structure, as demonstrated by increased amide absorption at the red edge for tau 2-19 gel and implies the involvement of water in gel structure. In aged, dehydrated tau 2-19 gel, proline carbonyls lose their bonds to water and tyrosine becomes deprotonated, as demonstrated by UVRR spectroscopy. The Fourier transform infrared (FTIR) amide I band shows that antiparallel beta-sheet structure increases with syneresis in the tau 2-19 hydrogel. The comparison of FTIR results for PHFs with collagen I gel and polyproline demonstrates that the secondary structure of PHFs is polyproline II. One implication of this assignment is that the fibrillation of hydrophilic tau is thermodynamically driven by the entropy gained as hydrogen-bonded water is freed, as for collagen I. The FTIR results also show that peptide domains culled from a longer protein do not necessarily fold into identical secondary structures. A pathological, sequential mechanism of gelation, syneresis, and fibrillation for tau in AD is suggested and is supported by the observation of amorphous neurofibrillary tangle development and fibrillation in vivo.

  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. Interfibrillar stiffening of echinoderm mutable collagenous tissue demonstrated at the nanoscale

    PubMed Central

    Mo, Jingyi; Blowes, Liisa M.; Egertová, Michaela; Terrill, Nicholas J.; Wang, Wen; Elphick, Maurice R.; Gupta, Himadri S.

    2016-01-01

    The mutable collagenous tissue (MCT) of echinoderms (e.g., sea cucumbers and starfish) is a remarkable example of a biological material that has the unique attribute, among collagenous tissues, of being able to rapidly change its stiffness and extensibility under neural control. However, the mechanisms of MCT have not been characterized at the nanoscale. Using synchrotron small-angle X-ray diffraction to probe time-dependent changes in fibrillar structure during in situ tensile testing of sea cucumber dermis, we investigate the ultrastructural mechanics of MCT by measuring fibril strain at different chemically induced mechanical states. By measuring a variable interfibrillar stiffness (EIF), the mechanism of mutability at the nanoscale can be demonstrated directly. A model of stiffness modulation via enhanced fibrillar recruitment is developed to explain the biophysical mechanisms of MCT. Understanding the mechanisms of MCT quantitatively may have applications in development of new types of mechanically tunable biomaterials. PMID:27708167

  10. Collagen esterification enhances the function and survival of pancreatic β cells in 2D and 3D culture systems.

    PubMed

    Ko, Jae Hyung; Kim, Yang Hee; Jeong, Seong Hee; Lee, Song; Park, Si-Nae; Shim, In Kyong; Kim, Song Cheol

    2015-08-01

    Collagen, one of the most important components of the extracellular matrix (ECM), may play a role in the survival of pancreatic islet cells. In addition, chemical modifications that change the collagen charge profile to a net positive charge by esterification have been shown to increase the adhesion and proliferation of various cell types. The purpose of this study was to characterize and compare the effects of native collagen (NC) and esterified collagen (EC) on β cell function and survival. After isolation by the collagenase digestion technique, rat islets were cultured with NC and EC in 2 dimensional (2D) and 3 dimensional (3D) environments for a long-term duration in vitro. The cells were assessed for islet adhesion, morphology, viability, glucose-induced insulin secretion, and mRNA expression of glucose metabolism-related genes, and visualized by scanning electron microscopy (SEM). Islet cells attached tightly in the NC group, but islet cell viability was similar in both the NC and EC groups. Glucose-stimulated insulin secretion was higher in the EC group than in the NC group in both 2D and 3D culture. Furthermore, the mRNA expression levels of glucokinase in the EC group were higher than those in the NC group and were associated with glucose metabolism and insulin secretion. Finally, SEM observation confirmed that islets had more intact component cells on EC sponges than on NC sponges. These results indicate that modification of collagen may offer opportunities to improve function and viability of islet cells.

  11. VLDL from Metabolic Syndrome Individuals Enhanced Lipid Accumulation in Atria with Association of Susceptibility to Atrial Fibrillation

    PubMed Central

    Lee, Hsiang-Chun; Lin, Hsin-Ting; Ke, Liang-Yin; Wei, Chi; Hsiao, Yi-Lin; Chu, Chih-Sheng; Lai, Wen-Ter; Shin, Shyi-Jang; Chen, Chu-Huang; Sheu, Sheng-Hsiung; Wu, Bin-Nan

    2016-01-01

    Metabolic syndrome (MetS) represents a cluster of metabolic derangements. Dyslipidemia is an important factor in MetS and is related to atrial fibrillation (AF). We hypothesized that very low density lipoproteins (VLDL) in MetS (MetS-VLDL) may induce atrial dilatation and vulnerability to AF. VLDL was therefore separated from normal (normal-VLDL) and MetS individuals. Wild type C57BL/6 male mice were divided into control, normal-VLDL (nVLDL), and MetS-VLDL (msVLDL) groups. VLDL (15 µg/g) and equivalent volumes of saline were injected via tail vein three times a week for six consecutive weeks. Cardiac chamber size and function were measured by echocardiography. MetS-VLDL significantly caused left atrial dilation (control, n = 10, 1.64 ± 0.23 mm; nVLDL, n = 7, 1.84 ± 0.13 mm; msVLDL, n = 10, 2.18 ± 0.24 mm; p < 0.0001) at week 6, associated with decreased ejection fraction (control, n = 10, 62.5% ± 7.7%, vs. msVLDL, n = 10, 52.9% ± 9.6%; p < 0.05). Isoproterenol-challenge experiment resulted in AF in young msVLDL mice. Unprovoked AF occurred only in elderly msVLDL mice. Immunohistochemistry showed excess lipid accumulation and apoptosis in msVLDL mice atria. These findings suggest a pivotal role of VLDL in AF pathogenesis for MetS individuals. PMID:26805814

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. Chitosan nanoparticles enhance the antibacterial activity of chlorhexidine in collagen membranes used for periapical guided tissue regeneration.

    PubMed

    Barreras, Uriel Soto; Méndez, Fernando Torres; Martínez, Rita Elizabeth Martínez; Valencia, Carolina Samano; Rodríguez, Panfilo Raymundo Martinez; Rodríguez, Juan Pablo Loyola

    2016-01-01

    Endodontic failure is mainly associated with the persistence of microbial infection in the root canal system and/or the periradicular area. Microorganisms and their toxins located in the root canal system may trigger apical periodontitis and tissue destruction. Tissue regeneration in periapical surgery by using membrane barriers has shown an improved healing and bone closure. However, bacterial membrane contamination is a main reason of failure. In this in vitro study, different brands of chlorhexidine, a combination of chitosan nanoparticles containing chlorhexidine were tested against Enterococcus faecalis on agar plate's cultures and infected collagen membranes. Our results indicated that chitosan nanoparticles acted synergistically with chlorhexidine, inhibiting and eliminating significantly a greater amount of colony former units in both BHI-agar cultures and infected collagen membranes. These results suggested that chitosan nanoparticles could be used to improve regenerative procedures in periapical surgery. PMID:26478419

  18. Chitosan nanoparticles enhance the antibacterial activity of chlorhexidine in collagen membranes used for periapical guided tissue regeneration.

    PubMed

    Barreras, Uriel Soto; Méndez, Fernando Torres; Martínez, Rita Elizabeth Martínez; Valencia, Carolina Samano; Rodríguez, Panfilo Raymundo Martinez; Rodríguez, Juan Pablo Loyola

    2016-01-01

    Endodontic failure is mainly associated with the persistence of microbial infection in the root canal system and/or the periradicular area. Microorganisms and their toxins located in the root canal system may trigger apical periodontitis and tissue destruction. Tissue regeneration in periapical surgery by using membrane barriers has shown an improved healing and bone closure. However, bacterial membrane contamination is a main reason of failure. In this in vitro study, different brands of chlorhexidine, a combination of chitosan nanoparticles containing chlorhexidine were tested against Enterococcus faecalis on agar plate's cultures and infected collagen membranes. Our results indicated that chitosan nanoparticles acted synergistically with chlorhexidine, inhibiting and eliminating significantly a greater amount of colony former units in both BHI-agar cultures and infected collagen membranes. These results suggested that chitosan nanoparticles could be used to improve regenerative procedures in periapical surgery.

  19. 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

  20. 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.

  1. A single topical dose of erythropoietin applied on a collagen carrier enhances calvarial bone healing in pigs

    PubMed Central

    2014-01-01

    Background and purpose The osteogenic potency of erythropoietin (EPO) has been documented. However, its efficacy in a large-animal model has not yet been investigated; nor has a clinically safe dosage. The purpose of this study was to overcome such limitations of previous studies and thereby pave the way for possible clinical application. Our hypothesis was that EPO increases calvarial bone healing compared to a saline control in the same subject. Methods We used a porcine calvarial defect model. In each of 18 pigs, 6 cylindrical defects (diameter: 1 cm; height: 1 cm) were drilled, allowing 3 pairwise comparisons. Treatment consisted of either 900 IU/mL EPO or an equal volume of saline in combination with either autograft, a collagen carrier, or a polycaprolactone (PCL) scaffold. After an observation time of 5 weeks, the primary outcome (bone volume fraction (BV/TV)) was assessed with high-resolution quantitative computed tomography. Secondary outcome measures were histomorphometry and blood samples. Results The median BV/TV ratio of the EPO-treated collagen group was 1.06 (CI: 1.02–1.11) relative to the saline-treated collagen group. Histomorphometry showed a similar median effect size, but it did not reach statistical significance. Autograft treatment had excellent healing potential and was able to completely regenerate the bone defect independently of EPO treatment. Bony ingrowth into the PCL scaffold was sparse, both with and without EPO. Neither a substantial systemic effect nor adverse events were observed. The number of blood vessels was similar in EPO-treated defects and saline-treated defects. Interpretation Topical administration of EPO on a collagen carrier moderately increased bone healing. The dosing regime was safe, and could have possible application in the clinical setting. However, in order to increase the clinical relevance, a more potent but still clinically safe dose should be investigated. PMID:24564750

  2. 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

  3. The effect on bone growth enhancement of implant coatings with hydroxyapatite and collagen deposited electrochemically and by plasma spray.

    PubMed

    Daugaard, Henrik; Elmengaard, Brian; Bechtold, Joan E; Jensen, Thomas; Soballe, Kjeld

    2010-03-01

    Skeletal bone consists of hydroxyapatite (HA) [Ca(10)(PO(4))(6)(OH)(2)] and collagen type I, both of which are osseoconductive. The goal of osseointegration of orthopedic and dental implants is the rapid achievement of a mechanically stable long-lasting fixation between bone and an implant surface. In this study, we evaluated the mechanical fixation and tissue distribution surrounding implants coated with three surfaces: plasma-sprayed HA coating, thinner coating of electrochemical-assisted deposition of HA, and an identical thin coating with a top layer of mineralized collagen. Uncoated plasma-sprayed titanium (Ti-6Al-4V) served as negative control. The electrochemical-assisted deposition was performed near physiological conditions. We used a canine experimental joint replacement model with four cylindrical implants (one of each treatment group) inserted in the humeri cancellous metaphyseal bone in a 1 mm gap. Observation time was 4 weeks. The mechanical fixation was quantified by push-out test to failure, and the peri-implant tissue formation by histomorphometric evaluation. HA coatings deposited by plasma spray technique or electrochemically, increased the mechanical fixation and bone ongrowth, but there was no statistical difference between the individual HA applications. Addition of collagen to the mineralized phase of the coating to create a more bone natural surface did not improve the osseoconductive effect of HA.

  4. [Skin collagen abnormalities in a Japanese patient with extracranial internal carotid artery dissection followed by extracranial vertebral artery dissection].

    PubMed

    Sengoku, Renpei; Sato, Hironori; Honda, Hidehiko; Inoue, Kiyoharu; Ono, Seiitsu

    2006-02-01

    A 41-year-old man with hypertension and hyperlipidemia who complained of left hemiparesis after a temporal headache was admitted to our hospital. A cervical MRI with gadolinium enhancement revealed an intramural hematoma is compatible with right extracranial internal carotid artery dissection. Two weeks later, he complained of sudden onset of pain in the right side of his neck. The right extracranial internal carotid artery dissection followed by the right extracranial vertebral artery dissection was diagnosed. Spontaneous cervical artery dissection (SCAD) is one of the causes of stroke in young adults. The pathogenesis of SCAD remains unknown. Minor trauma like an excessive sneeze, migraine, and connective tissue disorders such as fibromuscular dysplasia and Ehlers-Danlos syndrome are well-known as risk factors for SCAD. Pathologically skin collagen abnormalities have been seen in German patients with SCAD without clinical evidence for any specific connective tissue disorder. We examined the ultrastructural morphology of the Japanese patient's dermal connective tissue components by electron microscopy. The patient's collagen fibers contained fibrils with highly variable diameters, and there were other ultrastructural abnormalities, including flower-like fibrils and large-diameter composite fibrils. This is the first report of a case of ultrastructural abnormalities of dermal connective tissue in a Japanese patient with SCAD. PMID:16619839

  5. Enhancing structural support of the dermal microenvironment activates fibroblasts, endothelial cells, and keratinocytes in aged human skin in vivo.

    PubMed

    Quan, Taihao; Wang, Frank; Shao, Yuan; Rittié, Laure; Xia, Wei; Orringer, Jeffrey S; Voorhees, John J; Fisher, Gary J

    2013-03-01

    The dermal extracellular matrix (ECM) provides strength and resiliency to skin. The ECM consists mostly of type I collagen fibrils, which are produced by fibroblasts. Binding of fibroblasts to collagen fibrils generates mechanical forces, which regulate cellular morphology and function. With aging, collagen fragmentation reduces fibroblast-ECM binding and mechanical forces, resulting in fibroblast shrinkage and reduced function, including collagen production. Here, we report that these age-related alterations are largely reversed by enhancing the structural support of the ECM. Injection of dermal filler, cross-linked hyaluronic acid, into the skin of individuals over 70 years of age stimulates fibroblasts to produce type I collagen. This stimulation is associated with localized increase in mechanical forces, indicated by fibroblast elongation/spreading, and mediated by upregulation of type II TGF-β receptor and connective tissue growth factor. Interestingly, enhanced mechanical support of the ECM also stimulates fibroblast proliferation, expands vasculature, and increases epidermal thickness. Consistent with our observations in human skin, injection of filler into dermal equivalent cultures causes elongation of fibroblasts, coupled with type I collagen synthesis, which is dependent on the TGF-β signaling pathway. Thus, fibroblasts in aged human skin retain their capacity for functional activation, which is restored by enhancing structural support of the ECM.

  6. Human YKL39 (chitinase 3-like protein 2), an osteoarthritis-associated gene, enhances proliferation and type II collagen expression in ATDC5 cells

    SciTech Connect

    Miyatake, Kazumasa; Tsuji, Kunikazu; Yamaga, Mika; Yamada, Jun; Matsukura, Yu; Abula, Kahaer; Sekiya, Ichiro; Muneta, Takeshi

    2013-02-01

    Highlights: ► hYKL-39 expression is increased in osteoarthritic articular chondrocytes. ► To examine the molecular functions of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in chondrocytic ATDC5 cells. ► hYKL-39 enhanced proliferation and colony formation in ATDC5 cells. ► hYKL-39 increased type II collagen expression in ATDC5 cells treated with chondrogenic medium. -- Abstract: Human YKL39 (chitinase 3-like protein 2/CHI3L2) is a secreted 39 kDa protein produced by articular chondrocytes and synoviocytes. Recent studies showed that hYKL-39 expression is increased in osteoarthritic articular chondrocytes suggesting the involvement of hYKL-39 in the progression of osteoarthritis (OA). However little is known regarding the molecular function of hYKL-39 in joint homeostasis. Sequence analyses indicated that hYKL-39 has significant identity with the human chitotorisidase family molecules, although it is considered that hYKL-39 has no enzymatic activity since it lacks putative chitinase catalytic motif. In this study, to examine the molecular function of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in ATDC5 cells. Here we report that hYKL-39 enhances colony forming activity, cell proliferation, and type II collagen expression in these cells. These data suggest that hYKL-39 is a novel growth and differentiation factor involved in cartilage homeostasis.

  7. 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

  8. Curcumin Protects β-Lactoglobulin Fibril Formation and Fibril-Induced Neurotoxicity in PC12Cells

    PubMed Central

    Mazaheri, Mansooreh; Moosavi-Movahedi, Ali Akbar; Saboury, Ali Akbar; Khodagholi, Fariba; Shaerzadeh, Fatemeh; Sheibani, Nader

    2015-01-01

    In this study the β-lactoglobulin fibrillation, in the presence or absence of lead ions, aflatoxin M1 and curcumin, was evaluated using ThT fluorescence, Circular dichroism spectroscopy and atomic force microscopy. To investigate the toxicity of the different form of β-Lg fibrils, in the presence or absence of above toxins and curcumin, we monitored changes in the level of reactive oxygen species and morphology of the differentiated neuron-like PC12 cells. The cell viability, cell body area, average neurite length, neurite width, number of primary neurites, percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different form of β-Lg fibrils. Incubation of β-Lg with curcumin resulted in a significant decrease in ROS levels even in the presence of lead ions and aflatoxin M1. The β-Lg fibrils formed in the presence of lead ions and aflatoxin M1 attenuated the growth and complexity of PC12 cells compared with other form of β-Lg fibrils. However, the adverse effects of these toxins and protein fibrils were negated in the presence of curcumin. Furthermore, the antioxidant and inhibitory effects of curcumin protected PC12 cells against fibril neurotoxicity and enhanced their survival. Thus, curcumin may provide a protective effect toward β-Lg, and perhaps other protein, fibrils mediated neurotoxicity. PMID:26186474

  9. Collagen for bone tissue regeneration.

    PubMed

    Ferreira, Ana Marina; Gentile, Piergiorgio; Chiono, Valeria; Ciardelli, Gianluca

    2012-09-01

    In the last decades, increased knowledge about the organization, structure and properties of collagen (particularly concerning interactions between cells and collagen-based materials) has inspired scientists and engineers to design innovative collagen-based biomaterials and to develop novel tissue-engineering products. The design of resorbable collagen-based medical implants requires understanding the tissue/organ anatomy and biological function as well as the role of collagen's physicochemical properties and structure in tissue/organ regeneration. Bone is a complex tissue that plays a critical role in diverse metabolic processes mediated by calcium delivery as well as in hematopoiesis whilst maintaining skeleton strength. A wide variety of collagen-based scaffolds have been proposed for different tissue engineering applications. These scaffolds are designed to promote a biological response, such as cell interaction, and to work as artificial biomimetic extracellular matrices that guide tissue regeneration. This paper critically reviews the current understanding of the complex hierarchical structure and properties of native collagen molecules, and describes the scientific challenge of manufacturing collagen-based materials with suitable properties and shapes for specific biomedical applications, with special emphasis on bone tissue engineering. The analysis of the state of the art in the field reveals the presence of innovative techniques for scaffold and material manufacturing that are currently opening the way to the preparation of biomimetic substrates that modulate cell interaction for improved substitution, restoration, retention or enhancement of bone tissue function. PMID:22705634

  10. Loss of fibulin-4 disrupts collagen synthesis and maturation: implications for pathology resulting from EFEMP2 mutations.

    PubMed

    Papke, Christina L; Tsunezumi, Jun; Ringuette, Léa-Jeanne; Nagaoka, Hideaki; Terajima, Masahiko; Yamashiro, Yoshito; Urquhart, Greg; Yamauchi, Mitsuo; Davis, Elaine C; Yanagisawa, Hiromi

    2015-10-15

    Homozygous recessive mutations in either EFEMP2 (encoding fibulin-4) or FBLN5 (encoding fibulin-5), critical genes for elastogenesis, lead to autosomal recessive cutis laxa types 1B and 1A, respectively. Previously, fibulin-4 was shown to bind lysyl oxidase (LOX), an elastin/collagen cross-linking enzyme, in vitro. Consistently, reported defects in humans with EFEMP2 mutations are more severe and broad in range than those due to FBLN5 mutations and encompass both elastin-rich and collagen-rich tissues. However, the underlying disease mechanism in EFEMP2 mutations has not been fully addressed. Here, we show that fibulin-4 is important for the integrity of aortic collagen in addition to elastin. Smooth muscle-specific Efemp2 loss in mouse (termed SMKO) resulted in altered fibrillar collagen localization with larger, poorly organized fibrils. LOX activity was decreased in Efemp2-null cells, and collagen cross-linking was diminished in SMKO aortas; however, elastin cross-linking was unaffected and the level of mature LOX was maintained to that of wild-type aortas. Proteomic screening identified multiple proteins involved in procollagen processing and maturation as potential fibulin-4-binding partners. We showed that fibulin-4 binds procollagen C-endopeptidase enhancer 1 (Pcolce), which enhances proteolytic cleavage of the procollagen C-terminal propeptide during procollagen processing. Interestingly, however, procollagen cleavage was not affected by the presence or absence of fibulin-4 in vitro. Thus, our data indicate that fibulin-4 serves as a potential scaffolding protein during collagen maturation in the extracellular space. Analysis of collagen in other tissues affected by fibulin-4 loss should further increase our understanding of underlying pathologic mechanisms in patients with EFEMP2 mutations.

  11. 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

  12. Bioinspired Collagen/Glycosaminoglycan-Based Cellular Microenvironments for Tuning Osteoclastogenesis.

    PubMed

    Rother, Sandra; Salbach-Hirsch, Juliane; Moeller, Stephanie; Seemann, Thomas; Schnabelrauch, Matthias; Hofbauer, Lorenz C; Hintze, Vera; Scharnweber, Dieter

    2015-10-28

    Replicating the biocomplexity of native extracellular matrices (ECM) is critical for a deeper understanding of biochemical signals influencing bone homeostasis. This will foster the development of bioinspired biomaterials with adjustable bone-inducing properties. Collagen-based coatings containing single HA derivatives have previously been reported to promote osteogenic differentiation and modulate osteoclastogenesis and resorption depending on their sulfation degree. However, the potential impact of different GAG concentrations as well as the interplay of multiple GAGs in these coatings is not characterized in detail to date. These aspects were addressed in the current study by integrating HA and different sulfate-modified HA derivatives (sHA) during collagen in vitro fibrillogenesis. Besides cellular microenvironments with systematically altered single-GAG concentrations, matrices containing both low and high sHA (sHA1, sHA4) were characterized by biochemical analysis such as agarose gel electrophoresis, performed for the first time with sHA derivatives. The morphology and composition of the collagen coatings were altered in a GAG sulfation- and concentration-dependent manner. In multi-GAG microenvironments, atomic force microscopy revealed intermediate collagen fibril structures with thin fibrils and microfibrils. GAG sulfation altered the surface charge of the coatings as demonstrated by ζ-potential measurements revealed for the first time as well. This highlights the prospect of GAG-containing matrices to adjust defined surface charge properties. The sHA4- and the multi-GAG coatings alike significantly enhanced the viability of murine osteoclast-precursor-like RAW264.7 cells. Although in single-GAG matrices there was no dose-dependent effect on cell viability, osteoclastogenesis was significantly suppressed only on sHA4-coatings in a dose-dependent fashion. The multi-GAG coatings led to an antiosteoclastogenic effect in-between those with single-GAGs which

  13. Bioinspired Collagen/Glycosaminoglycan-Based Cellular Microenvironments for Tuning Osteoclastogenesis.

    PubMed

    Rother, Sandra; Salbach-Hirsch, Juliane; Moeller, Stephanie; Seemann, Thomas; Schnabelrauch, Matthias; Hofbauer, Lorenz C; Hintze, Vera; Scharnweber, Dieter

    2015-10-28

    Replicating the biocomplexity of native extracellular matrices (ECM) is critical for a deeper understanding of biochemical signals influencing bone homeostasis. This will foster the development of bioinspired biomaterials with adjustable bone-inducing properties. Collagen-based coatings containing single HA derivatives have previously been reported to promote osteogenic differentiation and modulate osteoclastogenesis and resorption depending on their sulfation degree. However, the potential impact of different GAG concentrations as well as the interplay of multiple GAGs in these coatings is not characterized in detail to date. These aspects were addressed in the current study by integrating HA and different sulfate-modified HA derivatives (sHA) during collagen in vitro fibrillogenesis. Besides cellular microenvironments with systematically altered single-GAG concentrations, matrices containing both low and high sHA (sHA1, sHA4) were characterized by biochemical analysis such as agarose gel electrophoresis, performed for the first time with sHA derivatives. The morphology and composition of the collagen coatings were altered in a GAG sulfation- and concentration-dependent manner. In multi-GAG microenvironments, atomic force microscopy revealed intermediate collagen fibril structures with thin fibrils and microfibrils. GAG sulfation altered the surface charge of the coatings as demonstrated by ζ-potential measurements revealed for the first time as well. This highlights the prospect of GAG-containing matrices to adjust defined surface charge properties. The sHA4- and the multi-GAG coatings alike significantly enhanced the viability of murine osteoclast-precursor-like RAW264.7 cells. Although in single-GAG matrices there was no dose-dependent effect on cell viability, osteoclastogenesis was significantly suppressed only on sHA4-coatings in a dose-dependent fashion. The multi-GAG coatings led to an antiosteoclastogenic effect in-between those with single-GAGs which

  14. Enhancement by PL 14736 of granulation and collagen organization in healing wounds and the potential role of egr-1 expression.

    PubMed

    Tkalcević, Vanesa Ivetić; Cuzić, Snjezana; Brajsa, Karmen; Mildner, Boris; Bokulić, Ana; Situm, Kristina; Perović, Daniela; Glojnarić, Ines; Parnham, Michael J

    2007-09-10

    Apart from becaplermin (recombinant human platelet-derived growth factor homodimer of B chains, PDGF-BB), for the treatment of lower extremity diabetic ulcers, few agents are available for pharmacological stimulation of wound healing. We have compared the mechanism of action of the potential wound healing agent, PL 14736 (G E P P P G K P A D D A G L V), with that of PDGF-BB on granulation tissue formation following sponge implantation in the normoglycemic rat and in healing full-thickness excisional wounds in db/db genetically diabetic mice. Expression of the immediate response gene, early growth response gene-1 (egr-1) was studied in Caco-2 cells in vitro. While PDGF-BB and PL 14736 had similar selectivity for stimulation of granulation tissue in both sponge granuloma and in healing wounds in db/db mice, PL 14736 was more active in stimulating early collagen organization. It also stimulated expression of egr-1 and its repressor nerve growth factor 1-A binding protein-2 (nab2) in non-differentiated Caco-2 cells more rapidly than PDGF-BB. EGR-1 induces cytokine and growth factor generation and early extracellular matrix (collagen) formation, offering an explanation for the beneficial effects of PL 14736 on wound healing. PMID:17628536

  15. Ixora coccinea Enhances Cutaneous Wound Healing by Upregulating the Expression of Collagen and Basic Fibroblast Growth Factor.

    PubMed

    Upadhyay, Aadesh; Chattopadhyay, Pronobesh; Goyary, Danswrang; Mitra Mazumder, Papiya; Veer, Vijay

    2014-01-01

    Background. Ixora coccinea L. (Rubiaceae) has been documented for traditional use in hypertension, menstrual irregularities, sprain, chronic ulcer, and skin diseases. In the present study, I. coccinea was subjected to in vitro and in vivo wound healing investigation. Methods. Petroleum ether, chloroform, methanol, and water sequential I. coccinea leaves extracts were evaluated for in vitro antioxidant, antimicrobial, and fibroblast proliferation activities. The promising I. coccinea methanol extract (IxME) was screened for in vivo wound healing activity in Wistar rat using circular excision model. Wound contraction measurement, hydroxyproline quantification, and western blot for collagen type III (COL3A1), basic fibroblast growth factor (bFGF), and Smad-2, -3, -4, and -7 was performed with 7-day postoperative wound granulation tissue. Gentamicin sulfate (0.01% w/w) hydrogel was used as reference standard. Results. IxME showed the potent antimicrobial, antioxidant activities, with significant fibroblast proliferation inducing activity, as compared to all other extracts. In vivo study confirmed the wound healing accelerating potential of IxME, as evidenced by faster wound contraction, higher hydroxyproline content, and improved histopathology of granulation tissue. Western blot analysis revealed that the topical application of I. coccinea methanol extract stimulates the fibroblast growth factor and Smad mediated collagen production in wound tissue. PMID:24624303

  16. 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...

  17. Age-Associated Increase in Skin Fibroblast-Derived Prostaglandin E2 Contributes to Reduced Collagen Levels in Elderly Human Skin.

    PubMed

    Li, Yong; Lei, Dan; Swindell, William R; Xia, Wei; Weng, Shinuo; Fu, Jianping; Worthen, Christal A; Okubo, Toru; Johnston, Andrew; Gudjonsson, Johann E; Voorhees, John J; Fisher, Gary J

    2015-09-01

    Production of type I collagen declines during aging, leading to skin thinning and impaired function. Prostaglandin E2 (PGE2) is a pleiotropic lipid mediator that is synthesized from arachidonic acid by the sequential actions of cyclooxygenases (COX) and PGE synthases (PTGES). PGE2 inhibits collagen production by fibroblasts in vitro. We report that PTGES1 and COX2 progressively increase with aging in sun-protected human skin. PTGES1 and COX2 mRNA were increased 3.4-fold and 2.7-fold, respectively, in the dermis of elderly (>80 years) versus young (21-30 years) individuals. Fibroblasts were the major cell source of both enzymes. PGE2 levels were increased 70% in elderly skin. Fibroblasts in aged skin display reduced spreading due to collagen fibril fragmentation. To investigate the relationship between spreading and PGE2 synthesis, fibroblasts were cultured on micropost arrays or hydrogels of varying mechanical compliance. Reduced spreading/mechanical force resulted in increased expression of both PTGES1 and COX2 and elevated levels of PGE2. Inhibition of PGE2 synthesis by diclofenac enhanced collagen production in skin organ cultures. These data suggest that reduced spreading/mechanical force of fibroblasts in aged skin elevates PGE2 production, contributing to reduced collagen production. Inhibition of PGE2 production may be therapeutically beneficial for combating age-associated collagen deficit in human skin.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. Matrix production and collagen structure are enhanced in two types of osteogenic progenitor cells by a simple fluid shear stress stimulus.

    PubMed

    Delaine-Smith, R M; MacNeil, S; Reilly, G C

    2012-08-03

    Mesenchymal progenitor cells play a vital role in bone regenerative medicine and tissue engineering strategies. To be clinically useful osteoprogenitors should be readily available with the potential to form bone matrix. While mesenchymal stromal cells from bone marrow have shown promise for tissue engineering, they are obtained in small numbers and there is risk of donor site morbidity. Osteogenic progenitor cells derived from dermal tissue may provide a more abundant and easily expandable source of cells. Bone turnover in vivo is regulated by mechanical forces, particularly oscillatory fluid shear stresses (FSS), and in vitro osteogenic progenitors have been shown to be regulated by mechanical stimuli. The aim of this study was to assess what effect osteogenic media and FSS, generated by a simple rocking platform, had on cell behaviour and matrix production in human progenitor dermal fibroblasts (HDFs) and the embryonic stem cell-derived mesenchymal progenitor cell line (hES-MP). Osteogenic media stimulated alkaline phosphatase activity (ALP) and calcium deposition in HDFs. The addition of FSS further enhanced ALP activity and mineralised matrix deposition in both progenitor cells cultured in osteogenic media. Both types of progenitor cell subjected to FSS showed increases in collagen secretion and apparent collagen organisation as imaged by second harmonic generation.

  3. Contact activation of blood coagulation on a defined kaolin/collagen surface in a microfluidic assay.

    PubMed

    Zhu, Shu; Diamond, Scott L

    2014-12-01

    Generation of active Factor XII (FXIIa) triggers blood clotting on artificial surfaces and may also enhance intravascular thrombosis. We developed a patterned kaolin (0 to 0.3 pg/μm(2))/type 1 collagen fibril surface for controlled microfluidic clotting assays. Perfusion of whole blood (treated only with a low level of 4 μg/mL of the XIIa inhibitor, corn trypsin inhibitor) drove platelet deposition followed by fibrin formation. At venous wall shear rate (100 s(-1)), kaolin accelerated onset of fibrin formation by ~100 sec when compared to collagen alone (250 sec vs. 350 sec), with little effect on platelet deposition. Even with kaolin present, arterial wall shear rate (1000 s(-1)) delayed and suppressed fibrin formation compared to venous wall shear rate. A comparison of surfaces for extrinsic activation (tissue factor TF/collagen) versus contact activation (kaolin/collagen) that each generated equal platelet deposition at 100 s(-1) revealed: (1) TF surfaces promoted much faster fibrin onset (at 100 sec) and more endpoint fibrin at 600 sec at either 100 s(-1) or 1000 s(-1), and (2) kaolin and TF surfaces had a similar sensitivity for reduced fibrin deposition at 1000 s(-1) (compared to fibrin formed at 100 s(-1)) despite differing coagulation triggers. Anti-platelet drugs inhibiting P2Y1, P2Y12, cyclooxygenase-1 or activating IP-receptor or guanylate cyclase reduced platelet and fibrin deposition on kaolin/collagen. Since FXIIa or FXIa inhibition may offer safe antithrombotic therapy, especially for biomaterial thrombosis, these defined collagen/kaolin surfaces may prove useful in drug screening tests or in clinical diagnostic assays of blood under flow conditions.

  4. Contact activation of blood coagulation on a defined kaolin/collagen surface in a microfluidic assay

    PubMed Central

    Zhu, Shu; Diamond, Scott L.

    2014-01-01

    Generation of active Factor XII (FXIIa) triggers blood clotting on artificial surfaces and may also enhance intravascular thrombosis. We developed a patterned kaolin (0 to 0.3 pg/μm2)/type 1 collagen fibril surface for controlled microfluidic clotting assays. Perfusion of whole blood (treated only with a low level of 4 μg/mL of the XIIa inhibitor, corn trypsin inhibitor) drove platelet deposition followed by fibrin formation. At venous wall shear rate (100 s−1), kaolin accelerated onset of fibrin formation by ~100 sec when compared to collagen alone (250 sec vs. 350 sec), with little effect on platelet deposition. Even with kaolin present, arterial wall shear rate (1000 s−1) delayed and suppressed fibrin formation compared to venous wall shear rate. A comparison of surfaces for extrinsic activation (tissue factor TF/collagen) versus contact activation (kaolin/collagen) that each generated equal platelet deposition at 100 s−1 revealed: (1) TF surfaces promoted much faster fibrin onset (at 100 sec) and more endpoint fibrin at 600 sec at either 100 s−1 or 1000 s−1, and (2) kaolin and TF surfaces had a similar sensitivity for reduced fibrin deposition at 1000 s−1 (compared to fibrin formed at 100 s−1) despite differing coagulation triggers. Anti-platelet drugs inhibiting P2Y1, P2Y12, cyclooxygenase-1 or activating IP-receptor or guanylate cyclase reduced platelet and fibrin deposition on kaolin/collagen. Since FXIIa or FXIa inhibition may offer safe antithrombotic therapy, especially for biomaterial thrombosis, these defined collagen/kaolin surfaces may prove useful in drug screening tests or in clinical diagnostic assays of blood under flow conditions. PMID:25303860

  5. Contact activation of blood coagulation on a defined kaolin/collagen surface in a microfluidic assay.

    PubMed

    Zhu, Shu; Diamond, Scott L

    2014-12-01

    Generation of active Factor XII (FXIIa) triggers blood clotting on artificial surfaces and may also enhance intravascular thrombosis. We developed a patterned kaolin (0 to 0.3 pg/μm(2))/type 1 collagen fibril surface for controlled microfluidic clotting assays. Perfusion of whole blood (treated only with a low level of 4 μg/mL of the XIIa inhibitor, corn trypsin inhibitor) drove platelet deposition followed by fibrin formation. At venous wall shear rate (100 s(-1)), kaolin accelerated onset of fibrin formation by ~100 sec when compared to collagen alone (250 sec vs. 350 sec), with little effect on platelet deposition. Even with kaolin present, arterial wall shear rate (1000 s(-1)) delayed and suppressed fibrin formation compared to venous wall shear rate. A comparison of surfaces for extrinsic activation (tissue factor TF/collagen) versus contact activation (kaolin/collagen) that each generated equal platelet deposition at 100 s(-1) revealed: (1) TF surfaces promoted much faster fibrin onset (at 100 sec) and more endpoint fibrin at 600 sec at either 100 s(-1) or 1000 s(-1), and (2) kaolin and TF surfaces had a similar sensitivity for reduced fibrin deposition at 1000 s(-1) (compared to fibrin formed at 100 s(-1)) despite differing coagulation triggers. Anti-platelet drugs inhibiting P2Y1, P2Y12, cyclooxygenase-1 or activating IP-receptor or guanylate cyclase reduced platelet and fibrin deposition on kaolin/collagen. Since FXIIa or FXIa inhibition may offer safe antithrombotic therapy, especially for biomaterial thrombosis, these defined collagen/kaolin surfaces may prove useful in drug screening tests or in clinical diagnostic assays of blood under flow conditions. PMID:25303860

  6. A titanium surface with nano-ordered spikes and pores enhances human dermal fibroblastic extracellular matrix production and integration of collagen fibers.

    PubMed

    Yamada, Masahiro; Kato, Eiji; Yamamoto, Akiko; Sakurai, Kaoru

    2016-02-02

    The acquisition of substantial dermal sealing determines the prognosis of percutaneous titanium-based medical devices or prostheses. A nano-topographic titanium surface with ordered nano-spikes and pores has been shown to induce periodontal-like connective tissue attachment and activate gingival fibroblastic functions. This in vitro study aimed to determine whether an alkali-heat (AH) treatment-created nano-topographic titanium surface could enhance human dermal fibroblastic functions and binding strength to the deposited collagen on the titanium surface. The surface topographies of commercially pure titanium machined discs exposed to two different AH treatments were evaluated. Human dermal fibroblastic cultures grown on the discs were evaluated in terms of cellular morphology, proliferation, extracellular matrix (ECM) and proinflammatory cytokine synthesis, and physicochemical binding strength of surface-deposited collagen. An isotropically-patterned, shaggy nano-topography with a sponge-like inner network and numerous well-organized, anisotropically-patterned fine nano-spikes and pores were observed on each nano-topographic surface type via scanning electron microscopy. In contrast to the typical spindle-shaped cells on the machined surfaces, the isotropically- and anisotropically-patterned nano-topographic titanium surfaces had small circular/angular cells containing contractile ring-like structures and elongated, multi-shaped cells with a developed cytoskeletal network and multiple filopodia and lamellipodia, respectively. These nano-topographic surfaces enhanced dermal-related ECM synthesis at both the protein and gene levels, without proinflammatory cytokine synthesis or reduced proliferative activity. Deposited collagen fibers were included in these surfaces and sufficiently bound to the nano-topographies to resist the physical, enzymatic and chemical detachment treatments, in contrast to machined surfaces. Well-organized, isotropically

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. Minocycline attenuates Aβ oligomers-induced pro-inflammatory phenotype in primary microglia while enhancingfibrils phagocytosis.

    PubMed

    El-Shimy, Ismail Amr; Heikal, Ola Ahmed; Hamdi, Nabila

    2015-11-16

    Microglia, the brain innate immune cells, are activated in response to amyloid beta (Aβ) resulting in neuroinflammation in AD brains. Recently, two phenotypes have been described for microglia: the pro-inflammatory classical and the anti-inflammatory alternative. Changes in microglia phenotype that control their phagocytic function are yet to be determined. The highly neurotoxic Aβ oligomers (oAβ) formed at an early disease stage induce pro-inflammatory microglia activation releasing neurotoxic mediators and contributing to neurodegeneration. A novel strategy for AD treatment is to attenuate microglia-induced inflammation while maintaining efficient Aβ clearance. Minocycline effectively crosses the blood-brain barrier and has widely reported neuroprotective effects. Yet, its exact mechanism of neuroprotection and its effects on microglia are still unknown. The aim of this study is to investigate the effect of minocycline on the phagocytic uptake of fAβ by primary microglia in relation to their activation state in an inflammatory milieu generated by oAβ or LPS. The study shows that minocycline is able to attenuate oAβ-induced neuroinflammatory response of microglia by inhibiting their pro-inflammatory phenotype activation. In addition, a significant enhancement of fAβ phagocytosis by minocycline- treated microglia is reported for the first time, providing novel insight into its neuroprotective role in AD.

  12. Enhanced Sarcoplasmic Reticulum Ca2+-leak and Increased Na+-Ca2+-Exchanger Function Underlie Delayed Afterdepolarizations in Patients with Chronic Atrial Fibrillation

    PubMed Central

    Voigt, Niels; Li, Na; Wang, Qiongling; Wang, Wei; Trafford, Andrew W.; Abu-Taha, Issam; Sun, Qiang; Wieland, Thomas; Ravens, Ursula; Nattel, Stanley; Wehrens, Xander HT.; Dobrev, Dobromir

    2015-01-01

    Background Delayed afterdepolarizations (DADs) carried by Na+-Ca2+-exchange current (INCX) in response to sarcoplasmic reticulum (SR) Ca2+-leak can promote atrial fibrillation (AF). The mechanisms leading to DADs in AF-patients have not been defined. Methods and Results Protein levels (Western-blot), membrane-currents and action-potentials (patch-clamp), and [Ca2+]i (Fluo-3) were measured in right-atrial samples from 77 sinus-rhythm (Ctl) and 69 chronic-AF (cAF) patients. Diastolic [Ca2+]i and SR-Ca2+-content (integrated INCX during caffeine-induced-Ca2+-transient [cCaT]) were unchanged, whereas diastolic SR Ca2+-leak, estimated by blocking RyR2 with tetracaine, was ~50% higher in cAF vs. Ctl. Single-channel recordings from atrial RyR2 reconstituted into lipid-bilayers revealed enhanced open-probability in cAF-samples, providing a molecular basis for increased SR Ca2+-leak. Calmodulin-expression (+60%), CaMKII-autophosphorylation at Thr287 (+40%) and RyR2-phosphorylation at Ser2808 (PKA/CaMKII-site, +236%) and Ser2814 (CaMKII-site, +77%) were increased in cAF. The selective CaMKII-blocker KN-93 decreased SR Ca2+-leak, the frequency of spontaneous Ca2+-release events and RyR2 open-probability in cAF, whereas PKA-inhibition with H-89 was ineffective. Knock-in mice with constitutively-phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca2+-sparks and increased susceptibility to pacing-induced AF vs. controls. The relationship between [Ca2+]i and INCX-density revealed INCX-upregulation in cAF. Spontaneous Ca2+-release events accompanied by inward INCX-currents and DADs/triggered-activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca2+]i (diastolic [Ca2+]i–voltage coupling gain) was higher in cAF vs. Ctl. Conclusions Enhanced SR Ca2+-leak through CaMKII-hyperphosphorylated RyR2, in combination with larger INCX for a given SR Ca2+-release and increased diastolic [Ca2+]i–voltage coupling gain, cause AF-promoting atrial

  13. 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

  14. 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

  15. 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.

  16. 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

  17. Sodium Nitroprusside-enhanced Cardiopulmonary Resuscitation Facilitates Intra-Arrest Therapeutic Hypothermia in a Porcine Model of Prolonged Ventricular Fibrillation

    PubMed Central

    Debaty, Guillaume; Matsuura, Timothy R.; Bartos, Jason A.; Rees, Jennifer N.; McKnite, Scott H.; Lick, Michael; Boucher, François; Yannopoulos, Demetris

    2014-01-01

    Objectives The aim of this study was to assess the effect of Sodium Nitroprusside-enhanced Cardiopulmonary Resuscitation (SNPeCPR) on heat exchange during surface cooling. We hypothesized that SNPeCPR would decrease the time required to reach brain temperature < 35 °C compared to Active Compression-Decompression plus Impedance Threshold Device (ACD-ITD) CPR alone, in the setting of intra-CPR cooling. We further hypothesized that the addition of epinephrine during SNPeCPR would mitigate heat exchange. Design Prospective randomized animal investigation. Setting Preclinical animal laboratory. Subjects Female farm pigs (n = 28) Interventions After 10 minutes of untreated VF, animals were randomized to 3 different protocols: SNPeCPR (n = 8), SNPeCPR plus epinephrine (SNPeCPR+EPI, n = 10), and ACD-ITD alone (Control, n = 10). All animals received surface cooling at the initiation of CPR. SNPeCPR included ACD-ITD plus abdominal binding and 2 mg of SNP at 1, 4 and 8 minutes of CPR. No epinephrine was used during CPR in the SNPeCPR group. Control and SNPeCPR+EPI groups received 0.5 mg of epinephrine at minute 4.5 and 9 of CPR. Defibrillation occurred after 10 minutes of CPR. After ROSC, an Arctic Sun® was applied at maximum cooling on all animals. The primary endpoint was the time required to reach brain temperature < 35 °C beginning from the time of CPR initiation. Data are presented as mean ± SEM. Results The time required to reach a brain temperature of 35°C was decreased with SNPeCPR vs. Control or SNPeCPR+EPI (24 ± 6 min, 63 ± 8 min, and 50 ± 9 min, respectively, p = 0.005). Carotid blood flow was higher during CPR in the SNPeCPR group (83 ± 15 ml/min versus 26 ± 7 and 35 ± 5 in the Control and SNPeCPR+EPI group, respectively, p=0.001). Conclusion This study demonstrates that SNPeCPR facilitates intra-CPR hypothermia. The addition of epinephrine to SNPeCPR during CPR reduced its improvement in heat exchange. PMID:25525755

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. Next generation bone tissue engineering: non-viral miR-133a inhibition using collagen-nanohydroxyapatite scaffolds rapidly enhances osteogenesis

    NASA Astrophysics Data System (ADS)

    Mencía Castaño, Irene; Curtin, Caroline M.; Duffy, Garry P.; O’Brien, Fergal J.

    2016-06-01

    Bone grafts are the second most transplanted materials worldwide at a global cost to healthcare systems valued over $30 billion every year. The influence of microRNAs in the regenerative capacity of stem cells offers vast therapeutic potential towards bone grafting; however their efficient delivery to the target site remains a major challenge. This study describes how the functionalisation of porous collagen-nanohydroxyapatite (nHA) scaffolds with miR-133a inhibiting complexes, delivered using non-viral nHA particles, enhanced human mesenchymal stem cell-mediated osteogenesis through the novel focus on a key activator of osteogenesis, Runx2. This study showed enhanced Runx2 and osteocalcin expression, as well as increased alkaline phosphatase activity and calcium deposition, thus demonstrating a further enhanced therapeutic potential of a biomaterial previously optimised for bone repair applications. The promising features of this platform offer potential for a myriad of applications beyond bone repair and tissue engineering, thus presenting a new paradigm for microRNA-based therapeutics.

  8. Next generation bone tissue engineering: non-viral miR-133a inhibition using collagen-nanohydroxyapatite scaffolds rapidly enhances osteogenesis

    PubMed Central

    Mencía Castaño, Irene; Curtin, Caroline M.; Duffy, Garry P.; O’Brien, Fergal J.

    2016-01-01

    Bone grafts are the second most transplanted materials worldwide at a global cost to healthcare systems valued over $30 billion every year. The influence of microRNAs in the regenerative capacity of stem cells offers vast therapeutic potential towards bone grafting; however their efficient delivery to the target site remains a major challenge. This study describes how the functionalisation of porous collagen-nanohydroxyapatite (nHA) scaffolds with miR-133a inhibiting complexes, delivered using non-viral nHA particles, enhanced human mesenchymal stem cell-mediated osteogenesis through the novel focus on a key activator of osteogenesis, Runx2. This study showed enhanced Runx2 and osteocalcin expression, as well as increased alkaline phosphatase activity and calcium deposition, thus demonstrating a further enhanced therapeutic potential of a biomaterial previously optimised for bone repair applications. The promising features of this platform offer potential for a myriad of applications beyond bone repair and tissue engineering, thus presenting a new paradigm for microRNA-based therapeutics. PMID:27297802

  9. Stellar fibril magnetic systems. I - Reduced energy state

    NASA Technical Reports Server (NTRS)

    Parker, E. N.

    1984-01-01

    The remarkable fibril structure of the magnetic fields at the surface of the sun (with fibrils compressed to 1,000-2,000 gauss) lies outside existing statistical theories of magnetohydrodynamic turbulence. The total energy of the fibril field is enhanced by a factor of more than 100 above the energy for the mean field in a continuum state. The magnetic energy density within a fibril is of the order of 100 times the local kinetic energy density, so that no simple application of equipartition principles is possible. It is pointed out that the total energy of the atmosphere (thermal + gravitational + magnetic) is reduced by the fibril state of the field by avoiding the magnetic inhibition of the convective overturning, suggesting that the formation of the observed intense fibril state may be in response to the associated energy reduction. Calculation of the minimum total energy of a polytropic atmosphere permeated by magnetic fibrils yields theoretical fibril fields of the order of 1-5 kilogauss when characteristics appropriate to the solar convective zone are introduced, in rough agreement with the actual fields of 1-2 kilogauss. The polytrope model, although crude, establishes that a large reduction in total energy is made possible by the fibril state.

  10. 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

  11. Blockade of interleukin-6 receptor enhances the anti-arthritic effect of glucocorticoids without decreasing bone mineral density in mice with collagen-induced arthritis.

    PubMed

    Suzuki, M; Yoshida, H; Hashizume, M; Tanaka, K; Matsumoto, Y

    2015-11-01

    In a mouse arthritis model, we investigated whether interleukin-6 receptor (IL-6R) blockade would enhance the anti-arthritic effect of glucocorticoids (GCs). DBA/1J mice were immunized with type II collagen (CII), and were treated with prednisolone (PSL) and/or anti-mouse IL-6R antibody (MR16-1). Also, the effects of IL-6 on gene expression and the nuclear translocation of glucocorticoid receptors (GRs) were examined in cultured cells treated with dexamethasone (DEX). PSL reduced the arthritis score dose-dependently in the collagen-induced arthritis (CIA) mouse model. The arthritis score in the PSL (3 mg/kg) + MR16-1 group was lower than in the PSL (3 mg/kg) group, and at the same level as in the PSL (6 mg/kg) group. Lumbar vertebra bone mineral density (BMD) was decreased significantly in CIA mice and was higher in the PSL (3 mg/kg) + MR16-1 group than in the PSL (6 mg/kg) group. In the in-vitro synovial cells, IL-6 pretreatment attenuated the inhibitory effect of DEX on cyclooxygenase (COX)-2 expression and inhibited the nuclear translocation of GR induced by DEX. In contrast, in MC3T3-E1 osteoblastic cells, IL-6 pretreatment exacerbated the decrease in expression of osteocalcin and the increase in expression of receptor activator of nuclear factor kappa-B ligand (RANKL) by DEX. We demonstrated that IL-6 signalling blockade by an anti-IL-6R antibody can augment the anti-arthritic effect of GCs and inhibit the bone loss they cause. PMID:26201536

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. Collagen matrix-induced expression of integrin αVβ3 in circulating angiogenic cells can be targeted by matricellular protein CCN1 to enhance their function.

    PubMed

    McNeill, Brian; Vulesevic, Branka; Ostojic, Aleksandra; Ruel, Marc; Suuronen, Erik J

    2015-04-01

    Circulating angiogenic cells (CACs) play an important role in vascular homeostasis and hold therapeutic promise for treating a variety of cardiovascular diseases. However, further improvements are needed because the effects of CAC therapy remain minimal or transient. The regenerative potential of these cells can be improved by culture on a collagen-based matrix through the up-regulation of key integrin proteins. We found that human CAC function was enhanced by using the matricellular protein CCN1 (CYR61/CTGF/NOV family member 1) to target integrin αV and β3, which are up-regulated on matrix. Compared to matrix-cultured CACs, CCN1-matrix CACs exhibited a 2.2-fold increase in cell proliferation, 1.8-fold greater migration toward VEGF, and 1.7-fold more incorporation into capillary-like structures in an angiogenesis assay. In vivo, intramuscular injection of CCN1-matrix-cultured CACs into ischemic hind limbs of CD-1 nude mice resulted in blood flow recovery to 80% of baseline, which was greater than matrix-cultured CACs (66%) and PBS (35%) treatment groups. Furthermore, transplanted CCN1-matrix CACs exhibited greater engraftment (11-fold) and stimulated the up-regulation of survival and angiogenic genes (>3-fold). These findings reveal the importance of cell-matrix interactions in regulating CAC function and also reveal a mechanism by which these may be exploited to enhance cell therapies for ischemic disease.

  17. Macromolecular crowding favors the fibrillization of β2-microglobulin by accelerating the nucleation step and inhibiting fibril disassembly.

    PubMed

    Luo, Xu-Dong; Kong, Fan-Lou; Dang, Hai-Bin; Chen, Jie; Liang, Yi

    2016-11-01

    Hemodialysis-associated amyloidosis (HAA) involves the fibrillization of β2-microglobulin (β2M) and occurs in crowded physiological environments. However, how macromolecular crowding affects amyloid formation of β2M remains elusive. Here we study the effects of macromolecular crowding on amyloid formation and fibril disassembly of wild-type human β2M and its pathogenic mutant ΔN6. At strongly acidic pH2.5, the presence of a strong crowding agent (Ficoll 70 or dextran 70) not only dramatically accelerates the fibrillization of both wild-type β2M and its ΔN6 variant by reducing the lag time to a large extent, indicating the acceleration of the nucleation phase, but also remarkably increases the amount of β2M fibrils. At weakly acidic pH6.2, such an enhancing effect of macromolecular crowding on fibril formation is only observed for pathogenic mutant ΔN6, but not for wild-type β2M which does not form amyloid fibrils in the absence and presence of a crowding agent. Thus, we propose that the monomers of β2M form the nuclei, which is enhanced by macromolecular crowding, followed by the step of fibril elongation. Furthermore, at physiological pH, macromolecular crowding remarkably inhibits β2M fibril disassembly by decreasing rate constants corresponding to fast and slow stages of fibril disaggregation. Our data demonstrate that macromolecular crowding favors the fibrillization of β2M by accelerating the nucleation step and inhibiting fibril disassembly. Our findings provide clear evidence for the pathology of HAA that macromolecular crowding should be taken into account. PMID:27481166

  18. Effects of macromolecular crowding and osmolyte on human Tau fibrillation.

    PubMed

    Wu, Yingying; Teng, Ningning; Li, Sen

    2016-09-01

    Tau fibrillation is reported to be involved in neurodegenerative disorders, such as Alzheimer's disease, in which the natural environment is very crowded in the cells. Understanding the role of crowding environments in regulating Tau fibrillation is of great importance for elucidating the etiology of these diseases. In this experiment, the effects of macromolecular crowding and osmolyte reagents in the crowding environment on Tau fibrillation were studied by thioflavin T binding, SDS-PAGE and TEM assays. Ficoll 70 and Dextran 70 of different concentrations were used as macromolecular crowding reagents inside the cells and showed a strong enhancing effect on the fibrillation of normal and hyperphosphorylated Tau. The enhancing effect of Dextran is stronger than that of Ficoll 70 at the same concentration. In addition, the cellular osmolyte sucrose was found to protect Tau against fibrillation, and inhibit the enhancing effect of macromolecular crowding on Tau fibrillation. A possible model for the fibrillation process of Tau and the effect of macromolecular crowding and osmolyte on this process was proposed based on these experimental results. The information obtained from our study can enhance the understanding of how proteins aggregate and avoid aggregation in crowded physiological environments and might lead to a better understanding of the molecular mechanisms of Alzheimer's disease in vivo.

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

    NASA Astrophysics Data System (ADS)

    Jee, Sang Soo

    Bone is an interpenetrating inorganic/organic composite that consists of mineralized collagen fibrils, which is hierarchically organized into various structures. The structure of mineralized collagen fibril, in which nano-crystals of hydroxyapatite are embedded within the collagen fibrils, provides remarkable mechanical and bio-resorptive properties. Therefore, there have been many attempts to produce collagen-hydroxyapatite composites having a bone-like structure. However, duplication of even the most fundamental level of bone structure has not been easily achieved by conventional nucleation and growth techniques, which are based on the most widely accepted hypothesis of bone mineralization. In nature, the collagen fibril is mineralized via intrafibrillar mineralization, which produces preferentially oriented hydroxyapatite nano-crystals occupying the interstices in collagen fibrils. Our group has demonstrated that intrafibrillar mineralization can be achieved by using a new method based on the Polymer-Induced Liquid-Precursor (PILP) mineralization process. In the PILP process, a poly-anionic additive can produce an amorphous calcium phosphate precursor which enables us to achieve intrafibrillar mineralization of collagen. It is thought that the precursor is pulled into the interstices of the collagen fibrils via capillary forces, and upon solidification and crystallization of the precursor produces an interpenetrating composite with the nanostructured architecture of bone. In this dissertation, to demonstrate the effectiveness of the PILP process on the intrafibrillar mineralization of collagen fibril, various collagen scaffolds, such as turkey tendon, bovine tendon and synthetic collagen sponge, were mineralized by the PILP process. Various poly-aspartates with different molecular weight were also used for the optimization of the PILP process for the mineralization of the collagen scaffolds. With the systematic researches, we discovered that the molecular weight

  20. 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

  1. 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.

  2. 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 ...

  3. 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

  4. The molecular structure of human tissue type XV presents a unique conformation among the collagens

    PubMed Central

    Myers, Jeanne C.; Amenta, Peter S.; Dion, Arnold S.; Sciancalepore, Justin P.; Nagaswami, Chandrasekaran; Weisel, John W.; Yurchenco, Peter D.

    2007-01-01

    Establishing the structure of the non-fibrillar collagens has provided a unique perspective to understanding their specialized functions in the extracellular matrix. These proteins exhibit very diverse conformations and supramolecular assemblies. Type XV collagen is a large macromolecule distinguished by a highly interrupted collagenous domain and many utilized sites of attachment for CS (chondroitin sulfate) and HS (heparan sulfate) glycosaminoglycan chains. It is present in most basement membrane zones of human tissues, where it is found closely associated with large collagen fibrils. To determine the molecular shape and organization of type XV, the protein was purified from human umbilical cords by salt extraction, and by ion-exchange and antibody-affinity chromatography. The representation of type XV in one of its most abundant tissue sources is estimated at only (1–2)×10−4% of dry weight. The molecules examined by transmission electron microscopy after rotary shadowing were visualized in multiple forms. Relatively few type XV monomers appeared elongated and kinked; most molecules were found in a knot/figure-of-eight/pretzel configuration not previously described for a collagen. Collective measurements of these populations revealed an average length of 193±16 nm. At the N-terminal end, identified by C-terminal antibody binding, were three 7.7 nm-diameter spheres, corresponding to TSPN-1 (N-terminal module of thrombospondin-1) modules, and attached to the collagen backbone by a short linker. The type XV monomers show the ability to self-assemble into higher-order structures. Some were arranged in complex clusters, but simpler oligomers, which may represent intermediates, were observed in a cruciform pattern with intermolecular binding sites that probably originate in the interruption sequences. The morphology of type XV is thus the antithesis of the fibrillar collagens, and the shape attains the required flexibility to form the spectrum of

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. Autologous circulating angiogenic cells treated with osteopontin and delivered via a collagen scaffold enhance wound healing in the alloxan-induced diabetic rabbit ear ulcer model

    PubMed Central

    2013-01-01

    Introduction Diabetic foot ulceration is the leading cause of amputation in people with diabetes mellitus. Peripheral vascular disease is present in the majority of patients with diabetic foot ulcers. Despite standard treatments there exists a high amputation rate. Circulating angiogenic cells previously known as early endothelial progenitor cells are derived from peripheral blood and support angiogenesis and vasculogenesis, providing a potential topical treatment for non-healing diabetic foot ulcers. Methods A scaffold fabricated from Type 1 collagen facilitates topical cell delivery to a diabetic wound. Osteopontin is a matricellular protein involved in wound healing and increases the angiogenic potential of circulating angiogenic cells. A collagen scaffold seeded with circulating angiogenic cells was developed. Subsequently the effect of autologous circulating angiogenic cells that were seeded in a collagen scaffold and topically delivered to a hyperglycemic cutaneous wound was assessed. The alloxan-induced diabetic rabbit ear ulcer model was used to determine healing in response to the following treatments: collagen seeded with autologous circulating angiogenic cells exposed to osteopontin, collagen seeded with autologous circulating angiogenic cells, collagen alone and untreated wound. Stereology was used to assess angiogenesis in wounds. Results The cells exposed to osteopontin and seeded on collagen increased percentage wound closure as compared to other groups. Increased angiogenesis was observed with the treatment of collagen and collagen seeded with circulating angiogenic cells. Conclusions These results demonstrate that topical treatment of full thickness cutaneous ulcers with autologous circulating angiogenic cells increases wound healing. Cells exposed to the matricellular protein osteopontin result in superior wound healing. The wound healing benefit is associated with a more efficient vascular network. This topical therapy provides a potential novel

  13. Segmentation and measurement of collagen fibers for shoulder and joint therapy studies

    SciTech Connect

    Mascio, L.

    1994-11-15

    Various shoulder instabilities are debilitating, especially in individuals who perform overhead activities. Thermal modification of soft tissues in joints may allow precise alteration of these tissues` mechanical and/or structural properties to enhance joint function without inducing cell death or an inflammatory response. Several studies have evaluated laser energy for tissue welding. The collective findings are promising, and the next step is to identify the mechanisms responsible for laser-induced capsular tissue alternation, and the short- and long-term effects of non-ablative laser energy on joint capsular tissue. One step toward this goal is to compare the effect of three laser energy densities on the histologic properties of the tissue evaluating the architecture of the collagen (including density, fibril diameter distribution, and interfibrillar space) in sheep at various time intervals after surgery. The specific computer algorithms that are being used to make these measurements will be described.

  14. Poly(NaSS) functionalization modulates the conformation of fibronectin and collagen type I to enhance osteoblastic cell attachment onto Ti6Al4V.

    PubMed

    Felgueiras, Helena P; Sommerfeld, Sven D; Murthy, N Sanjeeva; Kohn, Joachim; Migonney, Véronique

    2014-08-12

    Functionalization of surfaces with poly(sodium styrenesulfonate) (poly(NaSS)) has recently been found to enhance osteointegration of implantable materials. Radical polymerization of poly(NaSS) on titanium (Ti)-based substrates has been used to improve their long-term performance by preventing fibrosis and consequently implant loosening. However, the influence of the sulfonate groups on the early cell behavior and the associated molecular phenomena remains to be understood. In this work, we used quartz crystal microbalance with dissipation (QCM-D) to elucidate the role of poly(NaSS) in enhancing osteoblastic cell attachment. This was measured by following the cell attachment using the MC3T3-E1 cell line, on fetal bovine serum (FBS) preadsorbed surfaces and on substrates adsorbed with a series of relevant proteins, bovine serum albumin (BSA), fibronectin (Fn), and collagen type I (Col I). Comparison of the performance of poly(NaSS) with other clinically important substrates such as Ti alloy Ti6Al4V, gold, and poly(desamino-tyrosyl-tyrosine ethyl ester carbonate) (poly(DTEc)) indicates poly(NaSS) to be a superior substrate for MC3T3-E1 cells attachment. This attachment was found to be integrin mediated in the presence of Fn and Col I. Antibodies specific to the RGD peptide and the N- and C-terminal HB-binding domains reacted more intensively with Fn adsorbed on poly(NaSS). Fn adapts a conformation favorable to RGD mediated cell attachment when adsorbed onto poly(NaSS).

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. Collagen shield delivery of trifluorothymidine.

    PubMed

    Gussler, J R; Ashton, P; VanMeter, W S; Smith, T J

    1990-11-01

    Corneal and aqueous levels of topically applied trifluorothymidine (F3T) were compared with and without the collagen shield in normal and damaged rabbit eyes. Shields were presoaked in 1% F3T for 15 minutes prior to application. Rabbits received either a presoaked shield, 1% F3T drops every two hours, or both. Corneal and aqueous levels of F3T were measured at 30 minutes, two, four, and eight hours. If 5 mm epithelial defects were created, the collagen shield and topical F3T drops produced significantly higher levels of F3T than drops alone at all periods tested (P less than .05). A presoaked shield alone produced greater levels of F3T than drops alone at 30 minutes and two hours (P less than .05). Collagen shields did not enhance F3T levels in eyes with intact epithelium. Implications for treatment of herpetic keratouveitis are discussed.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

    Aspects of the ultrastructural interaction between collagen and mineral crystals in embryonic chick bone have been examined by the novel technique of high voltage electron microscopic tomography to obtain three-dimensional information concerning extracellular calcification in this tissue. Newly mineralizing osteoid along periosteal surfaces of mid-diaphyseal regions from normal chick tibiae was embedded, cut into 0.25 microns thick sections, and documented at 1.0 MV in the Albany AEI-EM7 high voltage electron microscope. The areas of the tissue studied contained electron dense mineral crystals associated with collagen fibrils, some marked by crystals disposed along their cylindrically shaped lengths. Tomographic reconstructions of one site with two mineralizing fibrils were computed from a 5 degrees tilt series of micrographs over a +/- 60 degrees range. Reconstructions showed that the mineral crystals were platelets of irregular shape. Their sizes were variable, measured here up to 80 x 30 x 8 nm in length, width, and thickness, respectively. The longest crystal dimension, corresponding to the c-axis crystallographically, was generally parallel to the collagen fibril long axis. Individual crystals were oriented parallel to one another in each fibril examined. They were also parallel in the neighboring but apparently spatially separate fibrils. Crystals were periodically (approximately 67 nm repeat distance) arranged along the fibrils and their location appeared to correspond to collagen hole and overlap zones defined by geometrical imaging techniques. The crystals appeared to be continuously distributed along a fibril, their size and number increasing in a tapered fashion from a relatively narrow tip containing smaller and infrequent crystals to wider regions having more densely packed and larger crystals. Defined for the first time by direct visual 3D imaging, these data describe the size, shape, location, orientation, and development of early crystals in normal

  8. 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

  9. 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.

  10. Single-Dose Electrospun Nanoparticles-in-Nanofibers Wound Dressings with Enhanced Epithelialization, Collagen Deposition, and Granulation Properties.

    PubMed

    Ali, Isra H; Khalil, Islam A; El-Sherbiny, Ibrahim M

    2016-06-15

    Phenytoin (Ph), an antiepileptic drug, was reported to exhibit high wound healing activity. However, its limited solubility, bioavailability, and inefficient distribution during topical administration limit its use. Therefore, this study aims to develop new single-dose electrospun nanoparticles-in-nanofibers (NPs-in-NFs) wound dressings that allow a well-controlled release of Ph. These NPs-in-NFs systems are based on enhanced chitosan (CS)/poly(ethylene oxide) (PEO) electrospun nanofibers (NFs) incorporating optimized Ph-loaded nanocarriers. First, a study was conducted to investigate Ph loading efficiency into polymeric nanocarriers of different types; pluronic nanomicelles and poly(lactic-co-glycolic) acids nanoparticles (PLGA NPs). The drug release profile from the nanocarriers was further optimized via lecithin coating. Second, different electrospinning parameters were manipulated to fabricate beads-free homogeneous NFs with optimized polymer ratios. Plain and Ph-loaded nanocarriers were characterized using Fourier transform infrared (FTIR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamic light scattering (DLS), and scanning electron microscopy (SEM). Both entrapment efficiency of Ph (EE%) and its release profile in phosphate buffer saline (PBS; pH 5.5), simulating the wound environment, were studied. Biodegradability, swelling, vapor permeability, and porosity of the developed Ph-loaded NPs-in-NFs wound dressings were investigated. Morphology of the NPs-in-NFs was also studied using SEM and confocal laser microscopy (CLSM). Besides, the release profiles of Ph from the optimized NPs-in-NFs were assessed. The newly developed wound dressings were evaluated in vitro for their cytotoxicity using human fibroblasts and in vivo using a wound healing mice model. Nanocarriers with particle size ranging from 100 to 180 nm were successfully prepared. All nanocarriers attained a high drug entrapment efficiency exceeding 94% and showed

  11. 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.

  12. 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

  13. Estrogen enhances the bone regeneration potential of periodontal ligament stem cells derived from osteoporotic rats and seeded on nano-hydroxyapatite/collagen/poly(L-lactide)

    PubMed Central

    E, LING-LING; XU, WEN-HUAN; FENG, LIN; LIU, YI; CAI, DONG-QING; WEN, NING; ZHENG, WEN-JIE

    2016-01-01

    This study investigated the effects of estrogen on the bone regeneration potential of periodontal ligament stem cells (PDLSCs) derived from osteoporotic rats and seeded on a collagen-based composite scaffold [nano-hydroxyapatite/collagen/poly(L-lactide) (nHAC/PLA)]. For this purpose, 48 healthy 3-month-old Sprague-Dawley female rats were divided into 2 groups as follows: the bilaterally ovariectomized (OVX) rats and sham-operated rats. The PDLSCs were isolated at 3 months after surgery (by which time postmenopausal osteoporosis had developed). The effects of estrogen on the characteristics of these cells seeded in a culture plate and of the cells seeded on nHAC/PLA were then investigated. The PDLSC + nHAC/PLA constructs were implanted subcutaneously into the backs of severe combined immunodeficient (SCID) mice for 12 weeks in order to examine the role of estrogen in the bone formation ability of PDLSCs derived from osteoporotic rats. The results from methyl thiazolyl tetrazolium (MTT) assay revealed that the proliferation of the cells derived from the rats in the OVX group was significantly higher than that of the cells derived from the rats in the sham-operated group at the stage of logarithmic growth. The staining intensity of alkaline phosphatase (ALP) and the mineralization of the cells derived from the rats in the OVX group was significantly weaker than that of the cells from the rats in the sham-operated group. When the PDLSCs were seeded on nHAC/PLA, ALP activity, osteocalcin (OCN) secretion, mineral formation and the mRNA expression levels of ALP, OCN, estrogen receptor (ER)α and ERβ in the cells derived from the rats in the OVX group were markedly decreased. Treatment with 17β-estradiol (E2) significantly weakened the proliferative ability of the cells derived from the OVX group rats, and enhanced their osteogenic differentiation ability and the mRNA expression levels of ALP, OCN, ERα and ERβ. When the constructs were implanted into the backs of SCID

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

    The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold because the surface of the scaffold can determine the fate of stem cells. A conductive surface is required for a scaffold to direct stem cells toward neural differentiation. However, most conductive polymers are toxic and not amenable to biological degradation, which restricts the design of neural tissue engineering scaffolds. In this study, we used a bioactive three-dimensional (3D) porcine acellular dermal matrix (PADM), which is mainly composed of type I collagen, as a basic material and successfully assembled a layer of reduced graphene oxide (rGO) nanosheets on the surface of the PADM channels to obtain a porous 3D, biodegradable, conductive and biocompatible PADM-rGO hybrid neural tissue engineering scaffold. Compared with the PADM scaffold, assembling the rGO into the scaffold did not induce a significant change in the microstructure but endowed the PADM-rGO hybrid scaffold with good conductivity. A comparison of the neural differentiation of rat bone-marrow-derived mesenchymal stem cells (MSCs) was performed by culturing the MSCs on PADM and PADM-rGO scaffolds in neuronal culture medium, followed by the determination of gene expression and immunofluorescence staining. The results of both the gene expression and protein level assessments suggest that the rGO-assembled PADM scaffold may promote the differentiation of MSCs into neuronal cells with higher protein and gene expression levels after 7 days under neural differentiation conditions. This study demonstrated that the PADM-rGO hybrid scaffold is a promising scaffold for neural tissue engineering; this scaffold can not only support the growth of MSCs at a high proliferation rate but also enhance the differentiation of MSCs into neural cells.The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold

  15. Effect of immobilized collagen type IV on biological properties of endothelial cells for the enhanced endothelialization of synthetic vascular graft materials.

    PubMed

    Heo, Yunhoe; Shin, Young Min; Lee, Yu Bin; Lim, Youn Mook; Shin, Heungsoo

    2015-10-01

    Regeneration of healthy endothelium onto vascular graft materials is imperative for prevention of intimal hyperplasia and thrombogenesis. In this study, we investigated the effect of collagen type IV (COL-IV) immobilized onto electrospun nanofibers on modulation of endothelial cell (EC) function, as a potential signal to rapid endothelialization of vascular grafts. COL-IV is assembled in basement membrane underneath intimal layer and regulates morphogenesis of blood vessels. For immobilization of COL-IV, poly(l-lactic acid) (PLLA) nanofibers (PL) were prepared as a model vascular graft substrate, onto which acrylic acid (AAc) was then grafted by using gamma-ray irradiation. AAc graft was dependent on irradiation doses and AAc concentrations, which allowed us to select the condition of 5% (v/v) AAc and 10 kGy for further conjugation of COL-IV. COL-IV immobilization was proportionally controlled as a function of its concentration. Atomic force microscope (AFM) analysis qualitatively supported immobilization of COL-IV, demonstrating increase in root mean square roughness of the PL from 665.37 ± 13.20 nm to 1440.74 ± 33.24. However, the Young's modulus of nanofibers was retained as approximately 1 MPa, regardless of surface modification. The number of ECs attached on the nanofibers with immobilized COL-IV was significantly increased by 5 times (1052 ± 138 cells/mm(2)) from pristine PL (234 ± 41 cells/mm(2)). In addition, the effect of immobilized COL-IV was profound for enhancing proliferation and up-regulation of markers implicated in rapid endothelialization. Collectively, our results suggest that COL-IV immobilized onto electrospun PLLA nanofibers may serve as a promising instructive cue used in vascular graft materials.

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

    PubMed

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

    2016-01-28

    The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold because the surface of the scaffold can determine the fate of stem cells. A conductive surface is required for a scaffold to direct stem cells toward neural differentiation. However, most conductive polymers are toxic and not amenable to biological degradation, which restricts the design of neural tissue engineering scaffolds. In this study, we used a bioactive three-dimensional (3D) porcine acellular dermal matrix (PADM), which is mainly composed of type I collagen, as a basic material and successfully assembled a layer of reduced graphene oxide (rGO) nanosheets on the surface of the PADM channels to obtain a porous 3D, biodegradable, conductive and biocompatible PADM-rGO hybrid neural tissue engineering scaffold. Compared with the PADM scaffold, assembling the rGO into the scaffold did not induce a significant change in the microstructure but endowed the PADM-rGO hybrid scaffold with good conductivity. A comparison of the neural differentiation of rat bone-marrow-derived mesenchymal stem cells (MSCs) was performed by culturing the MSCs on PADM and PADM-rGO scaffolds in neuronal culture medium, followed by the determination of gene expression and immunofluorescence staining. The results of both the gene expression and protein level assessments suggest that the rGO-assembled PADM scaffold may promote the differentiation of MSCs into neuronal cells with higher protein and gene expression levels after 7 days under neural differentiation conditions. This study demonstrated that the PADM-rGO hybrid scaffold is a promising scaffold for neural tissue engineering; this scaffold can not only support the growth of MSCs at a high proliferation rate but also enhance the differentiation of MSCs into neural cells. PMID:26750302

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

    PubMed

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

    2016-01-28

    The cell-material interface is one of the most important considerations in designing a high-performance tissue engineering scaffold because the surface of the scaffold can determine the fate of stem cells. A conductive surface is required for a scaffold to direct stem cells toward neural differentiation. However, most conductive polymers are toxic and not amenable to biological degradation, which restricts the design of neural tissue engineering scaffolds. In this study, we used a bioactive three-dimensional (3D) porcine acellular dermal matrix (PADM), which is mainly composed of type I collagen, as a basic material and successfully assembled a layer of reduced graphene oxide (rGO) nanosheets on the surface of the PADM channels to obtain a porous 3D, biodegradable, conductive and biocompatible PADM-rGO hybrid neural tissue engineering scaffold. Compared with the PADM scaffold, assembling the rGO into the scaffold did not induce a significant change in the microstructure but endowed the PADM-rGO hybrid scaffold with good conductivity. A comparison of the neural differentiation of rat bone-marrow-derived mesenchymal stem cells (MSCs) was performed by culturing the MSCs on PADM and PADM-rGO scaffolds in neuronal culture medium, followed by the determination of gene expression and immunofluorescence staining. The results of both the gene expression and protein level assessments suggest that the rGO-assembled PADM scaffold may promote the differentiation of MSCs into neuronal cells with higher protein and gene expression levels after 7 days under neural differentiation conditions. This study demonstrated that the PADM-rGO hybrid scaffold is a promising scaffold for neural tissue engineering; this scaffold can not only support the growth of MSCs at a high proliferation rate but also enhance the differentiation of MSCs into neural cells.

  18. Effect of immobilized collagen type IV on biological properties of endothelial cells for the enhanced endothelialization of synthetic vascular graft materials.

    PubMed

    Heo, Yunhoe; Shin, Young Min; Lee, Yu Bin; Lim, Youn Mook; Shin, Heungsoo

    2015-10-01

    Regeneration of healthy endothelium onto vascular graft materials is imperative for prevention of intimal hyperplasia and thrombogenesis. In this study, we investigated the effect of collagen type IV (COL-IV) immobilized onto electrospun nanofibers on modulation of endothelial cell (EC) function, as a potential signal to rapid endothelialization of vascular grafts. COL-IV is assembled in basement membrane underneath intimal layer and regulates morphogenesis of blood vessels. For immobilization of COL-IV, poly(l-lactic acid) (PLLA) nanofibers (PL) were prepared as a model vascular graft substrate, onto which acrylic acid (AAc) was then grafted by using gamma-ray irradiation. AAc graft was dependent on irradiation doses and AAc concentrations, which allowed us to select the condition of 5% (v/v) AAc and 10 kGy for further conjugation of COL-IV. COL-IV immobilization was proportionally controlled as a function of its concentration. Atomic force microscope (AFM) analysis qualitatively supported immobilization of COL-IV, demonstrating increase in root mean square roughness of the PL from 665.37 ± 13.20 nm to 1440.74 ± 33.24. However, the Young's modulus of nanofibers was retained as approximately 1 MPa, regardless of surface modification. The number of ECs attached on the nanofibers with immobilized COL-IV was significantly increased by 5 times (1052 ± 138 cells/mm(2)) from pristine PL (234 ± 41 cells/mm(2)). In addition, the effect of immobilized COL-IV was profound for enhancing proliferation and up-regulation of markers implicated in rapid endothelialization. Collectively, our results suggest that COL-IV immobilized onto electrospun PLLA nanofibers may serve as a promising instructive cue used in vascular graft materials. PMID:26196092

  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. 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

  4. 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.

  5. Genetic Mutations in African Patients with Atrial Fibrillation: Rationale and Design of the Study of Genetics of Atrial Fibrillation in an African Population (SIGNAL)

    PubMed Central

    Bloomfield, Gerald S.; Temu, Tecla; Akwanalo, Constantine O.; Chen, Peng-Sheng; Emonyi, Wilfred; Heckbert, Susan R.; Koech, Myra M.; Manji, Imran; Shen, Changyu; Vatta, Matteo; Velazquez, Eric J.; Wessel, Jennifer; Kimaiyo, Sylvester; Inui, Thomas S.

    2015-01-01

    Background There is an urgent need to understand genetic associations with atrial fibrillation in ethnically diverse populations. There are no such data from sub-Saharan Africa, despite the fact that atrial fibrillation is one of the fastest-growing diseases. Moreover, patients with valvular heart disease are under-represented in studies of the genetics of atrial fibrillation. Methods We designed a case-control study of patients with and without a history of atrial fibrillation in Kenya. Cases with atrial fibrillation included those with and without valvular heart disease. Patients underwent clinical phenotyping and will have laboratory analysis and genetic testing of >240 candidate genes associated with cardiovascular diseases. A 12-month follow-up assessment will determine the groups’ morbidity and mortality. The primary analyses will describe genetic and phenotypic associations with atrial fibrillation. Results We recruited 298 participants: 72 (24%) with non-valvular atrial fibrillation, 78 (26%) with valvular atrial fibrillation and 148 (50%) controls without atrial fibrillation. The mean age of cases and controls were 53 and 48 years, respectively. Most (69%) participants were female. Controls more often had hypertension (45%) than those with valvular atrial fibrillation (27%). Diabetes and current tobacco smoking were uncommon. A history of stroke was present in 25% of cases and in 5% of controls. Conclusion This is the first study determining genetic associations in valvular and non-valvular atrial fibrillation in sub-Saharan Africa with a control population. The results advance knowledge about atrial fibrillation and will enhance international efforts to decrease atrial fibrillation-related morbidity. PMID:26385028

  6. NF-κB accumulation associated with COL1A1 transactivators defects during chronological aging represses type I collagen expression through a -112/-61-bp region of the COL1A1 promoter in human skin fibroblasts.

    PubMed

    Bigot, Nicolas; Beauchef, Gallic; Hervieu, Magalie; Oddos, Thierry; Demoor, Magali; Boumediene, Karim; Galéra, Philippe

    2012-10-01

    The aging process, especially of the skin, is governed by changes in the epidermal, dermo-epidermal, and dermal compartments. Type I collagen, which is the major component of dermis extracellular matrix (ECM), constitutes a prime target for intrinsic and extrinsic aging-related alterations. In addition, under the aging process, pro-inflammatory signals are involved and collagens are fragmented owing to enhanced matrix metalloproteinase activities, and fibroblasts are no longer able to properly synthesize collagen fibrils. Here, we demonstrated that low levels of type I collagen detected in aged skin fibroblasts are attributable to an inhibition of COL1A1 transcription. Indeed, on one hand, we observed decreased binding activities of specific proteins 1 and 3, CCAAT-binding factor, and human collagen-Krüppel box, which are well-known COL1A1 transactivators acting through the -112/-61-bp promoter sequence. On the other hand, the aging process was accompanied by elevated amounts and binding activities of NF-κB (p65 and p50 subunits), together with an increased number of senescent cells. The forced expression of NF-κB performed in young fibroblasts was able to establish an old-like phenotype by repressing COL1A1 expression through the short -112/-61-bp COL1A1 promoter and by elevating the senescent cell distribution. The concomitant decrease of transactivator functions and increase of transinhibitor activity is responsible for ECM dysfunction, leading to aging/senescence in dermal fibroblasts.

  7. 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.

  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. Co-delivery and controlled release of stromal cell-derived factor-1α chemically conjugated on collagen scaffolds enhances bone morphogenetic protein-2-driven osteogenesis in rats

    PubMed Central

    SUN, HAIPENG; WANG, JINMING; DENG, FEILONG; LIU, YUN; ZHUANG, XIUMEI; XU, JIAYUN; LI, LONG

    2016-01-01

    There has been considerable focus in investigations on the delivery systems and clinical applications of bone morphogenetic protein-2 (BMP-2) for novel bone formation. However, current delivery systems require high levels of BMP-2 to exert a biological function. There are several concerns in using of high levels of BMP-2, including safety and the high cost of treatment. Therefore, the development of strategies to decrease the levels of BMP-2 required in these delivery systems is required. In our previous studies, a controlled-release system was developed, which used Traut's reagent and the cross-linker, 4-(N-maleimi-domethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (Sulfo-SMCC), to chemically conjugate BMP-2 directly on collagen discs. In the current study, retention efficiency and release kinetics of stromal cell-derived factor-1α (SDF-1α) cross-linked on collagen scaffolds were detected. In addition, the osteogenic activity of SDF-1α and suboptimal doses of BMP-2 cross-linked on collagen discs following subcutaneous implantation in rats were evaluated. Independent two-tailed t-tests and one-way analysis of variance were used for analysis. In the present study, the controlled release of SDF-1α chemically conjugated on collagen scaffolds was demonstrated. By optimizing the concentrations of Traut's reagent and the Sulfo-SMCC cross-linker, a significantly higher level of SDF-1α was covalently retained on the collagen scaffold, compared with that retained using a physical adsorption method. Mesenchymal stem cell homing indicated that the biological function of the SDF-1α cross-linked on the collagen scaffolds remained intact. In rats, co-treatment with SDF-1α and a suboptimal dose of BMP-2 cross-linked on collagen scaffolds using this chemically conjugated method induced higher levels of ectopic bone formation, compared with the physical adsorption method. No ectopic bone formation was observed following treatment with a

  11. 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.

  12. 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

  13. 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

  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. 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

  16. 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.

  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. 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.

  19. 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

  20. 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.

  1. 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.

  2. Ingestion of BioCell Collagen®, a novel hydrolyzed chicken sternal cartilage extract; enhanced blood microcirculation and reduced facial aging signs

    PubMed Central

    Schwartz, Stephen R; Park, Joosang

    2012-01-01

    Skin aging and its clinical manifestation is associated with altered molecular metabolism in the extracellular matrix of the dermis. In a pilot open-label study, we investigated the effect of a dietary supplement, BioCell Collagen® (BCC), which contains a naturally occurring matrix of hydrolyzed collagen type II and low-molecular-weight hyaluronic acid and chondroitin sulfate, in 26 healthy females who displayed visible signs of natural and photoaging in the face. Daily supplementation with 1 g of BCC for 12 weeks led to a significant reduction of skin dryness/scaling (76%, P = 0.002) and global lines/wrinkles (13.2%, P = 0.028) as measured by visual/tactile score. Additionally, a significant increase in the content of hemoglobin (17.7%, P = 0.018) and collagen (6.3%, P = 0.002) in the skin dermis was observed after 6 weeks of supplementation. At the end of the study, the increase in hemoglobin remained significant (15%, P = 0.008), while the increase in collagen content was maintained, but the difference from baseline was not significant (3.5%, P = 0.134). This study provides preliminary data suggesting that dietary supplementation with BCC elicits several physiological events which can be harnessed to counteract natural photoaging processes to reduce visible aging signs in the human face. A controlled study is necessary to verify these observations. PMID:22956862

  3. Enriched Astaxanthin Extract from Haematococcus pluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts

    PubMed Central

    Chou, Hsin-Yu; Lee, Chelsea; Pan, Jian-Liang; Wen, Zhi-Hong; Huang, Shu-Hung; Lan, Chi-Wei John; Liu, Wang-Ta; Hour, Tzyh-Chyuan; Hseu, You-Cheng; Hwang, Byeong Hee; Cheng, Kuo-Chen; Wang, Hui-Min David

    2016-01-01

    Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE) from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA), and doxycycline. With sirius red stain