Chitosan: collagen sponges. In vitro mineralization

International Nuclear Information System (INIS)

Martins, Virginia da C.A.; Silva, Gustavo M.; Plepis, Ana Maria G.

2011-01-01

The regeneration of bone tissue is a problem that affects many people and scaffolds for bone tissue growth has been widely studied. The aim of this study was the in vitro mineralization of chitosan, chitosan:native collagen and chitosan:anionic collagen sponges. The sponges were obtained by lyophilization and mineralization was made by soaking the sponges in alternating solutions containing Ca 2+ and PO 4 3- . The mineralization was confirmed by infrared spectroscopy, energy dispersive X-ray and X-ray diffraction observing the formation of phosphate salts, possibly a carbonated hydroxyapatite since Ca/P=1.80. The degree of mineralization was obtained by thermogravimetry calculating the amount of residue at 750 deg C. The chitosan:anionic collagen sponge showed the highest degree of mineralization probably due to the fact that anionic collagen provides additional sites for interaction with the inorganic phase. (author)

Identification and characterization of the human type II collagen gene (COL2A1).

NARCIS (Netherlands)

K.S.E. Cheah (Kathryn); N.G. Stoker; J.R. Griffin; F.G. Grosveld (Frank); E. Solomon

1985-01-01

textabstractThe gene contained in the human cosmid clone CosHcol1, previously designated an alpha 1(I) collagen-like gene, has now been identified. CosHcol1 hybridizes strongly to a single 5.9-kilobase mRNA species present only in tissue in which type II collagen is expressed. DNA sequence analysis

Enhanced stabilization of collagen by furfural.

Science.gov (United States)

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) (pFurfural 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. Copyright © 2014 Elsevier B.V. All rights reserved.

Changes of MMP-1 and collagen type Ialpha1 by UVA, UVB and IRA are differentially regulated by Trx-1.

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Buechner, Nicole; Schroeder, Peter; Jakob, Sascha; Kunze, Kerstin; Maresch, Tanja; Calles, Christian; Krutmann, Jean; Haendeler, Judith

2008-07-01

Exposure of human skin to solar radiation, which includes ultraviolet (UV) radiation (UVA and UVB) visible light and infrared radiation, induces skin aging. The effects of light have been attributed to irradiation-induced reactive oxygen species (ROS) formation, but the specific signaling pathways are not well understood. Detrimental effects of solar radiation are dermal diseases and photoaging. Exposure of cultured human dermal fibroblasts to UVA, UVB or IRA increased ROS formation in vitro. One important redox regulator is the oxidoreductase thioredoxin-1 (Trx). Trx is ubiquitously expressed and has anti-oxidative and anti-apoptotic properties. Besides its function to reduce H(2)O(2), Trx binds to and regulates transcription factors. The aim of this study was to investigate whether Trx influences the regulation of MMP-1 and collagen Ialpha1 by UVA, UVB and IRA. We irradiated human dermal fibroblasts with UVA, UVB and IRA. UVA, UVB and IRA upregulated MMP-1 expression. Trx inhibited UVA-induced MMP-1 upregulation in a NFkappaB dependent manner. UVA, UVB and IRA reduced collagen Ialpha1 expression. Incubation with Trx inhibited the effects of UVB and IRA on collagen Ialpha1 expression. In conclusion, MMP-1 and collagen Ialpha1, which play important roles in aging processes, seems to be regulated by different transcriptional mechanisms and Trx can only influence distinct signaling pathways induced by UVA, UVB and probably IRA. Thus, Trx may serve as an important contributor to an "anti-aging therapeutic cocktail".

Collagen-binding peptidoglycans inhibit MMP mediated collagen degradation and reduce dermal scarring.

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Kate Stuart

Full Text Available Scarring of the skin is a large unmet clinical problem that is of high patient concern and impact. Wound healing is complex and involves numerous pathways that are highly orchestrated, leaving the skin sealed, but with abnormal organization and composition of tissue components, namely collagen and proteoglycans, that are then remodeled over time. To improve healing and reduce or eliminate scarring, more rapid restoration of healthy tissue composition and organization offers a unique approach for development of new therapeutics. A synthetic collagen-binding peptidoglycan has been developed that inhibits matrix metalloproteinase-1 and 13 (MMP-1 and MMP-13 mediated collagen degradation. We investigated the synthetic peptidoglycan in a rat incisional model in which a single dose was delivered in a hyaluronic acid (HA vehicle at the time of surgery prior to wound closure. The peptidoglycan treatment resulted in a significant reduction in scar tissue at 21 days as measured by histology and visual analysis. Improved collagen architecture of the treated wounds was demonstrated by increased tensile strength and transmission electron microscopy (TEM analysis of collagen fibril diameters compared to untreated and HA controls. The peptidoglycan's mechanism of action includes masking existing collagen and inhibiting MMP-mediated collagen degradation while modulating collagen organization. The peptidoglycan can be synthesized at low cost with unique design control, and together with demonstrated preclinical efficacy in reducing scarring, warrants further investigation for dermal wound healing.

An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13.

Science.gov (United States)

Inanc, Seniz; Keles, Didem; Oktay, Gulgun

2017-10-01

Collagen zymography is an SDS-PAGE-based method for detecting both the proenzyme and active forms of collagenases. Although collagen zymography is used for assessment of the matrix metalloproteinases MMP-1 and MMP-13, it can be difficult to detect these collagenases due to technical issues. Moreover, it remains unclear whether the collagenase activity of MMP-8 can be detected by this method. Here, we present an improved collagen zymography method that allows quantification of the activities of MMP-1, MMP-8, and MMP-13. Activities of recombinant collagenases could be detected in collagen zymogram gels copolymerized with 0.3 mg/mL type I collagen extracted from rat tail tendon. This improved method is sensitive enough to detect the activity of as little as 1 ng of collagenase. We generated standard curves for the three collagenases to quantify the collagenolytic activity levels of unknown samples. To validate our improved method, we investigated MMP-1 activity levels in human thyroid cancer (8505C) and normal thyroid (Nthy-ori-3-1) cell lines, finding that the proenzyme and active MMP-1 levels were greater in 8505C cells than in Nthy-ori-3-1 cells. Taken together, our data show that collagen zymography can be used in both molecular and clinical investigations to evaluate collagenase activities in various pathological conditions.

Collagen XII myopathy with rectus femoris atrophy and collagen XII retention in fibroblasts

DEFF Research Database (Denmark)

Witting, Nanna; Krag, Thomas; Werlauff, Ulla

2018-01-01

INTRODUCTION: Mutation in the collagen XII gene (COL12A1) was recently reported to induce Bethlem myopathy. We describe a family affected by collagen XII-related myopathy in 3 generations. METHODS: Systematic interview, clinical examination, skin biopsies, and MRI of muscle were used. RESULTS...... affection and abnormal collagen XII retention in fibroblasts. MRI disclosed a selective wasting of the rectus femoris muscle. DISCUSSION: COL12A1 mutations should be considered in patients with a mild Bethlem phenotype who present with selective wasting of the rectus femoris, absence of the outside......-in phenomenon on MRI, and abnormal collagen XII retention in fibroblasts. Muscle Nerve, 2018....

Surface biology of collagen scaffold explains blocking of wound contraction and regeneration of skin and peripheral nerves.

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Yannas, I V; Tzeranis, D; So, P T

2015-12-23

We review the details of preparation and of the recently elucidated mechanism of biological (regenerative) activity of a collagen scaffold (dermis regeneration template, DRT) that has induced regeneration of skin and peripheral nerves (PN) in a variety of animal models and in the clinic. DRT is a 3D protein network with optimized pore size in the range 20-125 µm, degradation half-life 14 ± 7 d and ligand densities that exceed 200 µM α1β1 or α2β1 ligands. The pore has been optimized to allow migration of contractile cells (myofibroblasts, MFB) into the scaffold and to provide sufficient specific surface for cell-scaffold interaction; the degradation half-life provides the required time window for satisfactory binding interaction of MFB with the scaffold surface; and the ligand density supplies the appropriate ligands for specific binding of MFB on the scaffold surface. A dramatic change in MFB phenotype takes place following MFB-scaffold binding which has been shown to result in blocking of wound contraction. In both skin wounds and PN wounds the evidence has shown clearly that contraction blocking by DRT is followed by induction of regeneration of nearly perfect organs. The biologically active structure of DRT is required for contraction blocking; well-matched collagen scaffold controls of DRT, with structures that varied from that of DRT, have failed to induce regeneration. Careful processing of collagen scaffolds is required for adequate biological activity of the scaffold surface. The newly understood mechanism provides a relatively complete paradigm of regenerative medicine that can be used to prepare scaffolds that may induce regeneration of other organs in future studies.

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

International Nuclear Information System (INIS)

Zhang Yufeng; Cheng Xiangrong; Wang Jiawei; Wang Yining; Shi Bin; Huang Cui; Yang Xuechao; Liu Tongjun

2006-01-01

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

Aortic VCAM-1: an early marker of vascular inflammation in collagen-induced arthritis.

Science.gov (United States)

Denys, Anne; Clavel, Gaëlle; Lemeiter, Delphine; Schischmanoff, Olivier; Boissier, Marie-Christophe; Semerano, Luca

2016-05-01

Cardiovascular disease (CVD) is a major cause of morbidity and mortality in rheumatoid arthritis (RA). There are limited experimental data on vascular involvement in arthritis models. To study the link between CVD and inflammation in RA, we developed a model of vascular dysfunction and articular inflammation by collagen-induced arthritis (CIA) in C57Bl/6 (B6) mice. We studied the expression of vascular inflammatory markers in CIA with and without concomitant hyperlipidic diet (HD). Collagen-induced arthritis was induced with intradermal injection of chicken type-II collagen followed by a boost 21 days later. Mice with and without CIA were fed a standard diet or an HD for 12 weeks starting from the day of the boost. Arthritis severity was evaluated with a validated clinical score. Aortic mRNA levels of vascular cell adhesion molecule-1 (VCAM-1), inducible nitric oxide synthase (iNOS) and interleukin-17 were analysed by quantitative RT-PCR. Vascular cell adhesion molecule-1 localization in the aortic sinus was determined by immunohistochemistry. Atherosclerotic plaque presence was assessed in aortas. Collagen-induced arthritis was associated with increased expression of VCAM-1, independent of diet. VCAM-1 overexpression was detectable as early as 4 weeks after collagen immunization and persisted after 15 weeks. The HD induced atheroma plaque formation and aortic iNOS expression regardless of CIA. Concomitant CIA and HD had no additive effect on atheroma or VCAM-1 or iNOS expression. CIA and an HD diet induced a distinct and independent expression of large-vessel inflammation markers in B6 mice. This model may be relevant for the study of CVD in RA. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Helicase-like transcription factor (Hltf regulates G2/M transition, Wt1/Gata4/Hif-1a cardiac transcription networks, and collagen biogenesis.

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Rebecca A Helmer

Full Text Available HLTF/Hltf regulates transcription, remodels chromatin, and coordinates DNA damage repair. Hltf is expressed in mouse brain and heart during embryonic and postnatal development. Silencing Hltf is semilethal. Seventy-four percent of congenic C57BL/6J Hltf knockout mice died, 75% within 12-24 hours of birth. Previous studies in neonatal (6-8 hour postpartum brain revealed silencing Hltf disrupted cell cycle progression, and attenuated DNA damage repair. An RNA-Seq snapshot of neonatal heart transcriptome showed 1,536 of 20,000 total transcripts were altered (p < 0.05 - 10 up- and 1,526 downregulated. Pathway enrichment analysis with MetaCore™ showed Hltf's regulation of the G2/M transition (p=9.726E(-15 of the cell cycle in heart is nearly identical to its role in brain. In addition, Brca1 and 12 members of the Brca1 associated genome surveillance complex are also downregulated. Activation of caspase 3 coincides with transcriptional repression of Bcl-2. Hltf loss caused downregulation of Wt1/Gata4/Hif-1a signaling cascades as well as Myh7b/miR499 transcription. Hltf-specific binding to promoters and/or regulatory regions of these genes was authenticated by ChIP-PCR. Hif-1a targets for prolyl (P4ha1, P4ha2 and lysyl (Plod2 collagen hydroxylation, PPIase enzymes (Ppid, Ppif, Ppil3 for collagen trimerization, and lysyl oxidase (Loxl2 for collagen-elastin crosslinking were downregulated. However, transcription of genes for collagens, fibronectin, Mmps and their inhibitors (Timps was unaffected. The collective downregulation of genes whose protein products control collagen biogenesis caused disorganization of the interstitial and perivascular myocardial collagen fibrillar network as viewed with picrosirius red-staining, and authenticated with spectral imaging. Wavy collagen bundles in control hearts contrasted with collagen fibers that were thin, short and disorganized in Hltf null hearts. Collagen bundles in Hltf null hearts were tangled and

viking: identification and characterization of a second type IV collagen in Drosophila.

Science.gov (United States)

Yasothornsrikul, S; Davis, W J; Cramer, G; Kimbrell, D A; Dearolf, C R

1997-10-01

We have taken an enhancer trap approach to identify genes that are expressed in hematopoietic cells and tissues of Drosophila. We conducted a molecular analysis of two P-element insertion strains that have reporter gene expression in embryonic hemocytes, strain 197 and vikingICO. This analysis has determined that viking encodes a collagen type IV gene, alpha2(IV). The viking locus is located adjacent to the previously described DCg1, which encodes collagen alpha1(IV), and in the opposite orientation. The alpha2(IV) and alpha1(IV) collagens are structurally very similar to one another, and to vertebrate type IV collagens. In early development, viking and DCg1 are transcribed in the same tissue-specific pattern, primarily in the hemocytes and fat body cells. Our results suggest that both the alpha1 and alpha2 collagen IV chains may contribute to basement membranes in Drosophila. This work also provides the foundation for a more complete genetic dissection of collagen type IV molecules and their developmental function in Drosophila.

Surface modification of electrospun PLGA scaffold with collagen for bioengineered skin substitutes

Energy Technology Data Exchange (ETDEWEB)

Sadeghi, A.R., E-mail: sadeghi_av@ymail.com [Materials Research Group, Iranian Academic Center for Education, Culture and Research, (ACECR), Mashhad Branch, Mashhad (Iran, Islamic Republic of); Nokhasteh, S. [Materials Research Group, Iranian Academic Center for Education, Culture and Research, (ACECR), Mashhad Branch, Mashhad (Iran, Islamic Republic of); Molavi, A.M. [Materials Research Group, Iranian Academic Center for Education, Culture and Research, (ACECR), Mashhad Branch, Mashhad (Iran, Islamic Republic of); Materials Engineering Department, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Khorsand-Ghayeni, M. [Materials Research Group, Iranian Academic Center for Education, Culture and Research, (ACECR), Mashhad Branch, Mashhad (Iran, Islamic Republic of); Naderi-Meshkin, H. [Stem Cell and Regenerative Medicine Research Department, Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad Branch, Mashhad (Iran, Islamic Republic of); Mahdizadeh, A. [Nanotechnology Institute, University of Sistan and Baluchestan, Zahedan (Iran, Islamic Republic of)

2016-09-01

In skin tissue engineering, surface feature of the scaffolds plays an important role in cell adhesion and proliferation. In this study, non-woven fibrous substrate based on poly (lactic-co-glycolic acid) (PLGA) (75/25) were hydrolyzed in various concentrations of NaOH (0.05 N, 0.1 N, 0.3 N) to increase carboxyl and hydroxyl groups on the fiber surfaces. These functional groups were activated by EDC/NHS to create chemical bonding with collagen. To improve bioactivity, the activated substrates were coated with a collagen solution (2 mg/ml) and cross-linking was carried out using the EDC/NHS in MES buffer. The effectiveness of the method was evaluated by contact angle measurements, porosimetry, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), tensile and degradation tests as well as in vitro cell attachment and cytotoxicity assays. Cell culture results of human dermal fibroblasts (HDF) and keratinocytes cell line (HaCat) revealed that the cells could attach to the scaffold. Further investigation with MTT assay showed that the cell proliferation of HaCat significantly increases with collagen coating. It seems that sufficient stability of collagen on the surface due to proper chemical bonding and cross-linking has increased the bioactivity of surface remarkably which can be promising for bioengineered skin applications. - Highlights: • Surface activation was carried out by hydrolysis of PLGA fibers. • To improve bioactivity, the activated samples were coated with a collagen solution. • Functional groups were activated by EDC/NHS to create chemical bonding with collagen. • Cross-linking of collagen was carried out using EDC/NHS in MES buffer. • The coated samples exhibited better adhesion and proliferation of epidermal cells.

Surface modification of electrospun PLGA scaffold with collagen for bioengineered skin substitutes

International Nuclear Information System (INIS)

Sadeghi, A.R.; Nokhasteh, S.; Molavi, A.M.; Khorsand-Ghayeni, M.; Naderi-Meshkin, H.; Mahdizadeh, A.

2016-01-01

In skin tissue engineering, surface feature of the scaffolds plays an important role in cell adhesion and proliferation. In this study, non-woven fibrous substrate based on poly (lactic-co-glycolic acid) (PLGA) (75/25) were hydrolyzed in various concentrations of NaOH (0.05 N, 0.1 N, 0.3 N) to increase carboxyl and hydroxyl groups on the fiber surfaces. These functional groups were activated by EDC/NHS to create chemical bonding with collagen. To improve bioactivity, the activated substrates were coated with a collagen solution (2 mg/ml) and cross-linking was carried out using the EDC/NHS in MES buffer. The effectiveness of the method was evaluated by contact angle measurements, porosimetry, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), tensile and degradation tests as well as in vitro cell attachment and cytotoxicity assays. Cell culture results of human dermal fibroblasts (HDF) and keratinocytes cell line (HaCat) revealed that the cells could attach to the scaffold. Further investigation with MTT assay showed that the cell proliferation of HaCat significantly increases with collagen coating. It seems that sufficient stability of collagen on the surface due to proper chemical bonding and cross-linking has increased the bioactivity of surface remarkably which can be promising for bioengineered skin applications. - Highlights: • Surface activation was carried out by hydrolysis of PLGA fibers. • To improve bioactivity, the activated samples were coated with a collagen solution. • Functional groups were activated by EDC/NHS to create chemical bonding with collagen. • Cross-linking of collagen was carried out using EDC/NHS in MES buffer. • The coated samples exhibited better adhesion and proliferation of epidermal cells.

Susceptibility of Calcium-Deficient Hydroxapatite Collagen Composite to Irradiation

International Nuclear Information System (INIS)

Ohta, M.; Yasuda, M.; Okamura, H.

2001-01-01

The ESR spectrum of calcium-deficient hydroxyapatite (DAp) has the peaks assigned to the hyperfine structure of dangling H + (hyperfine coupling constant = 50.8 mT, I = 1/2) due to the HPO 2- 4 ion which captured the hole released by X ray irradiation. This ESR peak intensity in DAp with collagen (c-DAp) decreased in an increase in the amount of collagen added into Ca(H 2 PO 4 ) 2 .H 2 O (MCP) electrolytic solution, because dangling H + of HPO 2- 4 binds to the carboxyl group of collagen due to the hydrogen bond. The ESR signal intensities at near g=2 in DAp, c-DAp, and stoichiometric hydroxyapatite (HAp) after X ray irradiation, were proportional to the absorbed dose in the range from 6 to 380 Gy. These ESR signal intensities decreased when the X ray-irradiated DAp, c-DAp, and HAp was suspended in the simulated body fluid. This fact suggests that the surface layer contained high density of ESR active species in DAp, c-DAp, and HAp dissolved in the simulated body fluid. Therefore, with the dosemeter utilising such biomaterials as tooth and bone, sufficient care must be paid to the effect of body fluid. (author)

Study of collagen metabolism after β radiation injury

International Nuclear Information System (INIS)

Zhou Yinghui; Xulan; Wu Shiliang; Zhang Xueguang; Chen Liesong

2000-01-01

Objective: To investigate the change of collagen metabolism and it's regulation after β radiation. Method: The animal model of β radiation injury was established by the β radiation produced by the linear accelerator; and irradiated NIH 3T3 cells were studied. In the experiment the contents of total collagen, collagen type I and type III were measured. The activity of MMPs-1 was tested. The contents of TGF-β 1 , IL-6 were also detected. Results: After exposure to β radiation, little change was found in the content of total collagen, but the content of collagen I decreased and the content of collagen III, MMPs-1 activity increased; the expression of TGF-β 1 , IL-6 increased. Conclusion: The changes in the metabolism of collagen play an important role in the irradiated injury of the skin; TGF-β 1 and IL-6 may be essential in the regulation of the collagen metabolism

Collagen fibrillogenesis: fibronectin, integrins, and minor collagens as organizers and nucleators.

Science.gov (United States)

Kadler, Karl E; Hill, Adele; Canty-Laird, Elizabeth G

2008-10-01

Collagens are triple helical proteins that occur in the extracellular matrix (ECM) and at the cell-ECM interface. There are more than 30 collagens and collagen-related proteins but the most abundant are collagens I and II that exist as D-periodic (where D = 67 nm) fibrils. The fibrils are of broad biomedical importance and have central roles in embryogenesis, arthritis, tissue repair, fibrosis, tumor invasion, and cardiovascular disease. Collagens I and II spontaneously form fibrils in vitro, which shows that collagen fibrillogenesis is a selfassembly process. However, the situation in vivo is not that simple; collagen I-containing fibrils do not form in the absence of fibronectin, fibronectin-binding and collagen-binding integrins, and collagen V. Likewise, the thin collagen II-containing fibrils in cartilage do not form in the absence of collagen XI. Thus, in vivo, cellular mechanisms are in place to control what is otherwise a protein self-assembly process. This review puts forward a working hypothesis for how fibronectin and integrins (the organizers) determine the site of fibril assembly, and collagens V and XI (the nucleators) initiate collagen fibrillogenesis.

COLOSTRUM-COLLAGEN-HYDROXYAPATITE COMPOSITE, AN EXCELLENT CANDIDATE BIOMATERIAL FOR BONE REPAIR AND BONE INFECTION MANAGEMENT

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Dio Nurdin Setiawan

2014-05-01

Full Text Available In the case ofbone fracture or defect after surgery, which is common in patients with bone cancer (osteosarcoma, it takes a long time for closure and it may cause an infection problem. The use ofcollagen-hydroxyapatite composite with a blend ofcolostrum as a scaffold is aimed to accelerate the process of osteoblast growth, inhibite the emergence of infections, and act as bone tissue repair material. The method used was the hydrogel formation process and freeze dry process to remove the solvent and to form pores. The composition of scaffold composite manufactured was 15% collagen, 75% hydroxyapatite and 10% colostrum. Combination ofscaffold collagen-hydroxyapatite-colostrum has quite reliable properties because SEM test showed that scaffold could bind to both and could bind to both and could form sufficient pores to provide enough place for bone cells (osteoblats to grow. The results of MTT assay revealed percentage of above 60%, which indicates that the material is not toxic. In conclusion, collagen-hydroxyapatite-colostrum combination is an excellent biomaterial candidate for bone repair and bone infection management.

Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

Science.gov (United States)

Brachvogel, Bent; Zaucke, Frank; Dave, Keyur; Norris, Emma L; Stermann, Jacek; Dayakli, Münire; Koch, Manuel; Gorman, Jeffrey J; Bateman, John F; Wilson, Richard

2013-05-10

Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. Matrilin-4, collagen XII, thrombospondin-4, fibronectin, βig-h3, and epiphycan are components of the in vivo collagen IX interactome. We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level

Peroxidasin-mediated crosslinking of collagen IV is independent of NADPH oxidases

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Gábor Sirokmány

2018-06-01

Full Text Available Collagen IV is a major component of the basement membrane in epithelial tissues. The NC1 domains of collagen IV protomers are covalently linked together through sulfilimine bonds, the formation of which is catalyzed by peroxidasin. Although hydrogen peroxide is essential for this reaction, the exact source of the oxidant remains elusive. Members of the NOX/DUOX NADPH oxidase family are specifically devoted to the production of superoxide and hydrogen peroxide. Our aim in this study was to find out if NADPH oxidases contribute in vivo to the formation of collagen IV sulfilimine crosslinks. We used multiple genetically modified in vivo model systems to provide a detailed assessment of this question. Our data indicate that in various peroxidasin-expressing tissues sulfilimine crosslinks between the NC1 domains of collagen IV can be readily detected in the absence of functioning NADPH oxidases. We also analyzed how subatmospheric oxygen levels influence the collagen IV network in collagen-producing cultured cells with rapid matrix turnover. We showed that collagen IV crosslinks remain intact even under strongly hypoxic conditions. Our hypothesis is that during collagen IV network formation PXDN cooperates with a NOX/DUOX-independent H2O2 source that is functional also at very low ambient oxygen levels. Keywords: Peroxidasin, NADPH oxidase, Hydrogen peroxide, Collagen IV, Sulfilimine

Nanorod mediated collagen scaffolds as extra cellular matrix mimics

International Nuclear Information System (INIS)

Vedhanayagam, Mohan; Nair, Balachandran Unni; Sreeram, Kalarical Janardhanan; Mohan, Ranganathan

2015-01-01

Creating collagen scaffolds that mimic extracellular matrices without using toxic exogenous materials remains a big challenge. A new strategy to create scaffolds through end-to-end crosslinking through functionalized nanorods leading to well-designed architecture is presented here. Self-assembled scaffolds with a denaturation temperature of 110 °C, porosity of 70%, pore size of 0.32 μm and Young’s modulus of 231 MPa were developed largely driven by imine bonding between 3-mercapto-1-propanal (MPA) functionalized ZnO nanorods and collagen. The mechanical properties obtained were much higher than that of native collagen, collagen—MPA, collagen—3-mercapto-1-propanol (3MPOH) or collagen- 3-MPOH-ZnO, clearly bringing out the relevance of nanorod mediated assembly of fibrous networks. This new strategy has led to scaffolds with mechanical properties much higher than earlier reports and can provide support for cell growth and facilitation of cell attachment. (paper)

Collagen macromolecular drug delivery systems

International Nuclear Information System (INIS)

Gilbert, D.L.

1988-01-01

The objective of this study was to examine collagen for use as a macromolecular drug delivery system by determining the mechanism of release through a matrix. Collagen membranes varying in porosity, crosslinking density, structure and crosslinker were fabricated. Collagen characterized by infrared spectroscopy and solution viscosity was determined to be pure and native. The collagen membranes were determined to possess native vs. non-native quaternary structure and porous vs. dense aggregate membranes by electron microscopy. Collagen monolithic devices containing a model macromolecule (inulin) were fabricated. In vitro release rates were found to be linear with respect to t 1/2 and were affected by crosslinking density, crosslinker and structure. The biodegradation of the collagen matrix was also examined. In vivo biocompatibility, degradation and 14 C-inulin release rates were evaluated subcutaneously in rats

Collagenous sprue

DEFF Research Database (Denmark)

Soendergaard, Christoffer; Riis, Lene Buhl; Nielsen, Ole Haagen

2014-01-01

Collagenous sprue is a rare clinicopathological condition of the small bowel. It is characterised by abnormal subepithelial collagen deposition and is typically associated with malabsorption, diarrhoea and weight loss. The clinical features of collagenous sprue often resemble those of coeliac...... disease and together with frequent histological findings like mucosal thinning and intraepithelial lymphocytosis the diagnosis may be hard to reach without awareness of this condition. While coeliac disease is treated using gluten restriction, collagenous sprue is, however, not improved...... by this intervention. In cases of diet-refractory 'coeliac disease' it is therefore essential to consider collagenous sprue to initiate treatment at an early stage to prevent the fibrotic progression. Here, we report a case of a 78-year-old man with collagenous sprue and present the clinical and histological...

Crystal structure of the second fibronectin type III (FN3) domain from human collagen α1 type XX.

Science.gov (United States)

Zhao, Jingfeng; Ren, Jixia; Wang, Nan; Cheng, Zhong; Yang, Runmei; Lin, Gen; Guo, Yi; Cai, Dayong; Xie, Yong; Zhao, Xiaohong

2017-12-01

Collagen α1 type XX, which contains fibronectin type III (FN3) repeats involving six FN3 domains (referred to as the FN#1-FN#6 domains), is an unusual member of the fibril-associated collagens with interrupted triple helices (FACIT) subfamily of collagens. The results of standard protein BLAST suggest that the FN3 repeats might contribute to collagen α1 type XX acting as a cytokine receptor. To date, solution NMR structures of the FN#3, FN#4 and FN#6 domains have been determined. To obtain further structural evidence to understand the relationship between the structure and function of the FN3 repeats from collagen α1 type XX, the crystal structure of the FN#2 domain from human collagen α1 type XX (residues Pro386-Pro466; referred to as FN2-HCXX) was solved at 2.5 Å resolution. The crystal structure of FN2-HCXX shows an immunoglobulin-like fold containing a β-sandwich structure, which is formed by a three-stranded β-sheet (β1, β2 and β5) packed onto a four-stranded β-sheet (β3, β4, β6 and β7). Two consensus domains, tencon and fibcon, are structural analogues of FN2-HCXX. Fn8, an FN3 domain from human oncofoetal fibronectin, is the closest structural analogue of FN2-HCXX derived from a naturally occurring sequence. Based solely on the structural similarity of FN2-HCXX to other FN3 domains, the detailed functions of FN2-HCXX and the FN3 repeats in collagen α1 type XX cannot be identified.

Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

International Nuclear Information System (INIS)

Wang, Xianwei; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L.

2012-01-01

Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22 phox , p47 phox , p67 phox , NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H 2 O 2 . Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac fibroblast growth and collagen

Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

Energy Technology Data Exchange (ETDEWEB)

Wang, Xianwei, E-mail: XWang2@UAMS.edu; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L., E-mail: MehtaJL@UAMS.edu

2012-03-15

Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22{sup phox}, p47{sup phox}, p67{sup phox}, NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H{sub 2}O{sub 2}. Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac

Enhancement of Human Endothelial Cell Adhesion to Type I Collagen by Lysophosphatidic Acid (LPA and Sphingosine-1-Phosphate (S1P

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Hsinyu Lee

2004-06-01

Full Text Available The diverse cellular effects of lysophosphatidic acid (LPA and sphingosine-1-phosphate (S1P are transduced by two structurally homologous subfamilies of G protein-coupled receptors, which are encoded by endothelial differentiation genes (Edg Rs. Human umbilical cord vein endothelial cells (HUVECs express Edg Rs for LPA (Edg2 and S1P (Edg1 and 3, which transduce signals for migration of HUVECs through micropore filters coated with type I collagen. Since activation of integrins is essential for optimal migration of endothelial cells, we now examine the capacity of LPA and S1P to augment integrin mediation of endothelial cell binding to type I collagen. Lysophospholipid enhancement of HUVEC adhesion to type I collagen is detectable within 20 minutes. Enhancement of adhesion by both LPA and S1P is significant at 50 nM and optimal at 5µM. Pertussis toxin (PTx, a specific inhibitor of Gi, and C3 exotoxin, a specific inhibitor of Rho, both suppress LPA and S1P enhancement of HUVEC adhesion. In contrast, PD98059, which blocks MAP kinase kinase (MEK, and wortmannin, which inhibits phosphatidylinositol 3-kinase (PI3K, had no effect on LPA- or S1P-enhancement of HUVEC adhesion. Neutralizing monoclonal antibodies specific for α2 and β1 integrin chains, concomitantly decrease LPA and S1P enhancement of HUVEC adhesion to type I collagen. LPA and S1P thus promote type I collagen-dependent adhesion and migration of HUVECs by recruiting α2 and β1 integrin through both Gi and Rho pathways. Integrin α2/β1 therefore appears to be critical on the effects of LPA and S1P on endothelial cell physiology.

Collagen V-induced nasal tolerance downregulates pulmonary collagen mRNA gene and TGF-beta expression in experimental systemic sclerosis

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Parra Edwin R

2010-01-01

Full Text Available Abstract Background The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGF-beta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance. Methods Female New Zealand rabbits (N = 12 were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM. After 150 days, six immunized animals were tolerated by nasal administration of collagen V (25 μg/day (IM-TOL daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p Results IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 ± 0.118 vs. 0.874 ± 0.282, p p p = 0.026. The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 ± 0.07 vs. 1.0 ± 0.528, p = 0.002 and V (1.12 ± 0.42 vs. 4.74 ± 2.25, p = 0.009 collagen, in addition to decreased TGF-beta expression (p Conclusions Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.

Imaging collagen type I fibrillogenesis with high spatiotemporal resolution

International Nuclear Information System (INIS)

Stamov, Dimitar R; Stock, Erik; Franz, Clemens M; Jähnke, Torsten; Haschke, Heiko

2015-01-01

Fibrillar collagens, such as collagen type I, belong to the most abundant extracellular matrix proteins and they have received much attention over the last five decades due to their large interactome, complex hierarchical structure and high mechanical stability. Nevertheless, the collagen self-assembly process is still incompletely understood. Determining the real-time kinetics of collagen type I formation is therefore pivotal for better understanding of collagen type I structure and function, but visualising the dynamic self-assembly process of collagen I on the molecular scale requires imaging techniques offering high spatiotemporal resolution. Fast and high-speed scanning atomic force microscopes (AFM) provide the means to study such processes on the timescale of seconds under near-physiological conditions. In this study we have applied fast AFM tip scanning to study the assembly kinetics of fibrillar collagen type I nanomatrices with a temporal resolution reaching eight seconds for a frame size of 500 nm. By modifying the buffer composition and pH value, the kinetics of collagen fibrillogenesis can be adjusted for optimal analysis by fast AFM scanning. We furthermore show that amplitude-modulation imaging can be successfully applied to extract additional structural information from collagen samples even at high scan rates. Fast AFM scanning with controlled amplitude modulation therefore provides a versatile platform for studying dynamic collagen self-assembly processes at high resolution. - Highlights: • Continuous non-invasive time-lapse investigation of collagen I fibrillogenesis in situ. • Imaging of collagen I self-assembly with high spatiotemporal resolution. • Application of setpoint modulation to study the hierarchical structure of collagen I. • Observing real-time formation of the D-banding pattern in collagen I

Imaging collagen type I fibrillogenesis with high spatiotemporal resolution

Energy Technology Data Exchange (ETDEWEB)

Stamov, Dimitar R, E-mail: stamov@jpk.com [JPK Instruments AG, Bouchéstrasse 12, 12435 Berlin (Germany); Stock, Erik [JPK Instruments AG, Bouchéstrasse 12, 12435 Berlin (Germany); Franz, Clemens M [DFG-Center for Functional Nanostructures (CFN), Karlsruhe Institute of Technology (KIT), Wolfgang-Gaede-Strasse 1a, 76131 Karlsruhe (Germany); Jähnke, Torsten; Haschke, Heiko [JPK Instruments AG, Bouchéstrasse 12, 12435 Berlin (Germany)

2015-02-15

Fibrillar collagens, such as collagen type I, belong to the most abundant extracellular matrix proteins and they have received much attention over the last five decades due to their large interactome, complex hierarchical structure and high mechanical stability. Nevertheless, the collagen self-assembly process is still incompletely understood. Determining the real-time kinetics of collagen type I formation is therefore pivotal for better understanding of collagen type I structure and function, but visualising the dynamic self-assembly process of collagen I on the molecular scale requires imaging techniques offering high spatiotemporal resolution. Fast and high-speed scanning atomic force microscopes (AFM) provide the means to study such processes on the timescale of seconds under near-physiological conditions. In this study we have applied fast AFM tip scanning to study the assembly kinetics of fibrillar collagen type I nanomatrices with a temporal resolution reaching eight seconds for a frame size of 500 nm. By modifying the buffer composition and pH value, the kinetics of collagen fibrillogenesis can be adjusted for optimal analysis by fast AFM scanning. We furthermore show that amplitude-modulation imaging can be successfully applied to extract additional structural information from collagen samples even at high scan rates. Fast AFM scanning with controlled amplitude modulation therefore provides a versatile platform for studying dynamic collagen self-assembly processes at high resolution. - Highlights: • Continuous non-invasive time-lapse investigation of collagen I fibrillogenesis in situ. • Imaging of collagen I self-assembly with high spatiotemporal resolution. • Application of setpoint modulation to study the hierarchical structure of collagen I. • Observing real-time formation of the D-banding pattern in collagen I.

Activation of PPARs α, β/δ, and γ Impairs TGF-β1-Induced Collagens' Production and Modulates the TIMP-1/MMPs Balance in Three-Dimensional Cultured Chondrocytes

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Paul-Emile Poleni

2010-01-01

Full Text Available Background and Purpose. We investigated the potency of Peroxisome Proliferators-Activated Receptors (PPARs α, β/δ, and γ agonists to modulate Transforming Growth Factor-β1 (TGF-β1- induced collagen production or changes in Tissue Inhibitor of Matrix Metalloproteinase- (TIMP- 1/Matrix Metalloproteinase (MMP balance in rat chondrocytes embedded in alginate beads. Experimental Approach. Collagen production was evaluated by quantitative Sirius red staining, while TIMP-1 protein levels and global MMP (-1, -2, -3, -7, and -9 or specific MMP-13 activities were measured by ELISA and fluorigenic assays in culture media, respectively. Levels of mRNA for type II collagen, TIMP-1, and MMP-3 & 13 were quantified by real-time PCR. Key Results. TGF-β1 increased collagen deposition and type II collagen mRNA levels, while inducing TIMP-1 mRNA and protein expression. In contrast, it decreased global MMP or specific MMP-13 activities, while decreasing MMP-3 or MMP-13 mRNA levels. PPAR agonists reduced most of the effects of TGF-β1 on changes in collagen metabolism and TIMP-1/MMP balance in rat in a PPAR-dependent manner, excepted for Wy14643 on MMP activities. Conclusions and Implications. PPAR agonists reduce TGF-β1-modulated ECM turnover and inhibit chondrocyte activities crucial for collagen biosynthesis, and display a different inhibitory profile depending on selectivity for PPAR isotypes.

Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice: a comparative study using anti-TNFalpha, anti-IL-1alpha/beta and IL-1Ra.

NARCIS (Netherlands)

Joosten, L.A.B.; Helsen, M.M.A.; Loo, F.A.J. van de; Berg, W.B. van den

2008-01-01

OBJECTIVE: To examine the role of tumor necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha), and IL-1 beta in collagen-induced arthritis (CIA), immediately after onset and during the phase of established arthritis. METHODS: Male DBA/1 mice with collagen-induced arthritis were treated

Inhibition of microRNA-214-5p promotes cell survival and extracellular matrix formation by targeting collagen type IV alpha 1 in osteoblastic MC3T3-E1 cells.

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Li, Q S; Meng, F Y; Zhao, Y H; Jin, C L; Tian, J; Yi, X J

2017-08-01

This study aimed to investigate the functional effects of microRNA (miR)-214-5p on osteoblastic cells, which might provide a potential role of miR-214-5p in bone fracture healing. Blood samples were obtained from patients with hand fracture or intra-articular calcaneal fracture and from healthy controls (HCs). Expression of miR-214-5p was monitored by qRT-PCR at day 7, 14 and 21 post-surgery. Mouse osteoblastic MC3T3-E1 cells were transfected with antisense oligonucleotides (ASO)-miR-214-5p, collagen type IV alpha 1 (COL4A1) vector or their controls; thereafter, cell viability, apoptotic rate, and the expression of collagen type I alpha 1 (COL1A1), type II collagen (COL-II), and type X collagen (COL-X) were determined. Luciferase reporter assay, qRT-PCR, and Western blot were performed to ascertain whether COL4A1 was a target of miR-214-5p. Plasma miR-214-5p was highly expressed in patients with bone fracture compared with HCs after fracture (p extracellular matrix (ECM) formation of osteoblastic MC3T3-E1 cells by targeting COL4A1. Cite this article: Q. S. Li, F. Y. Meng, Y. H. Zhao, C. L. Jin, J. Tian, X. J. Yi. Inhibition of microRNA-214-5p promotes cell survival and extracellular matrix formation by targeting collagen type IV alpha 1 in osteoblastic MC3T3-E1 cells. Bone Joint Res 2017;6:464-471. DOI: 10.1302/2046-3758.68.BJR-2016-0208.R2. © 2017 Yi et al.

High-contrast multimodel nonlinear optical imaging of collagen and elastin

Energy Technology Data Exchange (ETDEWEB)

Zhuo, S M [Key Laboratory of Optoelectronic Science and Technology for Medicine (Fujian Normal University), Ministry of Education, Fuzhou 350007 (China); Chen, J X [Key Laboratory of Optoelectronic Science and Technology for Medicine (Fujian Normal University), Ministry of Education, Fuzhou 350007 (China); Luo, T S [Key Laboratory of Optoelectronic Science and Technology for Medicine (Fujian Normal University), Ministry of Education, Fuzhou 350007 (China); Chen, H L [Key Laboratory of Optoelectronic Science and Technology for Medicine (Fujian Normal University), Ministry of Education, Fuzhou 350007 (China); Zhao, J J [Department of Skin, Affiliated Xiehe Hospital, Fujian Medical University, Fuzhou 350001 (China)

2007-07-15

Collagen and elastin, as the major components in the extracellular matrix (ECM), are intrinsic indicators of physiological and pathological states. Here, we have developed a high-contrast multimodel nonlinear optical imaging technique to imaging collagen and elastin by detecting simultaneously two photon-excited fluorescence (TPEF) from elastin and second-harmonic generation (SHG) from collagen. Our results show that this technique can obtain a high-contrast TPEF/SHG image in human dermis and permit direct visualization of collagen and elastin. It was shown that the technique can provide collagen and elastin structural information to determine collagen and elastin fibril orientation and distribution and acquire some morphometric properties. It was found that the in-depth TPEF/SHG imaging and 3-D reconstruction of TPEF/SHG images can extract more collagen and elastin structural and biochemical information. The study results suggest that the high-contrast multimodel nonlinear imaging provides a powerful tool to study ECM intrinsic components and has the potential to provide more important information for the diagnosis of tissue.

High-contrast multimodel nonlinear optical imaging of collagen and elastin

International Nuclear Information System (INIS)

Zhuo, S M; Chen, J X; Luo, T S; Chen, H L; Zhao, J J

2007-01-01

Collagen and elastin, as the major components in the extracellular matrix (ECM), are intrinsic indicators of physiological and pathological states. Here, we have developed a high-contrast multimodel nonlinear optical imaging technique to imaging collagen and elastin by detecting simultaneously two photon-excited fluorescence (TPEF) from elastin and second-harmonic generation (SHG) from collagen. Our results show that this technique can obtain a high-contrast TPEF/SHG image in human dermis and permit direct visualization of collagen and elastin. It was shown that the technique can provide collagen and elastin structural information to determine collagen and elastin fibril orientation and distribution and acquire some morphometric properties. It was found that the in-depth TPEF/SHG imaging and 3-D reconstruction of TPEF/SHG images can extract more collagen and elastin structural and biochemical information. The study results suggest that the high-contrast multimodel nonlinear imaging provides a powerful tool to study ECM intrinsic components and has the potential to provide more important information for the diagnosis of tissue

Two-photon induced collagen cross-linking in bioartificial cardiac tissue

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Kuetemeyer, Kai; Kensah, George; Heidrich, Marko; Meyer, Heiko; Martin, Ulrich; Gruh, Ina; Heisterkamp, Alexander

2011-08-01

Cardiac tissue engineering is a promising strategy for regenerative therapies to overcome the shortage of donor organs for transplantation. Besides contractile function, the stiffness of tissue engineered constructs is crucial to generate transplantable tissue surrogates with sufficient mechanical stability to withstand the high pressure present in the heart. Although several collagen cross-linking techniques have proven to be efficient in stabilizing biomaterials, they cannot be applied to cardiac tissue engineering, as cell death occurs in the treated area. Here, we present a novel method using femtosecond (fs) laser pulses to increase the stiffness of collagen-based tissue constructs without impairing cell viability. Raster scanning of the fs laser beam over riboflavin-treated tissue induced collagen cross-linking by two-photon photosensitized singlet oxygen production. One day post-irradiation, stress-strain measurements revealed increased tissue stiffness by around 40% being dependent on the fibroblast content in the tissue. At the same time, cells remained viable and fully functional as demonstrated by fluorescence imaging of cardiomyocyte mitochondrial activity and preservation of active contraction force. Our results indicate that two-photon induced collagen cross-linking has great potential for studying and improving artificially engineered tissue for regenerative therapies.

The streptococcal collagen-like protein-1 (Scl1 is a significant determinant for biofilm formation by group a Streptococcus

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Oliver-Kozup Heaven A

2011-12-01

Full Text Available Abstract Background Group A Streptococcus (GAS is a human-specific pathogen responsible for a number of diseases characterized by a wide range of clinical manifestations. During host colonization GAS-cell aggregates or microcolonies are observed in tissues. GAS biofilm, which is an in vitro equivalent of tissue microcolony, has only recently been studied and little is known about the specific surface determinants that aid biofilm formation. In this study, we demonstrate that surface-associated streptococcal collagen-like protein-1 (Scl1 plays an important role in GAS biofilm formation. Results Biofilm formation by M1-, M3-, M28-, and M41-type GAS strains, representing an intraspecies breadth, were analyzed spectrophotometrically following crystal violet staining, and characterized using confocal and field emission scanning electron microscopy. The M41-type strain formed the most robust biofilm under static conditions, followed by M28- and M1-type strains, while the M3-type strains analyzed here did not form biofilm under the same experimental conditions. Differences in architecture and cell-surface morphology were observed in biofilms formed by the M1- and M41-wild-type strains, accompanied by varying amounts of deposited extracellular matrix and differences in cell-to-cell junctions within each biofilm. Importantly, all Scl1-negative mutants examined showed significantly decreased ability to form biofilm in vitro. Furthermore, the Scl1 protein expressed on the surface of a heterologous host, Lactococcus lactis, was sufficient to induce biofilm formation by this organism. Conclusions Overall, this work (i identifies variations in biofilm formation capacity among pathogenically different GAS strains, (ii identifies GAS surface properties that may aid in biofilm stability and, (iii establishes that the Scl1 surface protein is an important determinant of GAS biofilm, which is sufficient to enable biofilm formation in the heterologous host

Properties of Chitosan-Laminated Collagen Film

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Vera Lazić

2012-01-01

Full Text Available The objective of this study is to determine physical, mechanical and barrier properties of chitosan-laminated collagen film. Commercial collagen film, which is used for making collagen casings for dry fermented sausage production, was laminated with chitosan film layer in order to improve the collagen film barrier properties. Different volumes of oregano essential oil per 100 mL of filmogenic solution were added to chitosan film layer: 0, 0.2, 0.4, 0.6 and 0.8 mL to optimize water vapour barrier properties. Chitosan layer with 0.6 or 0.8 % of oregano essential oil lowered the water vapour transmission rate to (1.85±0.10·10–6 and (1.78±0.03·10–6 g/(m2·s·Pa respectively, compared to collagen film ((2.51±0.05·10–6 g/(m2·s·Pa. However, chitosan-laminated collagen film did not show improved mechanical properties compared to the collagen one. Tensile strength decreased from (54.0±3.8 MPa of the uncoated collagen film to (36.3±4.0 MPa when the film was laminated with 0.8 % oregano essential oil chitosan layer. Elongation at break values of laminated films did not differ from those of collagen film ((18.4±2.7 %. Oxygen barrier properties were considerably improved by lamination. Oxygen permeability of collagen film was (1806.8±628.0·10–14 cm3/(m·s·Pa and values of laminated films were below 35·10–14 cm3/(m·s·Pa. Regarding film appearance and colour, lamination with chitosan reduced lightness (L and yellowness (+b of collagen film, while film redness (+a increased. These changes were not visible to the naked eye.

Fibroblast Cluster Formation on 3D Collagen Matrices Requires Cell Contraction-Dependent Fibronectin Matrix Organization

Science.gov (United States)

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

2012-01-01

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

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

Science.gov (United States)

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

2013-02-15

Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin (FN) fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell-matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy. Copyright © 2012 Elsevier Inc. All rights reserved.

ACAT1 deletion in murine macrophages associated with cytotoxicity and decreased expression of collagen type 3A1

International Nuclear Information System (INIS)

Rodriguez, Annabelle; Ashen, M. Dominique; Chen, Edward S.

2005-01-01

In contrast to some published studies of murine macrophages, we previously showed that ACAT inhibitors appeared to be anti-atherogenic in primary human macrophages in that they decreased foam cell formation without inducing cytotoxicity. Herein, we examined foam cell formation and cytotoxicity in murine ACAT1 knockout (KO) macrophages in an attempt to resolve the discrepancies. Elicited peritoneal macrophages from normal C57BL6 and ACAT1 KO mice were incubated with DMEM containing acetylated LDL (acLDL, 100 μg protein/ml) for 48 h. Cells became cholesterol enriched and there were no differences in the total cholesterol mass. Esterified cholesterol mass was lower in ACAT1 KO foam cells compared to normal macrophages (p 14 C]adenine from macrophages, was approximately 2-fold greater in ACAT1 KO macrophages as compared to normal macrophages (p < 0.0001), and this was independent of cholesterol enrichment. cDNA microarray analysis showed that ACAT1 KO macrophages expressed substantially less collagen type 3A1 (26-fold), which was confirmed by RT-PCR. Total collagen content was also significantly reduced (57%) in lung homogenates isolated from ACAT1 KO mice (p < 0.02). Thus, ACAT1 KO macrophages show biochemical changes consistent with increased cytotoxicity and also a novel association with decreased expression of collagen type 3A1

Collagen-induced arthritis in C57BL/6 mice is associated with a robust and sustained T-cell response to type II collagen.

Science.gov (United States)

Inglis, Julia J; Criado, Gabriel; Medghalchi, Mino; Andrews, Melanie; Sandison, Ann; Feldmann, Marc; Williams, Richard O

2007-01-01

Many genetically modified mouse strains are now available on a C57BL/6 (H-2b) background, a strain that is relatively resistant to collagen-induced arthritis. To facilitate the molecular understanding of autoimmune arthritis, we characterised the induction of arthritis in C57BL/6 mice and then validated the disease as a relevant pre-clinical model for rheumatoid arthritis. C57BL/6 mice were immunised with type II collagen using different protocols, and arthritis incidence, severity, and response to commonly used anti-arthritic drugs were assessed and compared with DBA/1 mice. We confirmed that C57BL/6 mice are susceptible to arthritis induced by immunisation with chicken type II collagen and develop strong and sustained T-cell responses to type II collagen. Arthritis was milder in C57BL/6 mice than DBA/1 mice and more closely resembled rheumatoid arthritis in its response to therapeutic intervention. Our findings show that C57BL/6 mice are susceptible to collagen-induced arthritis, providing a valuable model for assessing the role of specific genes involved in the induction and/or maintenance of arthritis and for evaluating the efficacy of novel drugs, particularly those targeted at T cells.

Nonlinear optical response of the collagen triple helix and second harmonic microscopy of collagen liquid crystals

Science.gov (United States)

Deniset-Besseau, A.; De Sa Peixoto, P.; Duboisset, J.; Loison, C.; Hache, F.; Benichou, E.; Brevet, P.-F.; Mosser, G.; Schanne-Klein, M.-C.

2010-02-01

Collagen is characterized by triple helical domains and plays a central role in the formation of fibrillar and microfibrillar networks, basement membranes, as well as other structures of the connective tissue. Remarkably, fibrillar collagen exhibits efficient Second Harmonic Generation (SHG) and SHG microscopy proved to be a sensitive tool to score fibrotic pathologies. However, the nonlinear optical response of fibrillar collagen is not fully characterized yet and quantitative data are required to further process SHG images. We therefore performed Hyper-Rayleigh Scattering (HRS) experiments and measured a second order hyperpolarisability of 1.25 10-27 esu for rat-tail type I collagen. This value is surprisingly large considering that collagen presents no strong harmonophore in its amino-acid sequence. In order to get insight into the physical origin of this nonlinear process, we performed HRS measurements after denaturation of the collagen triple helix and for a collagen-like short model peptide [(Pro-Pro-Gly)10]3. It showed that the collagen large nonlinear response originates in the tight alignment of a large number of weakly efficient harmonophores, presumably the peptide bonds, resulting in a coherent amplification of the nonlinear signal along the triple helix. To illustrate this mechanism, we successfully recorded SHG images in collagen liquid solutions by achieving liquid crystalline ordering of the collagen triple helices.

The synthesis and coupling of photoreactive collagen-based peptides to restore integrin reactivity to an inert substrate, chemically-crosslinked collagen

Science.gov (United States)

Malcor, Jean-Daniel; Bax, Daniel; Hamaia, Samir W.; Davidenko, Natalia; Best, Serena M.; Cameron, Ruth E.; Farndale, Richard W.; Bihan, Dominique

2016-01-01

Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2β1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2β1 and α1β1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons. PMID:26854392

A PTEN-COL17A1 fusion gene and its novel regulatory role in Collagen XVII expression and GBM malignance.

Science.gov (United States)

Yan, Xiaoyan; Zhang, Chuanbao; Liang, Tingyu; Yang, Fan; Wang, Haoyuan; Wu, Fan; Wang, Wen; Wang, Zheng; Cheng, Wen; Xu, Jiangnan; Jiang, Tao; Chen, Jing; Ding, Yaozhong

2017-10-17

Collagen XVII expression has recently been demonstrated to be correlated with the tumor malignance. While Collagen XVII is known to be widely distributed in neurons of the human brain, its precise role in pathogenesis of glioblastoma multiforme (GBM) is unknown. In this study, we identified and characterized a new PTEN-COL17A1 fusion gene in GMB using transcriptome sequencing. Although fusion gene did not result in measurable fusion protein production, its presence is accompanied with high levels of COL17A1 expression, revealed a novel regulatory mechanism of Collagen XVII expression by PTEN-COL17A1 gene fusion. Knocked down Collagen XVII expression in glioma cell lines resulted in decreased tumor invasiveness, along with significant reduction of MMP9 expression, while increased Collagen XVII expression promotes invasive activities of glioma cells and associated with GBM recurrences. Together, our results uncovered a new PTEN-COL17A1 fusion gene and its novel regulatory role in Collagen XVII expression and GBM malignance, and demonstrated that COL17A1 could serve as a useful prognostic biomarker and therapeutic targets for GBM.

Osteoconductive properties of two different bioactive glass forms (powder and fiber) combined with collagen

Science.gov (United States)

Magri, Angela Maria Paiva; Fernandes, Kelly Rossetti; Ueno, Fabio Roberto; Kido, Hueliton Wilian; da Silva, Antonio Carlos; Braga, Francisco José Correa; Granito, Renata Neves; Gabbai-Armelin, Paulo Roberto; Rennó, Ana Claudia Muniz

2017-11-01

Bioactive Glasses (BG) is a group of synthetic silica-based materials with the unique ability to bond to living bone and can be used in bone repair. Although the osteogenic potential of BG, this material may have not present sufficient osteoconductive and osteoinductive properties to allow bone regeneration, especially in compromised situations. In order to overcome this limitation, it was proposed the combination the BG in two forms (powder and fiber) combined with collagen type I (COL-1). The aim of this study was to evaluate the BG/COL-based materials in terms of morphological characteristics, physicochemical features and mineralization. Additionally, the second objective was to investigate and compare the osteoconductive properties of two different bioactive glass forms (powder and fiber) enriched or not with collagen using a tibial bone defect model in rats. For this, four different formulations (BG powder - BGp, BG powder enriched with collagen - BGp/Col, BG fibers - BGf and BGp fibers enriched with collagen - BGf/Col) were developed. The physicochemical and morphological modifications were analyzed by SEM, FTIR, calcium assay and pH measurement. For in vivo evaluations, histopathology, morphometrical and immunohistochemistry were performed in a tibial defect in rats. The FTIR analysis indicated that BGp and BGf maintained the characteristic peaks for this class of material. Furthermore, the calcium assay showed an increased Ca uptake in the BG fibers. The pH measurements revealed that BGp (with or without collagen) presented higher pH values compared to BGf. In addition, the histological analysis demonstrated no inflammation for all groups at the site of the injury, besides a faster material degradation and higher bone ingrowth for groups with collagen. The immunohistochemistry analysis demonstrated Runx-2 and Rank-L expression for all the groups. Those findings support that BGp with collagen can be a promising alternative for treating fracture of difficult

Influence of oxazolidines and zirconium oxalate crosslinkers on the hydrothermal, enzymatic, and thermo mechanical stability of type 1 collagen fiber

International Nuclear Information System (INIS)

Haroun, Mahdi A.; Khirstova, Palmina K.; Gasmelseed, Gurashi A.; Covington, Antony D.

2009-01-01

Stabilization of type I rat tail tendon (RTT) collagen by crosslink agent oxazolidine and zirconium oxalate was studied to understand the effect on the thermal, enzymatic and mechanical stability of collagen. The results show that both oxazolidine and zirconium oxalate imparts thermal stability to collagen, and oxazolidine exhibits a marked increase in the peak temperature and enthalpy changes when compared to both native and zirconium oxalate tanned RTT. There is a decrease in the peak temperature and the enthalpy changes of oxazolidine tanned RTT fibers after treatment with urea, suggesting the possibility of alterations in the secondary structure of collagen after tanning. Oxazolidine, which forms carbocationic intermediates species in solution, have better crosslinking with collagen as seen from viscometry studies and hence provides better enzymatic stability to collagen than zirconium, which largely forms monomeric species in solution. Zirconium does not seem to change the tensile strength of RTT fibers significantly in wet condition as well as oxazolidine

Influence of oxazolidines and zirconium oxalate crosslinkers on the hydrothermal, enzymatic, and thermo mechanical stability of type 1 collagen fiber

Energy Technology Data Exchange (ETDEWEB)

Haroun, Mahdi A. [Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM, Serdang (Malaysia)], E-mail: Mahdiupm@hotmail.com; Khirstova, Palmina K. [People' s Hall 11113, P.O. Box 6272, Khartoum (Sudan); Gasmelseed, Gurashi A. [Juba University, Leather Dept. P.O. Box 12327 Khartoum (Sudan); Covington, Antony D. [Leather Centre, University College Northampton, Northampton (United Kingdom)

2009-02-20

Stabilization of type I rat tail tendon (RTT) collagen by crosslink agent oxazolidine and zirconium oxalate was studied to understand the effect on the thermal, enzymatic and mechanical stability of collagen. The results show that both oxazolidine and zirconium oxalate imparts thermal stability to collagen, and oxazolidine exhibits a marked increase in the peak temperature and enthalpy changes when compared to both native and zirconium oxalate tanned RTT. There is a decrease in the peak temperature and the enthalpy changes of oxazolidine tanned RTT fibers after treatment with urea, suggesting the possibility of alterations in the secondary structure of collagen after tanning. Oxazolidine, which forms carbocationic intermediates species in solution, have better crosslinking with collagen as seen from viscometry studies and hence provides better enzymatic stability to collagen than zirconium, which largely forms monomeric species in solution. Zirconium does not seem to change the tensile strength of RTT fibers significantly in wet condition as well as oxazolidine.

Mechanical response of collagen molecule under hydrostatic compression

International Nuclear Information System (INIS)

Saini, Karanvir; Kumar, Navin

2015-01-01

Proteins like collagen are the basic building blocks of various body tissues (soft and hard). Collagen molecules find their presence in the skeletal system of the body where they bear mechanical loads from different directions, either individually or along with hydroxy-apatite crystals. Therefore, it is very important to understand the mechanical behavior of the collagen molecule which is subjected to multi-axial state of loading. The estimation of strains of collagen molecule along different directions resulting from the changes in hydrostatic pressure magnitude, can provide us new insights into its mechanical behavior. In the present work, full atomistic simulations have been used to study global (volumetric) as well as local (along different directions) mechanical properties of the hydrated collagen molecule which is subjected to different hydrostatic pressure magnitudes. To estimate the local mechanical properties, the strains of collagen molecule along its longitudinal and transverse directions have been acquired at different hydrostatic pressure magnitudes. In spite of non-homogeneous distribution of atoms within the collagen molecule, the calculated values of local mechanical properties have been found to carry the same order of magnitude along the longitudinal and transverse directions. It has been demonstrated that the values of global mechanical properties like compressibility, bulk modulus, etc. as well as local mechanical properties like linear compressibility, linear elastic modulus, etc. are functions of magnitudes of applied hydrostatic pressures. The mechanical characteristics of collagen molecule based on the atomistic model have also been compared with that of the continuum model in the present work. The comparison showed up orthotropic material behavior for the collagen molecule. The information on collagen molecule provided in the present study can be very helpful in designing the future bio-materials.

Mechanical response of collagen molecule under hydrostatic compression.

Science.gov (United States)

Saini, Karanvir; Kumar, Navin

2015-04-01

Proteins like collagen are the basic building blocks of various body tissues (soft and hard). Collagen molecules find their presence in the skeletal system of the body where they bear mechanical loads from different directions, either individually or along with hydroxy-apatite crystals. Therefore, it is very important to understand the mechanical behavior of the collagen molecule which is subjected to multi-axial state of loading. The estimation of strains of collagen molecule along different directions resulting from the changes in hydrostatic pressure magnitude, can provide us new insights into its mechanical behavior. In the present work, full atomistic simulations have been used to study global (volumetric) as well as local (along different directions) mechanical properties of the hydrated collagen molecule which is subjected to different hydrostatic pressure magnitudes. To estimate the local mechanical properties, the strains of collagen molecule along its longitudinal and transverse directions have been acquired at different hydrostatic pressure magnitudes. In spite of non-homogeneous distribution of atoms within the collagen molecule, the calculated values of local mechanical properties have been found to carry the same order of magnitude along the longitudinal and transverse directions. It has been demonstrated that the values of global mechanical properties like compressibility, bulk modulus, etc. as well as local mechanical properties like linear compressibility, linear elastic modulus, etc. are functions of magnitudes of applied hydrostatic pressures. The mechanical characteristics of collagen molecule based on the atomistic model have also been compared with that of the continuum model in the present work. The comparison showed up orthotropic material behavior for the collagen molecule. The information on collagen molecule provided in the present study can be very helpful in designing the future bio-materials. Copyright © 2015 Elsevier B.V. All rights

Mechanical response of collagen molecule under hydrostatic compression

Energy Technology Data Exchange (ETDEWEB)

Saini, Karanvir, E-mail: karans@iitrpr.ac.in; Kumar, Navin

2015-04-01

Proteins like collagen are the basic building blocks of various body tissues (soft and hard). Collagen molecules find their presence in the skeletal system of the body where they bear mechanical loads from different directions, either individually or along with hydroxy-apatite crystals. Therefore, it is very important to understand the mechanical behavior of the collagen molecule which is subjected to multi-axial state of loading. The estimation of strains of collagen molecule along different directions resulting from the changes in hydrostatic pressure magnitude, can provide us new insights into its mechanical behavior. In the present work, full atomistic simulations have been used to study global (volumetric) as well as local (along different directions) mechanical properties of the hydrated collagen molecule which is subjected to different hydrostatic pressure magnitudes. To estimate the local mechanical properties, the strains of collagen molecule along its longitudinal and transverse directions have been acquired at different hydrostatic pressure magnitudes. In spite of non-homogeneous distribution of atoms within the collagen molecule, the calculated values of local mechanical properties have been found to carry the same order of magnitude along the longitudinal and transverse directions. It has been demonstrated that the values of global mechanical properties like compressibility, bulk modulus, etc. as well as local mechanical properties like linear compressibility, linear elastic modulus, etc. are functions of magnitudes of applied hydrostatic pressures. The mechanical characteristics of collagen molecule based on the atomistic model have also been compared with that of the continuum model in the present work. The comparison showed up orthotropic material behavior for the collagen molecule. The information on collagen molecule provided in the present study can be very helpful in designing the future bio-materials.

The degree of collagen crosslinks in medical collagen membranes determined by water absorption

International Nuclear Information System (INIS)

Braczko, M.; Tederko, A.; Grzybowski, J.

1994-01-01

Collagen membranes were crosslinked by using three agents: glutaraldehyde, hexametylenediisocyanate, and UV irradiation. The increasing concentrations of above chemical agents or longer time of UV exposition resulted in the higher cross-links degree and in the decrease of collagen membranes swelling (measured as water absorption), their elasticity and mechanical resistance. According to American standards, the degree of collagen biomaterial cross-links is determined by measuring of the digestion time by pepsin. However, that method is very time-consuming. In our study, we have that a simple, linear regression between logarithm of digestion time by pepsin exists and it was identical for all three cross-linking agents used. We have concluded that determination of water absorption can be an alternative, simple and fast method for examination of collagen membrane cross-links degree. (author). 16 refs, 7 figs, 1 tab

FGF-2 potently induces both proliferation and DSP expression in collagen type I gel cultures of adult incisor immature pulp cells

International Nuclear Information System (INIS)

Nakao, Kazuhisa; Itoh, Makoto; Tomita, Yusuke; Tomooka, Yasuhiro; Tsuji, Takashi

2004-01-01

We investigated the effects of both cytokines and extracellular matrices on the proliferation and differentiation of immature adult rat incisor dental pulp cells. These immature cells, which have a high-proliferative potency in vitro and do not express mRNAs for dentin non-collagenous proteins such as dentin sialoprotein (DSP), bone sialoprotein (BSP), and osteocalcin, exist in the root regions of adult rat incisors. Fibroblast growth factor-2 (FGF-2) stimulated the proliferation of these immature cells and the subsequent production of mineralized calcium was induced by β-glycerophosphate treatment. Additionally, FGF-2 dramatically induced the expression of DSP and BSP mRNAs, but only in collagen type I gel cultures, whereas neither plate-coated collagen type I nor fibronectin, laminin or collagen type IV cultures could produce this effect and generate sufficient physiological levels of these transcripts. Although bone morphogenetic protein-4 could not induce the proliferation of immature dental pulp cells nor upregulate DSP mRNA expression, it had a synergistic effect upon DSP transcript levels in conjunction with FGF-2. These results suggest that both the presence of FGF-2 and the three-dimensional formation of immature dental pulp cells in collagen type I gel cultures are essential for both DSP expression and odontoblast differentiation. These observations provide valuable information concerning the study of the commitment and differentiation of odontoblast lineages, and also provide a basis for the rational design of cytokine and extracellular matrix based compounds for regenerative therapies in new dental treatments

A 48 kDa collagen-binding phosphoprotein isolated from bovine aortic endothelial cells interacts with the collagenous domain, but not the globular domain, of collagen type IV.

Science.gov (United States)

Yannariello-Brown, J; Madri, J A

1990-01-15

We have identified collagen-binding proteins in detergent extracts of metabolically labelled bovine aortic endothelial cells (BAEC) by collagen type IV-Sepharose affinity chromatography. The major collagen type IV-binding protein identified by SDS/PAGE had a molecular mass of 48 kDa, which we term the 'collagen-binding 48 kDa protein' (CB48). The pI of CB48 was 8.0-8.3 in a two-dimensional gel system, running non-equilibrium pH gel electrophoresis in the first dimension and SDS/PAGE in the second dimension. Under these conditions CB48 separated into two major (a and b) and one minor isoform (c); a was the most basic of the three isoforms. Two-dimensional chymotryptic peptide maps derived from each individual isoform were virtually identical. The charge differences between the isoforms were due in part to differential H3(32)PO4 incorporation by the protein. CB48 bound to intact collagen type IV and the collagenous region of collagen type IV, but not to the globular NC1 domain. Cell-surface labelling and indirect immunofluorescence experiments localized the bulk of CB48 intracellularly in the endoplasmic reticulum Golgi region, with a minor population of molecules on the cell surface. A specific rabbit polyclonal anti-CB48 serum did not inhibit the attachment or spreading of BAEC to collagen type IV in an 'in vitro' adhesion assay, suggesting that the cell-surface population of CB48 is not involved in BAEC adhesion. We conclude that CB48 is a collagen-binding phosphoprotein that interacts with the collagenous domain of collagen type IV and may be involved in intracellular transport of collagen molecules.

[Polarized microscopic observation of the collagen change in bone healing during bone lengthening].

Science.gov (United States)

Zou, Pei; Li, Junhui; Li, Zhuyi

2006-01-01

To investigate the feature and regularity of the collagen change in bone healing during bone lengthening. Bone lengthening model was made in the middle segment of the rabbit tibia. Five days after the model was established, the bone was lengthened 1.5 mm per day for 14 days. The rabbits were put to death after elongation, 7, 14, 21, 30, 40, 50, 60 and 70 days after elongation. The distracted area of the bone was imbedded with paraffin. After being stained by the picric acid-sirius red staining, the slice was observed under polarized microscope. The features of the collagen change in the distracted bone were as follows: (1) In the fibrous tissue of the distracted area during lengthening period and the early stage after lengthening, there was not only collagen III but also much collagen I. (2) Collagen I , II and III were observed in the cartilage. (3) Collagen I, II and III were also observed in the pseudo-growth plate. (4) Collagen I took the dominance during lengthening period and the late stage after lengthening. New bone formation in bone lengthening is under the distracted force, so the collagen changes have different features compared with that in fracture healing. Collagen I, II and III can be identified by picric acid-sirius red staining and polarized microscope, so a new method for studying the collagen typing in bone repairing is provided.

Resliced image space construction for coronary artery collagen fibers.

Science.gov (United States)

Luo, Tong; Chen, Huan; Kassab, Ghassan S

2017-01-01

Collagen fibers play an important role in the biomechanics of the blood vessel wall. The objective of this study was to determine the 3D microstructure of collagen fibers in the media and adventitia of coronary arteries. We present a novel optimal angle consistence algorithm to reform image slices in the visualization and analysis of 3D collagen images. 3D geometry was reconstructed from resliced image space where the 3D skeleton was extracted as the primary feature for accurate reconstruction of geometrical parameters. Collagen fibers (range 80-200) were reconstructed from the porcine coronary artery wall for the measurement of various morphological parameters. Collagen waviness and diameters were 1.37 ± 0.19 and 2.61 ± 0.89 μm, respectively. The biaxial distributions of orientation had two different peaks at 110.7 ± 25.2° and 18.4 ± 19.3°. Results for width, waviness, and orientation were found to be in good agreement with manual measurements. In addition to accurately measuring 2D features more efficiently than the manual approach, the present method produced 3D features that could not be measured in the 2D manual approach. These additional parameters included the tilt angle (5.10 ± 2.95°) and cross-sectional area (CSA; 5.98 ± 3.79 μm2) of collagen fibers. These 3D collagen reconstructions provide accurate and reliable microstructure for biomechanical modeling of vessel wall mechanics.

Adherence, proliferation and collagen turnover by human fibroblasts seeded into different types of collagen sponges

NARCIS (Netherlands)

Middelkoop, E.; de Vries, H. J.; Ruuls, L.; Everts, V.; Wildevuur, C. H.; Westerhof, W.

1995-01-01

We describe an in vitro model that we have used to evaluate dermal substitutes and to obtain data on cell proliferation, the rate of degradation of the dermal equivalent, contractibility and de novo synthesis of collagen. We tested three classes of collagenous materials: (1) reconstituted

ADHERENCE, PROLIFERATION AND COLLAGEN TURNOVER BY HUMAN FIBROBLASTS SEEDED INTO DIFFERENT TYPES OF COLLAGEN SPONGES

NARCIS (Netherlands)

MIDDELKOOP, E; DEVRIES, HJC; RUULS, L; EVERTS, [No Value; WILDEVUUR, CHR; WESTERHOF, W

We describe an in vitro model that we have used to evaluate dermal substitutes and to obtain data on cell proliferation, the rate of degradation of the dermal equivalent, contractibility and de novo synthesis of collagen. We tested three classes of collagenous materials: (1) reconstituted

Collagen Structural Hierarchy and Susceptibility to Degradation by Ultraviolet Radiation.

Science.gov (United States)

Rabotyagova, Olena S; Cebe, Peggy; Kaplan, David L

2008-12-01

Collagen type I is the most abundant extracellular matrix protein in the human body, providing the basis for tissue structure and directing cellular functions. Collagen has complex structural hierarchy, organized at different length scales, including the characteristic triple helical feature. In the present study, the relationship between collagen structure (native vs. denatured) and sensitivity to UV radiation was assessed, with a focus on changes in primary structure, changes in conformation, microstructure and material properties. A brief review of free radical reactions involved in collagen degradation is also provided as a mechanistic basis for the changes observed in the study. Structural and functional changes in the collagens were related to the initial conformation (native vs. denatured) and the energy of irradiation. These changes were tracked using SDS-PAGE to assess molecular weight, Fourier transform infrared (FTIR) spectroscopy to study changes in the secondary structure, and atomic force microscopy (AFM) to characterize changes in mechanical properties. The results correlate differences in sensitivity to irradiation with initial collagen structural state: collagen in native conformation vs. heat-treated (denatured) collagen. Changes in collagen were found at all levels of the hierarchical structural organization. In general, the native collagen triple helix is most sensitive to UV-254nm radiation. The triple helix delays single chain degradation. The loss of the triple helix in collagen is accompanied by hydrogen abstraction through free radical mechanisms. The results received suggest that the effects of electromagnetic radiation on biologically relevant extracellular matrices (collagen in the present study) are important to assess in the context of the state of collagen structure. The results have implications in tissue remodeling, wound repair and disease progression.

Modern collagen wound dressings: function and purpose.

Science.gov (United States)

Fleck, Cynthia Ann; Simman, Richard

2010-09-01

Collagen, which is produced by fibroblasts, is the most abundant protein in the human body. A natural structural protein, collagen is involved in all 3 phases of the wound-healing cascade. It stimulates cellular migration and contributes to new tissue development. Because of their chemotactic properties on wound fibroblasts, collagen dressings encourage the deposition and organization of newly formed collagen, creating an environment that fosters healing. Collagen-based biomaterials stimulate and recruit specific cells, such as macrophages and fibroblasts, along the healing cascade to enhance and influence wound healing. These biomaterials can provide moisture or absorption, depending on the delivery system. Collagen dressings are easy to apply and remove and are conformable. Collagen dressings are usually formulated with bovine, avian, or porcine collagen. Oxidized regenerated cellulose, a plant-based material, has been combined with collagen to produce a dressing capable of binding to and protecting growth factors by binding and inactivating matrix metalloproteinases in the wound environment. The increased understanding of the biochemical processes involved in chronic wound healing allows the design of wound care products aimed at correcting imbalances in the wound microenvironment. Traditional advanced wound care products tend to address the wound's macroenvironment, including moist wound environment control, fluid management, and controlled transpiration of wound fluids. The newer class of biomaterials and wound-healing agents, such as collagen and growth factors, targets specific defects in the chronic wound environment. In vitro laboratory data point to the possibility that these agents benefit the wound healing process at a biochemical level. Considerable evidence has indicated that collagen-based dressings may be capable of stimulating healing by manipulating wound biochemistry.

Study of collagen metabolism and regulation after β radiation injury

International Nuclear Information System (INIS)

Zhou Yinghui; Xu Lan; Wu Shiliang; Qiu Hao; Jiang Zhi; Tu Youbin; Zhang Xueguang

2001-01-01

The animal model of β radiation injury was established by the β radiation produced by the linear accelerator; and irradiated NIH 3T3 cells were studied. In the experiment the contents of total collagen, collagen type I and type III were measured. The activity of MMPs-1 were tested. The contents of TGF-β 1 , IL-6 were also detected. The results showed that after exposure to β radiation, little change was found in the content of total collagen, but the content of collagen I decreased and the content of collagen III, MMPs-1 activity increased; the expression of TGF-β 1 , IL-6 increased. The results suggest that changes in the metabolism of collagen play an important role in the irradiated injury of the skin; TGF-β 1 , IL-6 may be essential in the regulation of the collagen metabolism

Proteolysis breaks tolerance toward intact α345(IV) collagen, eliciting novel anti-GBM autoantibodies specific for α345NC1 hexamers

Science.gov (United States)

Olaru, Florina; Wang, Xu-Ping; Luo, Wentian; Ge, Linna; Miner, Jeffrey H; Kleinau, Sandra; Geiger, Xochiquetzal J.; Wasiluk, Andrew; Heidet, Laurence; Kitching, A. Richard; Borza, Dorin-Bogdan

2012-01-01

Goodpasture disease is an autoimmune kidney disease mediated by autoAbs against NC1 monomers of α3(IV) collagen that bind to the glomerular basement membrane (GBM), usually causing rapidly progressive glomerulonephritis. We identified a novel type of human IgG4-restricted anti-GBM autoAbs associated with mild non-progressive glomerulonephritis, which specifically targeted α345NC1 hexamers but not α3NC1 monomers. The mechanisms eliciting these anti-GBM autoAbs were investigated in mouse models recapitulating this phenotype. Wild type and FcγRIIB−/− mice immunized with autologous murine GBM NC1 hexamers produced mouse IgG1-restricted autoAbs specific for α345NC1 hexamers, which bound to the GBM in vivo but did not cause glomerulonephritis. In these mice, intact collagen IV from murine GBM was not immunogenic. However, in Col4a3−/− Alport mice, both intact collagen IV and NC1 hexamers from murine GBM elicited IgG antibodies specific for α3α4α5NC1 hexamers, which were not subclass restricted. As heterologous antigen in COL4A3-humanized mice, murine GBM NC1 hexamers elicited mouse IgG1, IgG2a and IgG2b autoAbs specific for α345NC1 hexamers and induced anti-GBM Ab glomerulonephritis. These findings indicate that tolerance toward autologous intact α3α4α5(IV) collagen is established in hosts expressing this antigen, even though autoreactive B cells specific for α345NC1 hexamers are not purged from their repertoire. Proteolysis selectively breaches this tolerance by generating autoimmunogenic α3α4α5NC1 hexamers. This provides a mechanism eliciting autoAbs specific for α345NC1 hexamers, which are restricted to non-inflammatory IgG subclasses and non-nephritogenic. In Alport syndrome, lack of tolerance toward α3α4α5(IV) collagen promotes production of alloantibodies to α345NC1 hexamers, including pro-inflammatory IgG subclasses which mediate post-transplant anti-GBM nephritis. PMID:23303673

Transforming growth factor β1 induces the expression of collagen type I by DNA methylation in cardiac fibroblasts.

Directory of Open Access Journals (Sweden)

Xiaodong Pan

Full Text Available Transforming growth factor-beta (TGF-β, a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-β induces collagen type I alpha 1 (COL1A1 expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-β-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-β1. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-β1 upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-β1 for 48 h. TGF-β1 treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-β1 can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-β1-induced COL1A1 gene expression.

The non-phagocytic route of collagen uptake

DEFF Research Database (Denmark)

Madsen, Daniel H; Ingvarsen, Signe; Jürgensen, Henrik J

2011-01-01

The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments......, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional...... mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include β1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down...

Collagen type IV at the fetal-maternal interface.

Science.gov (United States)

Oefner, C M; Sharkey, A; Gardner, L; Critchley, H; Oyen, M; Moffett, A

2015-01-01

Extracellular matrix proteins play a crucial role in influencing the invasion of trophoblast cells. However the role of collagens and collagen type IV (col-IV) in particular at the implantation site is not clear. Immunohistochemistry was used to determine the distribution of collagen types I, III, IV and VI in endometrium and decidua during the menstrual cycle and the first trimester of pregnancy. Expression of col-IV alpha chains during the reproductive cycle was determined by qPCR and protein localisation by immunohistochemistry. The structure of col-IV in placenta was examined using transmission electron microscopy. Finally, the expression of col-IV alpha chain NC1 domains and collagen receptors was localised by immunohistochemistry. Col-IV alpha chains were selectively up-regulated during the menstrual cycle and decidualisation. Primary extravillous trophoblast cells express collagen receptors and secrete col-IV in vitro and in vivo, resulting in the increased levels found in decidua basalis compared to decidua parietalis. A novel expression pattern of col-IV in the mesenchyme of placental villi, as a three-dimensional network, was found. NC1 domains of col-IV alpha chains are known to regulate tumour cell migration and the selective expression of these domains in decidua basalis compared to decidua parietalis was determined. Col-IV is expressed as novel forms in the placenta. These findings suggest that col-IV not only represents a structural protein providing tissue integrity but also influences the invasive behaviour of trophoblast cells at the implantation site. Copyright © 2014 Elsevier Ltd. All rights reserved.

Study of collagen metabolism and regulation after {beta} radiation injury

Energy Technology Data Exchange (ETDEWEB)

Yinghui, Zhou; Lan, Xu; Shiliang, Wu; Hao, Qiu; Zhi, Jiang; Youbin, Tu; Xueguang, Zhang [Suzhou Medical College (China)

2001-04-01

The animal model of {beta} radiation injury was established by the {beta} radiation produced by the linear accelerator; and irradiated NIH 3T3 cells were studied. In the experiment the contents of total collagen, collagen type I and type III were measured. The activity of MMPs-1 were tested. The contents of TGF-{beta}{sub 1}, IL-6 were also detected. The results showed that after exposure to {beta} radiation, little change was found in the content of total collagen, but the content of collagen I decreased and the content of collagen III, MMPs-1 activity increased; the expression of TGF-{beta}{sub 1}, IL-6 increased. The results suggest that changes in the metabolism of collagen play an important role in the irradiated injury of the skin; TGF-{beta}{sub 1}, IL-6 may be essential in the regulation of the collagen metabolism.

Fibrous mini-collagens in hydra nematocysts.

Science.gov (United States)

Holstein, T W; Benoit, M; Herder, G V; David, C N; Wanner, G; Gaub, H E

1994-07-15

Nematocysts (cnidocysts) are exocytotic organelles found in all cnidarians. Here, atomic force microscopy and field emission scanning electron microscopy reveal the structure of the nematocyst capsule wall. The outer wall consists of globular proteins of unknown function. The inner wall consists of bundles of collagen-like fibrils having a spacing of 50 to 100 nanometers and cross-striations at intervals of 32 nanometers. The fibrils consist of polymers of "mini-collagens," which are abundant in the nematocysts of Hydra. The distinct pattern of mini-collagen fibers in the inner wall can provide the tensile strength necessary to withstand the high osmotic pressure (15 megapascals) in the capsules.

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

OpenAIRE

Hui Liang; Xiaoran Li; Bin Wang; Bing Chen; Yannan Zhao; Jie Sun; Yan Zhuang; Jiajia Shi; He Shen; Zhijun Zhang; Jianwu Dai

2016-01-01

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of ...

Collagen-Induced Arthritis: A model for Murine Autoimmune Arthritis.

Science.gov (United States)

Pietrosimone, K M; Jin, M; Poston, B; Liu, P

2015-10-20

Collagen-induced arthritis (CIA) is a common autoimmune animal model used to study rheumatoid arthritis (RA). The development of CIA involves infiltration of macrophages and neutrophils into the joint, as well as T and B cell responses to type II collagen. In murine CIA, genetically susceptible mice (DBA/1J) are immunized with a type II bovine collagen emulsion in complete Freund's adjuvant (CFA), and receive a boost of type II bovine collagen in incomplete Freund's adjuvant (IFA) 21 days after the first injection. These mice typically develop disease 26 to 35 days after the initial injection. C57BL/6J mice are resistant to arthritis induced by type II bovine collagen, but can develop arthritis when immunized with type II chicken collagen in CFA, and receive a boost of type II chicken collagen in IFA 21 days after the first injection. The concentration of heat-killed Mycobacterium tuberculosis H37RA (MT) in CFA also differs for each strain. DBA/1J mice develop arthritis with 1 mg/ml MT, while C57BL/6J mice require and 3-4 mg/ml MT in order to develop arthritis. CIA develops slowly in C57BL/6J mice and cases of arthritis are mild when compared to DBA/1J mice. This protocol describes immunization of DBA/1J mice with type II bovine collagen and the immunization of C57BL/6J mice with type II chicken collagen.

Effects of tissue fixation and dehydration on tendon collagen nanostructure.

Science.gov (United States)

Turunen, Mikael J; Khayyeri, Hanifeh; Guizar-Sicairos, Manuel; Isaksson, Hanna

2017-09-01

Collagen is the most prominent protein in biological tissues. Tissue fixation is often required for preservation or sectioning of the tissue. This may affect collagen nanostructure and potentially provide incorrect information when analyzed after fixation. We aimed to unravel the effect of 1) ethanol and formalin fixation and 2) 24h air-dehydration on the organization and structure of collagen fibers at the nano-scale using small and wide angle X-ray scattering. Samples were divided into 4 groups: ethanol fixed, formalin fixed, and two untreated sample groups. Samples were allowed to air-dehydrate in handmade Kapton pockets during the measurements (24h) except for one untreated group. Ethanol fixation affected the collagen organization and nanostructure substantially and during 24h of dehydration dramatic changes were evident. Formalin fixation had minor effects on the collagen organization but after 12h of air-dehydration the spatial variation increased substantially, not evident in the untreated samples. Generally, collagen shrinkage and loss of alignment was evident in all samples during 24h of dehydration but the changes were subtle in all groups except the ethanol fixed samples. This study shows that tissue fixation needs to be chosen carefully in order to preserve the features of interest in the tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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.

Do sufficient vitamin D levels at the end of summer in children and adolescents provide an assurance of vitamin D sufficiency at the end of winter? A cohort study.

Science.gov (United States)

Shakeri, Habibesadat; Pournaghi, Seyed-Javad; Hashemi, Javad; Mohammad-Zadeh, Mohammad; Akaberi, Arash

2017-10-26

The changes in serum 25-hydroxyvitamin D (25(OH)D) in adolescents from summer to winter and optimal serum vitamin D levels in the summer to ensure adequate vitamin D levels at the end of winter are currently unknown. This study was conducted to address this knowledge gap. The study was conducted as a cohort study. Sixty-eight participants aged 7-18 years and who had sufficient vitamin D levels at the end of the summer in 2011 were selected using stratified random sampling. Subsequently, the participants' vitamin D levels were measured at the end of the winter in 2012. A receiver operating characteristic (ROC) curve was used to determine optimal cutoff points for vitamin D at the end of the summer to predict sufficient vitamin D levels at the end of the winter. The results indicated that 89.7% of all the participants had a decrease in vitamin D levels from summer to winter: 14.7% of them were vitamin D-deficient, 36.8% had insufficient vitamin D concentrations and only 48.5% where able to maintain sufficient vitamin D. The optimal cutoff point to provide assurance of sufficient serum vitamin D at the end of the winter was 40 ng/mL at the end of the summer. Sex, age and vitamin D levels at the end of the summer were significant predictors of non-sufficient vitamin D at the end of the winter. In this age group, a dramatic reduction in vitamin D was observed over the follow-up period. Sufficient vitamin D at the end of the summer did not guarantee vitamin D sufficiency at the end of the winter. We found 40 ng/mL as an optimal cutoff point.

Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome.

Science.gov (United States)

O'Donoghue, Anthony J; Knudsen, Giselle M; Beekman, Chapman; Perry, Jenna A; Johnson, Alexander D; DeRisi, Joseph L; Craik, Charles S; Bennett, Richard J

2015-06-16

Pseudogymnoascus destructans is the causative agent of white-nose syndrome, a disease that has caused the deaths of millions of bats in North America. This psychrophilic fungus proliferates at low temperatures and targets hibernating bats, resulting in their premature arousal from stupor with catastrophic consequences. Despite the impact of white-nose syndrome, little is known about the fungus itself or how it infects its mammalian host. P. destructans is not amenable to genetic manipulation, and therefore understanding the proteins involved in infection requires alternative approaches. Here, we identify hydrolytic enzymes secreted by P. destructans, and use a novel and unbiased substrate profiling technique to define active peptidases. These experiments revealed that endopeptidases are the major proteolytic activities secreted by P. destructans, and that collagen, the major structural protein in mammals, is actively degraded by the secretome. A serine endopeptidase, hereby-named Destructin-1, was subsequently identified, and a recombinant form overexpressed and purified. Biochemical analysis of Destructin-1 showed that it mediated collagen degradation, and a potent inhibitor of peptidase activity was identified. Treatment of P. destructans-conditioned media with this antagonist blocked collagen degradation and facilitated the detection of additional secreted proteolytic activities, including aminopeptidases and carboxypeptidases. These results provide molecular insights into the secretome of P. destructans, and identify serine endopeptidases that have the clear potential to facilitate tissue invasion and pathogenesis in the mammalian host.

Prolyl 3-hydroxylase 1 and CRTAP are mutually stabilizing in the endoplasmic reticulum collagen prolyl 3-hydroxylation complex.

Science.gov (United States)

Chang, Weizhong; Barnes, Aileen M; Cabral, Wayne A; Bodurtha, Joann N; Marini, Joan C

2010-01-15

Null mutations in cartilage-associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1/LEPRE1) cause types VII and VIII OI, respectively, two novel recessive forms of osteogenesis imperfecta (OI) with severe to lethal bone dysplasia and overmodification of the type I collagen helical region. CRTAP and P3H1 form a complex with cyclophilin B (CyPB) in the endoplasmic reticulum (ER) which 3-hydroxylates the Pro986 residue of alpha1(I) and alpha1(II) collagen chains. We investigated the interaction of complex components in fibroblasts from types VII and VIII OI patients. Both CRTAP and P3H1 are absent or reduced on western blots and by immunofluorescence microscopy in cells containing null mutations in either gene. Levels of LEPRE1 or CRTAP transcripts, however, are normal in CRTAP- or LEPRE1-null cells, respectively. Stable transfection of a CRTAP or LEPRE1 expression construct into cells with null mutations for the transfected cDNA restored both CRTAP and P3H1 protein levels. Normalization of collagen helical modification in transfected CRTAP-null cells demonstrated that the restored proteins functioned effectively as a complex. These data indicate that CRTAP and P3H1 are mutually stabilized in the collagen prolyl 3-hydroxylation complex. CyPB levels were unaffected by mutations in either CRTAP or LEPRE1. Proteasomal inhibitors partially rescue P3H1 protein in CRTAP-null cells. In LEPRE1-null cells, secretion of CRTAP is increased compared with control cells and accounts for 15-20% of the decreased CRTAP detected in cells. Thus, mutual stabilization of P3H1 and CRTAP in the ER collagen modification complex is an underlying mechanism for the overlapping phenotype of types VII and VIII OI.

Proximal collagenous gastroenteritides:

DEFF Research Database (Denmark)

Nielsen, Ole Haagen; Riis, Lene Buhl; Danese, Silvio

2014-01-01

AIM: While collagenous colitis represents the most common form of the collagenous gastroenteritides, the collagenous entities affecting the proximal part of the gastrointestinal tract are much less recognized and possibly overlooked. The aim was to summarize the latest information through a syste...

Endocytic collagen degradation

DEFF Research Database (Denmark)

Madsen, Daniel H.; Jürgensen, Henrik J.; Ingvarsen, Signe Ziir

2012-01-01

it crucially important to understand both the collagen synthesis and turnover mechanisms in this condition. Here we show that the endocytic collagen receptor, uPARAP/Endo180, is a major determinant in governing the balance between collagen deposition and degradation. Cirrhotic human livers displayed a marked...... up-regulation of uPARAP/Endo180 in activated fibroblasts and hepatic stellate cells located close to the collagen deposits. In a hepatic stellate cell line, uPARAP/Endo180 was shown to be active in, and required for, the uptake and intracellular degradation of collagen. To evaluate the functional...... groups of mice clearly revealed a fibrosis protective role of uPARAP/Endo180. This effect appeared to directly reflect the activity of the collagen receptor, since no compensatory events were noted when comparing the mRNA expression profiles of the two groups of mice in an array system focused on matrix-degrading...

Characterization of Genipin-Modified Dentin Collagen

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Hiroko Nagaoka

2014-01-01

Full Text Available Application of biomodification techniques to dentin can improve its biochemical and biomechanical properties. Several collagen cross-linking agents have been reported to strengthen the mechanical properties of dentin. However, the characteristics of collagen that has undergone agent-induced biomodification are not well understood. The objective of this study was to analyze the effects of a natural cross-linking agent, genipin (GE, on dentin discoloration, collagen stability, and changes in amino acid composition and lysyl oxidase mediated natural collagen cross-links. Dentin collagen obtained from extracted bovine teeth was treated with three different concentrations of GE (0.01%, 0.1%, and 0.5% for several treatment times (0–24 h. Changes in biochemical properties of NaB3H4-reduced collagen were characterized by amino acid and cross-link analyses. The treatment of dentin collagen with GE resulted in a concentration- and time-dependent pigmentation and stability against bacterial collagenase. The lysyl oxidase-mediated trivalent mature cross-link, pyridinoline, showed no difference among all groups while the major divalent immature cross-link, dehydro-dihydroxylysinonorleucine/its ketoamine in collagen treated with 0.5% GE for 24 h, significantly decreased compared to control (P< 0.05. The newly formed GE-induced cross-links most likely involve lysine and hydroxylysine residues of collagen in a concentration-dependent manner. Some of these cross-links appear to be reducible and stabilized with NaB3H4.

Arterial stiffening provides sufficient explanation for primary hypertension.

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Klas H Pettersen

2014-05-01

Full Text Available Hypertension is one of the most common age-related chronic disorders, and by predisposing individuals for heart failure, stroke, and kidney disease, it is a major source of morbidity and mortality. Its etiology remains enigmatic despite intense research efforts over many decades. By use of empirically well-constrained computer models describing the coupled function of the baroreceptor reflex and mechanics of the circulatory system, we demonstrate quantitatively that arterial stiffening seems sufficient to explain age-related emergence of hypertension. Specifically, the empirically observed chronic changes in pulse pressure with age and the impaired capacity of hypertensive individuals to regulate short-term changes in blood pressure arise as emergent properties of the integrated system. The results are consistent with available experimental data from chemical and surgical manipulation of the cardio-vascular system. In contrast to widely held opinions, the results suggest that primary hypertension can be attributed to a mechanogenic etiology without challenging current conceptions of renal and sympathetic nervous system function.

Binding of collagens to an enterotoxigenic strain of Escherichia coli

International Nuclear Information System (INIS)

Visai, L.; Speziale, P.; Bozzini, S.

1990-01-01

An enterotoxigenic strain of Escherichia coli, B34289c, has been shown to bind the N-terminal region of fibronectin with high affinity. We now report that this strain also binds collagen. The binding of 125I-labeled type II collagen to bacteria was time dependent and reversible. Bacteria expressed a limited number of collagen receptors (2.2 x 10(4) per cell) and bound collagen with a Kd of 20 nM. All collagen types tested (I to V) as well as all tested cyanogen bromide-generated peptides [alpha 1(I)CB2, alpha 1(I)CB3, alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB4] were recognized by bacterial receptors, as demonstrated by the ability of these proteins to inhibit the binding of 125I-labeled collagen to bacteria. Of several unlabeled proteins tested in competition experiments, fibronectin and its N-terminal region strongly inhibited binding of the radiolabeled collagen to E. coli cells. Conversely, collagen competed with an 125I-labeled 28-kilodalton fibronectin fragment for bacterial binding. Collagen bound to bacteria could be displaced by excess amounts of either unlabeled fibronectin or its N-terminal fragment. Similarly, collagen could displace 125I-labeled N-terminal peptide of fibronectin bound to the bacterial cell surface. Bacteria grown at 41 degrees C or in the presence of glucose did not express collagen or fibronectin receptors. These results indicate the presence of specific binding sites for collagen on the surface of E. coli cells and furthermore that the collagen and fibronectin binding sites are located in close proximity, possibly on the same structure

Collagen Quantification in Tissue Specimens.

Science.gov (United States)

Coentro, João Quintas; Capella-Monsonís, Héctor; Graceffa, Valeria; Wu, Zhuning; Mullen, Anne Maria; Raghunath, Michael; Zeugolis, Dimitrios I

2017-01-01

Collagen is the major extracellular protein in mammals. Accurate quantification of collagen is essential in the biomaterials (e.g., reproducible collagen scaffold fabrication), drug discovery (e.g., assessment of collagen in pathophysiologies, such as fibrosis), and tissue engineering (e.g., quantification of cell-synthesized collagen) fields. Although measuring hydroxyproline content is the most widely used method to quantify collagen in biological specimens, the process is very laborious. To this end, the Sircol™ Collagen Assay is widely used due to its inherent simplicity and convenience. However, this method leads to overestimation of collagen content due to the interaction of Sirius red with basic amino acids of non-collagenous proteins. Herein, we describe the addition of an ultrafiltration purification step in the process to accurately determine collagen content in tissues.

ZEB1 induces LOXL2-mediated collagen stabilization and deposition in the extracellular matrix to drive lung cancer invasion and metastasis.

Science.gov (United States)

Peng, D H; Ungewiss, C; Tong, P; Byers, L A; Wang, J; Canales, J R; Villalobos, P A; Uraoka, N; Mino, B; Behrens, C; Wistuba, I I; Han, R I; Wanna, C A; Fahrenholtz, M; Grande-Allen, K J; Creighton, C J; Gibbons, D L

2017-04-06

Lung cancer is the leading cause of cancer-related deaths, primarily due to distant metastatic disease. Metastatic lung cancer cells can undergo an epithelial-to-mesenchymal transition (EMT) regulated by various transcription factors, including a double-negative feedback loop between the microRNA-200 (miR-200) family and ZEB1, but the precise mechanisms by which ZEB1-dependent EMT promotes malignancy remain largely undefined. Although the cell-intrinsic effects of EMT are important for tumor progression, the reciprocal dynamic crosstalk between mesenchymal cancer cells and the extracellular matrix (ECM) is equally critical in regulating invasion and metastasis. Investigating the collaborative effect of EMT and ECM in the metastatic process reveals increased collagen deposition in metastatic tumor tissues as a direct consequence of amplified collagen gene expression in ZEB1-activated mesenchymal lung cancer cells. In addition, collagen fibers in metastatic lung tumors exhibit greater linearity and organization as a result of collagen crosslinking by the lysyl oxidase (LOX) family of enzymes. Expression of the LOX and LOXL2 isoforms is directly regulated by miR-200 and ZEB1, respectively, and their upregulation in metastatic tumors and mesenchymal cell lines is coordinated to that of collagen. Functionally, LOXL2, as opposed to LOX, is the principal isoform that crosslinks and stabilizes insoluble collagen deposition in tumor tissues. In turn, focal adhesion formation and FAK/SRC signaling is activated in mesenchymal tumor cells by crosslinked collagen in the ECM. Our study is the first to validate direct regulation of LOX and LOXL2 by the miR-200/ZEB1 axis, defines a novel mechanism driving tumor metastasis, delineates collagen as a prognostic marker, and identifies LOXL2 as a potential therapeutic target against tumor progression.

Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells

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Hisatomi Keiko

2012-06-01

Full Text Available Abstract Background Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF. We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro. Methods The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining. Results TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1. Conclusion We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

Collagen Type I as a Ligand for Receptor-Mediated Signaling

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Iris Boraschi-Diaz

2017-05-01

Full Text Available Collagens form the fibrous component of the extracellular matrix in all multi-cellular animals. Collagen type I is the most abundant collagen present in skin, tendons, vasculature, as well as the organic portion of the calcified tissue of bone and teeth. This review focuses on numerous receptors for which collagen acts as a ligand, including integrins, discoidin domain receptors DDR1 and 2, OSCAR, GPVI, G6b-B, and LAIR-1 of the leukocyte receptor complex (LRC and mannose family receptor uPARAP/Endo180. We explore the process of collagen production and self-assembly, as well as its degradation by collagenases and gelatinases in order to predict potential temporal and spatial sites of action of different collagen receptors. While the interactions of the mature collagen matrix with integrins and DDR are well-appreciated, potential signals from immature matrix as well as collagen degradation products are possible but not yet described. The role of multiple collagen receptors in physiological processes and their contribution to pathophysiology of diseases affecting collagen homeostasis require further studies.

Calcium hydroxylapatite treatment of human skin: evidence of collagen turnover through picrosirius red staining and circularly polarized microscopy

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Zerbinati N

2018-01-01

Full Text Available Nicola Zerbinati,1 Alberto Calligaro2 1Department of Surgical and Morphological Sciences, University of Insubria (Varese and Polyspecialist Medical Center, Pavia, 2Department of Public Health, Experimental and Forensic Medicine, Unit of Histology and Embryology, University of Pavia, Pavia, Italy Background: Calcium hydroxylapatite (CaHA, Radiesse® is a biocompatible, injectable filler for facial soft-tissue augmentation that provides volume to tissues, followed by a process of neocollagenesis for improved skin quality. Objective: To examine the effects of CaHA treatment on the molecular organization of collagen using a combination of picrosirius red staining and circularly polarized light microscopy.Methods: Five subjects received subdermal injection of 0.3 mL of CaHA in tissues scheduled for removal during abdominoplasty 2 months later. Tissue specimens from the CaHA injection site and a control untreated area were obtained from excised skin at the time of surgery. Processed tissue sections were stained with picrosirius red solution 0.1% and visualized under circularly polarized light microscopy for identification of thick mature (type I and thin newly formed (type III collagen fibers. Pixel signals from both the control and CaHA-treated areas were extracted from the images, and morphometric computerized hue analysis was performed to provide a quantitative evaluation of mature and newly formed collagen fibers.Results: Under picrosirius red staining and circularly polarized light microscopy, green/yellow areas (thin newly formed collagen type III were visible among the collagen fibers in tissue sections from the area of CaHA injection. In contrast, the majority of the collagen fibers appeared red (thick mature collagen type I in control tissues. Morphometric analysis confirmed that, following CaHA treatment, the proportion of fibers represented by thin newly formed collagen type III increased significantly (p<0.01 in comparison with the

Role of TGF-beta1 in relation to exercise-induced type I collagen synthesis in human tendinous tissue

DEFF Research Database (Denmark)

Heinemeier, Katja; Langberg, Henning; Olesen, Jens L

2003-01-01

synthesis, is released from cultured tendon fibroblasts in response to mechanical loading. Thus TGF-beta1 could link mechanical loading and collagen synthesis in tendon tissue in vivo. Tissue levels of TGF-beta1 and type I collagen metabolism markers [procollagen I COOH-terminal propeptide (PICP) and COOH...... exercise (P insertion was markedly delayed by exercise compared with the decay seen in resting subjects...

Collagen crosslink location: a molecular marker for fibrosis in lungs of rats with experimental silicosis

International Nuclear Information System (INIS)

Gerriets, J.E.; Reiser, K.M.; Last, J.A.

1986-01-01

Collagen content is increased in lungs of animals with experimental silicosis. They hypothesize that the collagen deposited in such fibrotic lungs differs structurally from normal lung collagen. Silicotic lung collagen shows an increase in lysine hydroxylation. In addition, the ratio of the difunctional crosslinks DHLNL (dihydroxylysinonorleucine) to HLNL (hydroxylysinonorleucine) is sharply elevated compared to that in control lungs. The peptide α1(I)CB7 x α2(I)CB1 crosslinked by HLNL was demonstrated in NaB 3 H 4 -reduced, CNBr-digested collagen from rat tail tendon by peptide purification, followed by periodate oxidation and amino acid analysis. Further structural analysis of this peptide was obtained by digestion of the crosslinked peptide with trypsin and purification of the tryptic peptide containing this crosslink followed by amino acid analysis. They then examined the analogous collagenous peptide in normal and silicotic lungs and analyzed the crosslink it contained. They observed that DHLNL was present at specific sites previously containing HLNL; that is, the collagen in fibrotic lungs is altered at specific sites by post-translational modification of a lysine residue by hydroxylation in a predictable way. They conclude that such unusual hydroxylation of a specific lysine residue in the α2 chain provides a molecular marker for fibrotic lung collagen

Apneic oxygenation combined with extracorporeal arteriovenous carbon dioxide removal provides sufficient gas exchange in experimental lung injury

DEFF Research Database (Denmark)

Nielsen, Niels Dalsgaard; Kjærgaard, Benedict; Nielsen, Jakob Koefoed

In this porcine lung injury model, apneic oxygenation with arteriovenous CO2 removal provided sufficient gas exchange and stable hemodynamics, indicating that the method might have a potential in the treatment of severe ARDS.   Acknowledgements The membrane lungs were kindly provided by Novalung GmbH, Germany.......Background and aim of study We hypothesized that continuous high airway pressure without ventilatory movements (apneic oxygenation), using an open lung approach, combined with extracorporeal, pumpless, arterio-venous, carbon dioxide (CO2) removal would provide adequate gas exchange in acute lung...

Release of antibiotics from collagen dressing.

Science.gov (United States)

Grzybowski, J; Antos-Bielska, M; Ołdak, E; Trafny, E A

1997-01-01

Our new collagen dressing has been developed recently. Three types (A, B, and C) of the dressing were prepared in this study. Each type contained bacitracin, neomycin or colistin. The antibiotic was input into: i. collagen sponge (CS)--type A, ii. layer of limited hydrophobicity (LLH)--type B, and iii. into both CS and LLH layers--type C. The final concentration of the antibiotic that resulted from the loading level was 2 mg/cm2 for the dressings of type A and B and 4 mg/cm2 for the dressing of type C. The antibiotics were then extracted from the pieces of dressings for two days through dialysis membrane. Susceptibility of 54 bacterial strains (S. aureus, P. aeruginosa, and Acinetobacter) isolated from burn wounds were tested to the three antibiotics used for preparation of the dressings. The results of the study evidenced that efficiency of released of antibiotics into the extracts depended on the kind of antibiotic and on the type of dressing. The concentration of the antibiotics proved to be much higher than MIC90 values of the bacterial isolates tested in respect to their susceptibility. The dressing containing mixture of the three antibiotics in two layers--CS and LLH is now considered as potentially effective for care of infected wounds. It may be useful for the treatment of infected wounds or for profilaxis of contaminated wounds, ensuring: i. sufficient antimicrobial activity in wound, and ii. optimal wound environment for the presence of collagenic biomaterial on the damaged tissue.

Effects of isopropanol on collagen fibrils in new parchment

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Gonzalez Lee G

2012-03-01

Full Text Available Abstract 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.

The collagen receptor uPARAP/Endo180

DEFF Research Database (Denmark)

Engelholm, Lars H; Ingvarsen, Signe; Jürgensen, Henrik J

2009-01-01

The uPAR-associated protein (uPARAP/Endo180), a type-1 membrane protein belonging to the mannose receptor family, is an endocytic receptor for collagen. Through this endocytic function, the protein takes part in a previously unrecognized mechanism of collagen turnover. uPARAP/Endo180 can bind...... and internalize both intact and partially degraded collagens. In some turnover pathways, the function of the receptor probably involves an interplay with certain matrix-degrading proteases whereas, in other physiological processes, redundant mechanisms involving both endocytic and pericellular collagenolysis seem...... in collagen breakdown seems to be involved in invasive tumor growth Udgivelsesdato: 2009...

Biological effect of hydrolyzed collagen on bone metabolism.

Science.gov (United States)

Daneault, Audrey; Prawitt, Janne; Fabien Soulé, Véronique; Coxam, Véronique; Wittrant, Yohann

2017-06-13

Osteoporosis is a chronic and asymptomatic disease characterized by low bone mass and skeletal microarchitectural deterioration, increased risk of fracture, and associated comorbidities most prevalent in the elderly. Due to an increasingly aging population, osteoporosis has become a major health issue requiring innovative disease management. Proteins are important for bone by providing building blocks and by exerting specific regulatory function. This is why adequate protein intake plays a considerable role in both bone development and bone maintenance. More specifically, since an increase in the overall metabolism of collagen can lead to severe dysfunctions and a more fragile bone matrix and because orally administered collagen can be digested in the gut, cross the intestinal barrier, enter the circulation, and become available for metabolic processes in the target tissues, one may speculate that a collagen-enriched diet provides benefits for the skeleton. Collagen-derived products such as gelatin or hydrolyzed collagen (HC) are well acknowledged for their safety from a nutritional point of view; however, what is their impact on bone biology? In this manuscript, we critically review the evidence from literature for an effect of HC on bone tissues in order to determine whether HC may represent a relevant alternative in the design of future nutritional approaches to manage osteoporosis prevention.

Surface characterization of collagen/elastin based biomaterials for tissue regeneration

International Nuclear Information System (INIS)

Skopinska-Wisniewska, J.; Sionkowska, A.; Kaminska, A.; Kaznica, A.; Jachimiak, R.; Drewa, T.

2009-01-01

Collagen and elastin are the main proteins of extracellular matrix. Collagen plays a crucial role in tensile strength of tissues, whereas elastin provides resilience to many organs. Both biopolymers are readily available and biocompatible. These properties point out that collagen and elastin are good components of materials for many potential medical applications. The surface properties of biomaterials play an important role in biomedicine as the majority of biological reactions occur on the surface of implanted materials. One of the methods of surface modification is UV-irradiation. The exposition of the biomaterial on ultraviolet light can alterate surface properties of the materials, their chemical stability, swelling properties and mechanical properties as well. The aim of our work was to study the surface properties and biocompatibility of new collagen/elastin based biomaterials and consideration of the influence of ultraviolet light on these properties. The surface properties of collagen/elastin based biomaterials modified by UV-irradiation were studied using the technique of atomic force microscopy (AFM) and contact angle measurements. On the basis of the results the surface free energy and its polar component was calculated using Owens-Wendt method. To assess the biological performance of films based on collagen, elastin and their blends, the response of 3T3 cell was investigated. It was found that the surface of collagen/elastin film is enriched in less polar component - collagen. Exposition on UV light increases polarity of collagen/elastin based films, due to photooxidation process. The AFM images have shown that topography and roughness of the materials had been also affected by UV-irradiation. The changes in surface properties influence on interaction between the material's surface and cells. The investigation of 3T3 cells grown on films based on collagen, elastin and their blends, leads to the conclusion that higher content of elastin in biomaterial

Surface characterization of collagen/elastin based biomaterials for tissue regeneration

Energy Technology Data Exchange (ETDEWEB)

Skopinska-Wisniewska, J., E-mail: joanna@chem.uni.torun.pl [Faculty of Chemistry, Nicolaus Copernicus University, Gagarin 7, 87-100 Torun (Poland); Sionkowska, A.; Kaminska, A. [Faculty of Chemistry, Nicolaus Copernicus University, Gagarin 7, 87-100 Torun (Poland); Kaznica, A.; Jachimiak, R.; Drewa, T. [Collegium Medicum, Nicolaus Copernicus University, Karlowicz 24, 85-092 Bydgoszcz (Poland)

2009-07-15

Collagen and elastin are the main proteins of extracellular matrix. Collagen plays a crucial role in tensile strength of tissues, whereas elastin provides resilience to many organs. Both biopolymers are readily available and biocompatible. These properties point out that collagen and elastin are good components of materials for many potential medical applications. The surface properties of biomaterials play an important role in biomedicine as the majority of biological reactions occur on the surface of implanted materials. One of the methods of surface modification is UV-irradiation. The exposition of the biomaterial on ultraviolet light can alterate surface properties of the materials, their chemical stability, swelling properties and mechanical properties as well. The aim of our work was to study the surface properties and biocompatibility of new collagen/elastin based biomaterials and consideration of the influence of ultraviolet light on these properties. The surface properties of collagen/elastin based biomaterials modified by UV-irradiation were studied using the technique of atomic force microscopy (AFM) and contact angle measurements. On the basis of the results the surface free energy and its polar component was calculated using Owens-Wendt method. To assess the biological performance of films based on collagen, elastin and their blends, the response of 3T3 cell was investigated. It was found that the surface of collagen/elastin film is enriched in less polar component - collagen. Exposition on UV light increases polarity of collagen/elastin based films, due to photooxidation process. The AFM images have shown that topography and roughness of the materials had been also affected by UV-irradiation. The changes in surface properties influence on interaction between the material's surface and cells. The investigation of 3T3 cells grown on films based on collagen, elastin and their blends, leads to the conclusion that higher content of elastin in

Sufficiency Grounded as Sufficiently Free: A Reply to Shlomi Segall

DEFF Research Database (Denmark)

Nielsen, Lasse

2016-01-01

be grounded on (i) any personal value, nor (ii) any impersonal value. Consequently, sufficientarianism is groundless. This article contains a rejoinder to this critique. Its main claim is that the value of autonomy holds strong potential for grounding sufficiency. It argues, firstly, that autonomy carries...... both personal value for its recipient as well as impersonal value, and that both of these values are suitable for grounding sufficiency. It thus follows that we should reject both (i) and (ii). Secondly, although autonomy is presumably the strongest candidate for grounding sufficiency, the article...... provides some counterargument to Segall’s rejection of the other candidates—the impersonal value of virtue; the personal value for the allocator; and the personal value for others. If the arguments are sound, they show that we need not worry about sufficientarianism being groundless....

Effect of radiation on rat skin collagen

International Nuclear Information System (INIS)

Nogami, Akira

1980-01-01

I. Albino male rats were exposed for 16 weeks to ultraviolet light (UVL) which has principle emission at 305 nm. There were no significant changes between control and UVL-exposed skins in the total hydroxyproline content. However, a little increase of citrate-soluble collagen, a little decrease of insoluble collagen and a decrease of aldehyde content in soluble collagen were observed with UVL exposure. Total acid glycosaminoglycan in skin increased 30% or more from control. These results show that the effect of UVL on rat skin in vivo was merely inflammation phenomenon and that the 'aging' process of skin was not caused in our experimental conditions. II. The effects of radiation on the solubility of rat skin collagen were examined under various conditions. 1) When intact rats were exposed to a single dose of radiation from 43 kVp X-ray source, the solubility in skin collagen did not change at 4,000 R dosage, while in irradiation of 40,000 R a decreased solubility in collagen was observed. When rats were given 400 R a week for 12 weeks, there was no changes in the solubility of collagen during experimental period. 2) In vitro exposure to skins, an irradiation of 40,000 R from 43 kVp X-ray source caused a decrease in the solubility of collagen. While an irradiation of 40,000 R of dosage from 200 kVp X-ray source resulted in the increase in soluble collagen and the decrease in insoluble collagen. 3) When intact rats were given a single dose of 40,000 R from 60 Co- gamma -ray, insoluble collagen decreased in both young and adult rats. Similar changes in collagen solubility were observed in vitro gamma -irradiation. (author)

Promotion of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium

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Xin-Yu Li

2014-02-01

Full Text Available AIM:To investigate the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium (Ti surface.METHODS:The chimeric peptide RKLPDAPRGDN (minTBP-1-PRGDN was synthesized by connecting RKLPDA (minTBP-1 to the N-terminal of PRGDN , the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on Ti surface were tested using PRGDN and minTBP-1as controls. The keratocytes attached to the surface of Ti were either stained with FITC-labeled phalloidin and viewed with fluorescence microscope or quantified with alamar Blue method. The proliferation of keratocytes on Ti were quantified with 3-(4,5-dim- ethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide up-taking methods. The secretion of type I collagen were determined using an ELISA kit.RESULTS:The results showed that minTBP-1-PRGDN at a concentration of 100ng/mL was the most potent peptide to enhance the attachment of human keratocytes to the surface of Ti (1.40±0.03 folds, P=0.003, to promote the proliferation (1.26±0.05 folds, P=0.014 and the synthesis of type I collagen (1.530±0.128, P=0.008. MinTBP-1 at the same concentration could only promote the attachment (1.13±0.04 folds, P=0.020 and proliferation(1.15±0.06 folds, P=0.021, while PRGDN had no significant influence (P＞0.05.CONCLUSION:Our data shows that the novel chimeric peptide minTBP-1-PRGDN could promote the attachment, proliferation and type I collagen synthesis of human keratocytes on the surface of Ti.

Mechanisms of lamellar collagen formation in connective tissues.

Science.gov (United States)

Ghazanfari, Samaneh; Khademhosseini, Ali; Smit, Theodoor H

2016-08-01

The objective of tissue engineering is to regenerate functional tissues. Engineering functional tissues requires an understanding of the mechanisms that guide the formation and evolution of structure in the extracellular matrix (ECM). In particular, the three-dimensional (3D) collagen fiber arrangement is important as it is the key structural determinant that provides mechanical integrity and biological function. In this review, we survey the current knowledge on collagen organization mechanisms that can be applied to create well-structured functional lamellar tissues and in particular intervertebral disc and cornea. Thus far, the mechanisms behind the formation of cross-aligned collagen fibers in the lamellar structures is not fully understood. We start with cell-induced collagen alignment and strain-stabilization behavior mechanisms which can explain a single anisotropically aligned collagen fiber layer. These mechanisms may explain why there is anisotropy in a single layer in the first place. However, they cannot explain why a consecutive collagen layer is laid down with an alternating alignment. Therefore, we explored another mechanism, called liquid crystal phasing. While dense concentrations of collagen show such behavior, there is little evidence that the conditions for liquid crystal phasing are actually met in vivo. Instead, lysyl aldehyde-derived collagen cross-links have been found essential for correct lamellar matrix deposition. Furthermore, we suggest that supra-cellular (tissue-level) shear stress may be instrumental in the alignment of collagen fibers. Understanding the potential mechanisms behind the lamellar collagen structure in connective tissues will lead to further improvement of the regeneration strategies of functional complex lamellar tissues. Copyright © 2016 Elsevier Ltd. All rights reserved.

Circular RNA profiling reveals that circCOL3A1-859267 regulate type I collagen expression in photoaged human dermal fibroblasts

International Nuclear Information System (INIS)

Peng, Yating; Song, Xiaojing; Zheng, Yue; Wang, Xinyi; Lai, Wei

2017-01-01

Production of type I collagen declines is a main characteristic during photoaging, but the mechanism is still not fully understood. Circular RNAs (circRNAs) are a class of newly identified non-coding RNAs with regulatory potency by sequestering miRNAs like a sponge. It's more stable than linear RNAs, and would be a useful tool for regulation of gene expression. However, the role of circRNAs in collagen expression during photoaging is still unclear. Here we performed deep sequencing of RNA generated from UVA irradiated and no irradiated human dermal fibroblasts (HDFs) and identified 29 significantly differentially expressed circRNAs (fold change ≥ 1.5, P < 0.05), 12 circRNAs were up-regulated and 17 circRNAs were down-regulated.3 most differentially expressed circRNAs were verified by qRT-PCR and the down-regulated circCOL3A1-859267 exhibited the most significantly altered in photoaged HDFs. Overexpression of circCOL3A1-859267 inhibited UVA-induced decrease of type I collagen expression and silencing of it reduced type I collagen intensity. Via a bioinformatic method, 44 miRNAs were predicted to binding with circCOL3A1-859267, 5 of them have been confirmed or predicted to interact with type I collagen. This study show that circCOL3A1-859267 regulate type I collagen expression in photoaged HDFs, suggesting it may be a novel target for interfering photoaging.

Hyaluronan in aged collagen matrix increases prostate epithelial cell proliferation

Science.gov (United States)

Damodarasamy, Mamatha; Vernon, Robert B.; Chan, Christina K.; Plymate, Stephen R.; Wight, Thomas N.

2015-01-01

The extracellular matrix (ECM) of the prostate, which is comprised primarily of collagen, becomes increasingly disorganized with age, a property that may influence the development of hyperplasia and cancer. Collageous ECM extracted from the tails of aged mice exhibits many characteristics of collagen in aged tissues, including the prostate. When polymerized into a 3-dimensional (3D) gel, these collagen extracts can serve as models for the study of specific cell-ECM interactions. In the present study, we examined the behaviors of human prostatic epithelial cell lines representing normal prostate epithelial cells (PEC), benign prostatic hyperplasia (BPH-1), and adenocarcinoma (LNCaP) cultured in contact with 3D gels made from collagen extracts of young and aged mice. We found that proliferation of PEC, BPH-1, and LNCaP cells were all increased by culture on aged collagen gels relative to young collagen gels. In examining age-associated differences in the composition of the collagen extracts, we found that aged and young collagen had a similar amount of several collagen-associated ECM components, but aged collagen had a much greater content of the glycosaminoglycan hyaluronan (HA) than young collagen. The addition of HA (of similar size and concentration to that found in aged collagen extracts) to cells placed in young collagen elicited significantly increased proliferation in BPH-1 cells, but not in PEC or LNCaP cells, relative to controls not exposed to HA. Of note, histochemical analyses of human prostatic tissues showed significantly higher expression of HA in BPH and prostate cancer stroma relative to stroma of normal prostate. Collectively, these results suggest that changes in ECM involving increased levels of HA contribute to the growth of prostatic epithelium with aging. PMID:25124870

Stimulation of type I collagen activity in healing of pulp perforation

OpenAIRE

Kunarti, Sri

2008-01-01

Background: TGF-β1 is a connective tissue stimulant, potential regulator for tissue repair, and promoter in wound healing. The healing of pulp perforation is decided by quantity and quality of new collagen deposition. TGF-β1 upregulates collagen transcription. However, after several weeks production of type I collagen synthesis is stopped and enzymatic degradation of collagen matrix will occur. Purpose: Observe synthesis type I collagen during the process of pulp perforation healing in 7, 14,...

Characterization of Fibrin and Collagen Gels for Engineering Wound Healing Models

Directory of Open Access Journals (Sweden)

Oihana Moreno-Arotzena

2015-04-01

Full Text Available Hydrogels are used for 3D in vitro assays and tissue engineering and regeneration purposes. For a thorough interpretation of this technology, an integral biomechanical characterization of the materials is required. In this work, we characterize the mechanical and functional behavior of two specific hydrogels that play critical roles in wound healing, collagen and fibrin. A coherent and complementary characterization was performed using a generalized and standard composition of each hydrogel and a combination of techniques. Microstructural analysis was performed by scanning electron microscopy and confocal reflection imaging. Permeability was measured using a microfluidic-based experimental set-up, and mechanical responses were analyzed by rheology. We measured a pore size of 2.84 and 1.69 μm for collagen and fibrin, respectively. Correspondingly, the permeability of the gels was 1.00·10−12 and 5.73·10−13 m2. The shear modulus in the linear viscoelastic regime was 15 Pa for collagen and 300 Pa for fibrin. The gels exhibited strain-hardening behavior at ca. 10% and 50% strain for fibrin and collagen, respectively. This consistent biomechanical characterization provides a detailed and robust starting point for different 3D in vitro bioapplications, such as collagen and/or fibrin gels. These features may have major implications for 3D cellular behavior by inducing divergent microenvironmental cues.

MT1-MMP and type II collagen specify skeletal stem cells and their bone and cartilage progeny

DEFF Research Database (Denmark)

Szabova, L.; Yamada, S.S.; Wimer, H.

2009-01-01

-expressing cells of the skeleton rescues not only diminished chondrocyte proliferation, but surprisingly, also results in amelioration of the severe skeletal dysplasia associated with MT1-MMP deficiency through enhanced bone formation. Consistent with this increased bone formation, type II collagen was identified...... from nontransgenic MT1-MMP-deficient littermates. These observations show that type II collagen is not stringently confined to the chondrocyte but is expressed in skeletal stem/progenitor cells (able to regenerate bone, cartilage, myelosupportive stroma, marrow adipocytes) and in the chondrogenic...

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

Directory of Open Access Journals (Sweden)

Chanjuan Dong

2016-02-01

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

Development of a novel collagen-GAG nanofibrous scaffold via electrospinning

Energy Technology Data Exchange (ETDEWEB)

Zhong Shaoping [Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent 119260 (Singapore); Teo, Wee Eong [Division of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent 119260 (Singapore); Zhu Xiao [Singapore Eye Research Institute, Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751 (Singapore); Beuerman, Roger [Singapore Eye Research Institute, Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751 (Singapore); Ramakrishna, Seeram [Division of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent 119260 (Singapore); Yung, Lin Yue Lanry [Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent 119260 (Singapore)]. E-mail: cheyly@nus.edu.sg

2007-03-15

Collagen and glycosaminoglycan (GAG) are native constituents of human tissues and are widely utilized to fabricate scaffolds serving as an analog of native extracellular matrix (ECM).The development of blended collagen and GAG scaffolds may potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native ECM. In this study, we were able to obtain a novel nanofibrous collagen-GAG scaffold by electrospinning with collagen and chondroitin sulfate (CS), a widely used GAG. The electrospun collagen-GAG scaffold exhibited a uniform fiber structure in nano-scale diameter. By crosslinking with glutaraldehyde vapor, the collagen-GAG scaffolds could resist from collagenase degradation and enhance the biostability of the scaffolds. This led to the increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without crosslinking did not increase the biostability but still promoted cell growth. In conclusion, the electrospun collagen-GAG scaffolds, with high surface-to-volume ratio, may potentially provide a better environment for tissue formation/biosynthesis compared with the traditional scaffolds.

Development of a novel collagen-GAG nanofibrous scaffold via electrospinning

International Nuclear Information System (INIS)

Zhong Shaoping; Teo, Wee Eong; Zhu Xiao; Beuerman, Roger; Ramakrishna, Seeram; Yung, Lin Yue Lanry

2007-01-01

Collagen and glycosaminoglycan (GAG) are native constituents of human tissues and are widely utilized to fabricate scaffolds serving as an analog of native extracellular matrix (ECM).The development of blended collagen and GAG scaffolds may potentially be used in many soft tissue engineering applications since the scaffolds mimic the structure and biological function of native ECM. In this study, we were able to obtain a novel nanofibrous collagen-GAG scaffold by electrospinning with collagen and chondroitin sulfate (CS), a widely used GAG. The electrospun collagen-GAG scaffold exhibited a uniform fiber structure in nano-scale diameter. By crosslinking with glutaraldehyde vapor, the collagen-GAG scaffolds could resist from collagenase degradation and enhance the biostability of the scaffolds. This led to the increased proliferation of rabbit conjunctiva fibroblast on the scaffolds. Incorporation of CS into collagen nanofibers without crosslinking did not increase the biostability but still promoted cell growth. In conclusion, the electrospun collagen-GAG scaffolds, with high surface-to-volume ratio, may potentially provide a better environment for tissue formation/biosynthesis compared with the traditional scaffolds

Collagen reorganization at the tumor-stromal interface facilitates local invasion

Directory of Open Access Journals (Sweden)

Inman David R

2006-12-01

Full Text Available Abstract Background Stromal-epithelial interactions are of particular significance in breast tissue as misregulation of these interactions can promote tumorigenesis and invasion. Moreover, collagen-dense breast tissue increases the risk of breast carcinoma, although the relationship between collagen density and tumorigenesis is not well understood. As little is known about epithelial-stromal interactions in vivo, it is necessary to visualize the stroma surrounding normal epithelium and mammary tumors in intact tissues to better understand how matrix organization, density, and composition affect tumor formation and progression. Methods Epithelial-stromal interactions in normal mammary glands, mammary tumors, and tumor explants in three-dimensional culture were studied with histology, electron microscopy, and nonlinear optical imaging methodologies. Imaging of the tumor-stromal interface in live tumor tissue ex vivo was performed with multiphoton laser-scanning microscopy (MPLSM to generate multiphoton excitation (MPE of endogenous fluorophores and second harmonic generation (SHG to image stromal collagen. Results We used both laser-scanning multiphoton and second harmonic generation microscopy to determine the organization of specific collagen structures around ducts and tumors in intact, unfixed and unsectioned mammary glands. Local alterations in collagen density were clearly seen, allowing us to obtain three-dimensional information regarding the organization of the mammary stroma, such as radiating collagen fibers that could not have been obtained using classical histological techniques. Moreover, we observed and defined three tumor-associated collagen signatures (TACS that provide novel markers to locate and characterize tumors. In particular, local cell invasion was found predominantly to be oriented along certain aligned collagen fibers, suggesting that radial alignment of collagen fibers relative to tumors facilitates invasion. Consistent

Diffusion of MMPs on the surface of collagen fibrils: the mobile cell surface-collagen substratum interface.

Directory of Open Access Journals (Sweden)

Ivan E Collier

Full Text Available Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP(2/TIMP-2/MMP-2 can initiate (MT1-MMP and complete (MMP-2 degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP(2/TIMP-2/MMP-2 represents a Mobile Cell Surface-Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions.

Nucleotide sequence of a cDNA coding for the amino-terminal region of human prepro. alpha. 1(III) collagen

Energy Technology Data Exchange (ETDEWEB)

Toman, P D; Ricca, G A [Rorer Biotechnology, Inc., Springfield, VA (USA); de Crombrugghe, B [National Institutes of Health, Bethesda, MD (USA)

1988-07-25

Type III Collagen is synthesized in a variety of tissues as a precursor macromolecule containing a leader sequence, a N-propeptide, a N-telopeptide, the triple helical region, a C-telopeptide, and C-propeptide. To further characterize the human type III collagen precursor, a human placental cDNA library was constructed in gt11 using an oligonucleotide derived from a partial cDNA sequence corresponding to the carboxy-terminal part of the 1(III) collagen. A cDNA was identified which contains the leader sequence, the N-propeptide and N-telopeptide regions. The DNA sequence of these regions are presented here. The triple helical, C-telopeptide and C-propeptide amino acid sequence for human type III collagen has been determined previously. A comparison of the human amino acid sequence with mouse, chicken, and calf sequence shows 81%, 81%, and 92% similarity, respectively. At the DNA level, the sequence similarity between human and mouse or chicken type III collagen sequences in this area is 82% and 77%, respectively.

The collagenic architecture of human dura mater.

Science.gov (United States)

Protasoni, Marina; Sangiorgi, Simone; Cividini, Andrea; Culuvaris, Gloria Tiffany; Tomei, Giustino; Dell'Orbo, Carlo; Raspanti, Mario; Balbi, Sergio; Reguzzoni, Marcella

2011-06-01

Human dura mater is the most external meningeal sheet surrounding the CNS. It provides an efficient protection to intracranial structures and represents the most important site for CSF turnover. Its intrinsic architecture is made up of fibrous tissue including collagenic and elastic fibers that guarantee the maintenance of its biophysical features. The recent technical advances in the repair of dural defects have allowed for the creation of many synthetic and biological grafts. However, no detailed studies on the 3D microscopic disposition of collagenic fibers in dura mater are available. The authors report on the collagenic 3D architecture of normal dura mater highlighting the orientation, disposition in 3 dimensions, and shape of the collagen fibers with respect to the observed layer. Thirty-two dura mater specimens were collected during cranial decompressive surgical procedures, fixed in 2.5% Karnovsky solution, and digested in 1 N NaOH solution. After a routine procedure, the specimens were observed using a scanning electron microscope. The authors distinguished the following 5 layers in the fibrous dura mater of varying thicknesses, orientation, and structures: bone surface, external median, vascular, internal median, and arachnoid layers. The description of the ultrastructural 3D organization of the different layers of dura mater will give us more information for the creation of synthetic grafts that are as similar as possible to normal dura mater. This description will be also related to the study of the neoplastic invasion.

Greener synthesis of electrospun collagen/hydroxyapatite composite fibers with an excellent microstructure for bone tissue engineering

Directory of Open Access Journals (Sweden)

Zhou YY

2015-04-01

Full Text Available Yuanyuan Zhou,1,2 Hongchang Yao,1 Jianshe Wang,1 Dalu Wang,1 Qian Liu,1 Zhongjun Li11College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, People’s Republic of China; 2Institute of Enviromental and Municipal Engineering, North China University of Water Resources and Electric Power, Zhengzhou, People’s Republic of ChinaAbstract: In bone tissue engineering, collagen/hydroxyapatite (HAP fibrous composite obtained via electrospinning method has been demonstrated to support the cells’ adhesion and bone regeneration. However, electrospinning of natural collagen often requires the use of cytotoxic organic solvents, and the HAP crystals were usually aggregated and randomly distributed within a fibrous matrix of collagen, limiting their clinical potential. Here, an effective and greener method for the preparation of collagen/HAP composite fibers was developed for the first time, and this green product not only had 40 times higher mechanical properties than that previously reported, but also had an excellent microstructure similar to that of natural bone. By dissolving type I collagen in environmentally friendly phosphate buffered saline/ethanol solution instead of the frequently-used cytotoxic organic solvents, followed with the key step of desalination, co-electrospinning the collagen solution with the HAP sol, generates a collagen/HAP composite with a uniform and continuous fibrous morphology. Interestingly, the nano-HAP needles were found to preferentially orient along the longitudinal direction of the collagen fibers, which mimicked the nanostructure of natural bones. Based on the characterization of the related products, the formation mechanism for this novel phenomenon was proposed. After cross-linking with 1-ethyl-3-(3-dimethyl-aminopropyl-1-carbodiimide hydrochloride/N-hydroxysuccinimide, the obtained composite exhibited a significant enhancement in mechanical properties. In addition, the biocompatibility of the

A BMP responsive transcriptional region in the chicken type X collagen gene.

Science.gov (United States)

Volk, S W; Luvalle, P; Leask, T; Leboy, P S

1998-10-01

Bone morphogenetic proteins (BMPs) were originally identified by their ability to induce ectopic bone formation and have been shown to promote both chondrogenesis and chondrocyte hypertrophy. BMPs have recently been found to activate a membrane serine/threonine kinase signaling mechanism in a variety of cell types, but the downstream effectors of BMP signaling in chondrocyte differentiation remain unidentified. We have previously reported that BMP-2 markedly stimulates type X collagen expression in prehypertrophic chick sternal chondrocytes, and that type X collagen mRNA levels in chondrocytes cultured under serum-free (SF) conditions are elevated 3- to 5-fold within 24 h. To better define the molecular mechanisms of induction of chondrocyte hypertrophy by BMPs, we examined the effect of BMPs on type X collagen production by 15-day chick embryo sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. Two populations of chondrocytes were used: one representing resting cartilage isolated from the caudal third of the sterna and the second representing prehypertrophic cartilage from the cephalic third of the sterna. BMP-2, BMP-4, and BMP-7 all effectively promoted chondrocyte maturation of cephalic sternal chondrocytes as measured by high levels of alkaline phosphatase, diminished levels of type II collagen, and induction of the hypertrophic chondrocyte-specific marker, type X collagen. To test whether BMP control of type X collagen expression occurs at the transcriptional level, we utilized plasmid constructs containing the chicken collagen X promoter and 5' flanking regions fused to a reporter gene. Constructs were transiently transfected into sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. A 533 bp region located 2.4-2.9 kb upstream from the type X collagen transcriptional start site was both necessary and sufficient for strong BMP responsiveness

[Collagen nephritis].

Science.gov (United States)

Lago, N R; Bulos, M J; Monserrat, A J

1997-01-01

Fibrillar collagen in the glomeruli is considered specific of the nail-patella syndrome. A new nephropathy with diffuse intraglomerular deposition of type III collagen without nail and skeletal abnormalities has been described. We report the case of a 26-year-old woman who presented persistent proteinuria, hematuria, deafness without nail and skeletal abnormalities. The renal biopsy showed focal and segmental glomerulosclerosis by light microscopy. The electron microscopy revealed the presence of massive fibrillar collagen within the mesangial matriz and the basement membrane. This is the first patient reported in our country. We emphasize the usefulness of electron microscopy in the study of glomerular diseases.

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix.

Science.gov (United States)

Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

2016-02-17

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn't showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody-drug conjugates (ADC) or immunotoxins.

H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen

International Nuclear Information System (INIS)

Collier, I.E.; Wilhelm, S.M.; Eisen, A.Z.

1988-01-01

H-ras transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on this ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors. Type IV collagenase consists of three domains. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin

Nanomechanical mapping of hydrated rat tail tendon collagen I fibrils.

Science.gov (United States)

Baldwin, Samuel J; Quigley, Andrew S; Clegg, Charlotte; Kreplak, Laurent

2014-10-21

Collagen fibrils play an important role in the human body, providing tensile strength to connective tissues. These fibrils are characterized by a banding pattern with a D-period of 67 nm. The proposed origin of the D-period is the internal staggering of tropocollagen molecules within the fibril, leading to gap and overlap regions and a corresponding periodic density fluctuation. Using an atomic force microscope high-resolution modulus maps of collagen fibril segments, up to 80 μm in length, were acquired at indentation speeds around 10(5) nm/s. The maps revealed a periodic modulation corresponding to the D-period as well as previously undocumented micrometer scale fluctuations. Further analysis revealed a 4/5, gap/overlap, ratio in the measured modulus providing further support for the quarter-staggered model of collagen fibril axial structure. The modulus values obtained at indentation speeds around 10(5) nm/s are significantly larger than those previously reported. Probing the effect of indentation speed over four decades reveals two distinct logarithmic regimes of the measured modulus and point to the existence of a characteristic molecular relaxation time around 0.1 ms. Furthermore, collagen fibrils exposed to temperatures between 50 and 62°C and cooled back to room temperature show a sharp decrease in modulus and a sharp increase in fibril diameter. This is also associated with a disappearance of the D-period and the appearance of twisted subfibrils with a pitch in the micrometer range. Based on all these data and a similar behavior observed for cross-linked polymer networks below the glass transition temperature, we propose that collagen I fibrils may be in a glassy state while hydrated.

Collagenous sprue: a clinicopathologic study of 12 cases.

LENUS (Irish Health Repository)

Maguire, Aoife A

2012-02-01

Collagenous sprue is a rare form of small bowel enteropathy characterized by chronic diarrhea and progressive malabsorption with little data available on its natural history. The pathologic lesion consists of subepithelial collagen deposition associated with variable alterations in villous architecture. The small bowel biopsies of 12 cases were reviewed. Clinical details, celiac serology, and T-cell receptor gene rearrangement study results, when available, were collated. There were 8 females and 4 males (age ranged from 41 to 84 y) who presented with chronic diarrhea and weight loss. Small intestinal biopsies showed subepithelial collagen deposition with varying degrees of villous atrophy and varying numbers of intraepithelial lymphocytes. Four patients had previous biopsies showing enteropathic changes without collagen deposition. Seven cases were associated with collagenous colitis and 1 also had features of lymphocytic colitis. Three patients also had collagen deposition in gastric biopsies. One case was associated with lymphocytic gastritis. Celiac disease (CD, gluten-sensitive enteropathy) was documented in 4 patients. Five patients made a clinical improvement with combinations of a gluten-free diet and immunosuppressive therapy. Two patients died of complications of malnutrition and 1 of another illness. Clonal T-cell populations were identified in 5 of 6 cases tested. Four of these patients improved clinically after treatment but 1 has died. Collagenous sprue evolved on a background of CD in 4 cases. There was no history of CD in others and these cases may be the result of a biologic insult other than gluten sensitivity. None has developed clinical evidence of lymphoma to date.

A microscopic evaluation of collagen-bilirubin interactions: in vitro surface phenomenon.

Science.gov (United States)

Usharani, N; Jayakumar, G C; Rao, J R; Chandrasekaran, B; Nair, B U

2014-02-01

This study is carried out to understand the morphology variations of collagen I matrices influenced by bilirubin. The characteristics of bilirubin interaction with collagen ascertained using various techniques like XRD, CLSM, fluorescence, SEM and AFM. These techniques are used to understand the distribution, expression and colocalization patterns of collagen-bilirubin complexes. The present investigation mimic the in vivo mechanisms created during the disorder condition like jaundice. Fluorescence technique elucidates the crucial role played by bilirubin deposition and interaction during collagen organization. Influence of bilirubin during collagen fibrillogenesis and banding patterns are clearly visualize using SEM. As a result, collagen-bilirubin complex provides different reconstructed patterns because of the influence of bilirubin concentration. Selectivity, specificity and spatial organization of collagen-bilirubin are determined through AFM imaging. Consequently, it is observed that the morphology and quantity of the bilirubin binding to collagen varied by the concentrations and the adsorption rate in protein solutions. Microscopic studies of collagen-bilirubin interaction confirms that bilirubin influence the fibrillogenesis and alter the rate of collagen organization depending on the bilirubin concentration. This knowledge helps to develop a novel drug to inhibit the interface point of interaction between collagen and bilirubin. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

Stabilization and anomalous hydration of collagen fibril under heating.

Directory of Open Access Journals (Sweden)

Sasun G Gevorkian

Full Text Available BACKGROUND: Type I collagen is the most common protein among higher vertebrates. It forms the basis of fibrous connective tissues (tendon, chord, skin, bones and ensures mechanical stability and strength of these tissues. It is known, however, that separate triple-helical collagen macromolecules are unstable at physiological temperatures. We want to understand the mechanism of collagen stability at the intermolecular level. To this end, we study the collagen fibril, an intermediate level in the collagen hierarchy between triple-helical macromolecule and tendon. METHODOLOGY/PRINCIPAL FINDING: When heating a native fibril sample, its Young's modulus decreases in temperature range 20-58°C due to partial denaturation of triple-helices, but it is approximately constant at 58-75°C, because of stabilization by inter-molecular interactions. The stabilization temperature range 58-75°C has two further important features: here the fibril absorbs water under heating and the internal friction displays a peak. We relate these experimental findings to restructuring of collagen triple-helices in fibril. A theoretical description of the experimental results is provided via a generalization of the standard Zimm-Bragg model for the helix-coil transition. It takes into account intermolecular interactions of collagen triple-helices in fibril and describes water adsorption via the Langmuir mechanism. CONCLUSION/SIGNIFICANCE: We uncovered an inter-molecular mechanism that stabilizes the fibril made of unstable collagen macromolecules. This mechanism can be relevant for explaining stability of collagen.

Second-harmonic generation imaging of collagen in ancient bone.

Science.gov (United States)

Thomas, B; McIntosh, D; Fildes, T; Smith, L; Hargrave, F; Islam, M; Thompson, T; Layfield, R; Scott, D; Shaw, B; Burrell, C L; Gonzalez, S; Taylor, S

2017-12-01

Second-harmonic generation imaging (SHG) captures triple helical collagen molecules near tissue surfaces. Biomedical research routinely utilizes various imaging software packages to quantify SHG signals for collagen content and distribution estimates in modern tissue samples including bone. For the first time using SHG, samples of modern, medieval, and ice age bones were imaged to test the applicability of SHG to ancient bone from a variety of ages, settings, and taxa. Four independent techniques including Raman spectroscopy, FTIR spectroscopy, radiocarbon dating protocols, and mass spectrometry-based protein sequencing, confirm the presence of protein, consistent with the hypothesis that SHG imaging detects ancient bone collagen. These results suggest that future studies have the potential to use SHG imaging to provide new insights into the composition of ancient bone, to characterize ancient bone disorders, to investigate collagen preservation within and between various taxa, and to monitor collagen decay regimes in different depositional environments.

Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen.

Science.gov (United States)

Terajima, Masahiko; Taga, Yuki; Chen, Yulong; Cabral, Wayne A; Hou-Fu, Guo; Srisawasdi, Sirivimol; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Kurie, Jonathan M; Marini, Joan C; Yamauchi, Mitsuo

2016-04-29

Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1-3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cyclophilin-B Modulates Collagen Cross-linking by Differentially Affecting Lysine Hydroxylation in the Helical and Telopeptidyl Domains of Tendon Type I Collagen*

Science.gov (United States)

Terajima, Masahiko; Taga, Yuki; Chen, Yulong; Cabral, Wayne A.; Hou-Fu, Guo; Srisawasdi, Sirivimol; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Kurie, Jonathan M.; Marini, Joan C.; Yamauchi, Mitsuo

2016-01-01

Covalent intermolecular cross-linking provides collagen fibrils with stability. The cross-linking chemistry is tissue-specific and determined primarily by the state of lysine hydroxylation at specific sites. A recent study on cyclophilin B (CypB) null mice, a model of recessive osteogenesis imperfecta, demonstrated that lysine hydroxylation at the helical cross-linking site of bone type I collagen was diminished in these animals (Cabral, W. A., Perdivara, I., Weis, M., Terajima, M., Blissett, A. R., Chang, W., Perosky, J. E., Makareeva, E. N., Mertz, E. L., Leikin, S., Tomer, K. B., Kozloff, K. M., Eyre, D. R., Yamauchi, M., and Marini, J. C. (2014) PLoS Genet. 10, e1004465). However, the extent of decrease appears to be tissue- and molecular site-specific, the mechanism of which is unknown. Here we report that although CypB deficiency resulted in lower lysine hydroxylation in the helical cross-linking sites, it was increased in the telopeptide cross-linking sites in tendon type I collagen. This resulted in a decrease in the lysine aldehyde-derived cross-links but generation of hydroxylysine aldehyde-derived cross-links. The latter were absent from the wild type and heterozygous mice. Glycosylation of hydroxylysine residues was moderately increased in the CypB null tendon. We found that CypB interacted with all lysyl hydroxylase isoforms (isoforms 1–3) and a putative lysyl hydroxylase-2 chaperone, 65-kDa FK506-binding protein. Tendon collagen in CypB null mice showed severe size and organizational abnormalities. The data indicate that CypB modulates collagen cross-linking by differentially affecting lysine hydroxylation in a site-specific manner, possibly via its interaction with lysyl hydroxylases and associated molecules. This study underscores the critical importance of collagen post-translational modifications in connective tissue formation. PMID:26934917

TGF-β1-elevated TRPM7 channel regulates collagen expression in hepatic stellate cells via TGF-β1/Smad pathway

International Nuclear Information System (INIS)

Fang, Ling; Huang, Cheng; Meng, Xiaoming; Wu, Baoming; Ma, Taotao; Liu, Xuejiao; Zhu, Qian; Zhan, Shuxiang; Li, Jun

2014-01-01

Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts plays a critical role in the development of liver fibrosis, since myofibroblasts are the key cells responsible for excessive deposition of ECM proteins. Transient receptor potential melastatin 7 (TRPM7), a non-selective cation channel with protein serine/threonine kinase activity, has been demonstrated to function in the proliferation of activated HSCs. Here, we investigated the functional role of TRPM7 in collagen deposition in activated HSC-T6 cells (a rat hepatic stellate cell line). TRPM7 mRNA and protein were measured by Real-time PCR and Western blot in TGF-β1-activated HSC-T6 cells in vitro. Results demonstrated that TRPM7 protein was dramatically increased in fibrotic human livers. Stimulation of HSC-T6 cells with TGF-β1 increased TRPM7 mRNA and protein level in a time-dependent manner. Nevertheless, TGF-β1-elicited upregulation of TRPM7 in HSC-T6 cells was abrogated by SB431542 (TGF-β1 receptor blocker) or SIS3 (inhibitor of Smad3 phosphorylation). Additionally, blockade of TRPM7 channels with non-specific TRPM7 blocker 2-APB or synthetic siRNA targeting TRPM7 attenuated TGF-β1-induced expression of myofibroblast markers, as measured by the induction of α-SMA and Col1α1. Silencing TRPM7 also increased the ratio of MMPs/TIMPs by increasing MMP-13 expression and decreasing TIMP-1 and TIMP-2 levels. Strikingly, phosphorylation of p-Smad2 and p-Smad3, associated with collagen production, was decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TGF-β1 elevates TRPM7 expression in HSCs via Smad3-dependant mechanisms, which in turn contributes Smad protein phosphorylation, and subsequently increases fibrous collagen expression. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis. - Highlights: • Upregulation of TRPM7 protein in human fibrotic livers • Upregulation of TRPM7 by TGF-β1 elicited Smad signaling in HSC-T6 cells

TGF-β1-elevated TRPM7 channel regulates collagen expression in hepatic stellate cells via TGF-β1/Smad pathway

Energy Technology Data Exchange (ETDEWEB)

Fang, Ling, E-mail: fangling_1984@126.com [School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Institute for Liver Diseases of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Medical University, Ministry of Education, Mei Shan Road, Hefei, Anhui Province 230032 (China); The First Affiliated Hospital of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Huang, Cheng; Meng, Xiaoming; Wu, Baoming; Ma, Taotao; Liu, Xuejiao; Zhu, Qian [School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Institute for Liver Diseases of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Medical University, Ministry of Education, Mei Shan Road, Hefei, Anhui Province 230032 (China); Zhan, Shuxiang [School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Institute for Liver Diseases of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Medical University, Ministry of Education, Mei Shan Road, Hefei, Anhui Province 230032 (China); The First Affiliated Hospital of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Li, Jun, E-mail: lj@ahmu.edu.cn [School of Pharmacy, Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Institute for Liver Diseases of Anhui Medical University, Mei Shan Road, Hefei, Anhui Province 230032 (China); Key Laboratory of Anti-inflammatory and Immune Medicine, Anhui Medical University, Ministry of Education, Mei Shan Road, Hefei, Anhui Province 230032 (China)

2014-10-15

Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts plays a critical role in the development of liver fibrosis, since myofibroblasts are the key cells responsible for excessive deposition of ECM proteins. Transient receptor potential melastatin 7 (TRPM7), a non-selective cation channel with protein serine/threonine kinase activity, has been demonstrated to function in the proliferation of activated HSCs. Here, we investigated the functional role of TRPM7 in collagen deposition in activated HSC-T6 cells (a rat hepatic stellate cell line). TRPM7 mRNA and protein were measured by Real-time PCR and Western blot in TGF-β1-activated HSC-T6 cells in vitro. Results demonstrated that TRPM7 protein was dramatically increased in fibrotic human livers. Stimulation of HSC-T6 cells with TGF-β1 increased TRPM7 mRNA and protein level in a time-dependent manner. Nevertheless, TGF-β1-elicited upregulation of TRPM7 in HSC-T6 cells was abrogated by SB431542 (TGF-β1 receptor blocker) or SIS3 (inhibitor of Smad3 phosphorylation). Additionally, blockade of TRPM7 channels with non-specific TRPM7 blocker 2-APB or synthetic siRNA targeting TRPM7 attenuated TGF-β1-induced expression of myofibroblast markers, as measured by the induction of α-SMA and Col1α1. Silencing TRPM7 also increased the ratio of MMPs/TIMPs by increasing MMP-13 expression and decreasing TIMP-1 and TIMP-2 levels. Strikingly, phosphorylation of p-Smad2 and p-Smad3, associated with collagen production, was decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TGF-β1 elevates TRPM7 expression in HSCs via Smad3-dependant mechanisms, which in turn contributes Smad protein phosphorylation, and subsequently increases fibrous collagen expression. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis. - Highlights: • Upregulation of TRPM7 protein in human fibrotic livers • Upregulation of TRPM7 by TGF-β1 elicited Smad signaling in HSC-T6 cells

Diffusion of MMPs on the Surface of Collagen Fibrils: The Mobile Cell Surface – Collagen Substratum Interface

Science.gov (United States)

Collier, Ivan E.; Legant, Wesley; Marmer, Barry; Lubman, Olga; Saffarian, Saveez; Wakatsuki, Tetsuro; Elson, Elliot; Goldberg, Gregory I.

2011-01-01

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions. PMID:21912660

Bi-allelic Alterations in AEBP1 Lead to Defective Collagen Assembly and Connective Tissue Structure Resulting in a Variant of Ehlers-Danlos Syndrome

KAUST Repository

Blackburn, Patrick R.; Xu, Zhi; Tumelty, Kathleen E.; Zhao, Rose W.; Monis, William J.; Harris, Kimberly G.; Gass, Jennifer M.; Cousin, Margot A.; Boczek, Nicole J.; Mitkov, Mario V.; Cappel, Mark A.; Francomano, Clair A.; Parisi, Joseph E.; Klee, Eric W.; Faqeih, Eissa; Alkuraya, Fowzan S.; Layne, Matthew D.; McDonnell, Nazli B.; Atwal, Paldeep S.

2018-01-01

AEBP1 encodes the aortic carboxypeptidase-like protein (ACLP) that associates with collagens in the extracellular matrix (ECM) and has several roles in development, tissue repair, and fibrosis. ACLP is expressed in bone, the vasculature, and dermal tissues and is involved in fibroblast proliferation and mesenchymal stem cell differentiation into collagen-producing cells. Aebp1 mice have abnormal, delayed wound repair correlating with defects in fibroblast proliferation. In this study, we describe four individuals from three unrelated families that presented with a unique constellation of clinical findings including joint laxity, redundant and hyperextensible skin, poor wound healing with abnormal scarring, osteoporosis, and other features reminiscent of Ehlers-Danlos syndrome (EDS). Analysis of skin biopsies revealed decreased dermal collagen with abnormal collagen fibrils that were ragged in appearance. Exome sequencing revealed compound heterozygous variants in AEBP1 (c.1470delC [p.Asn490_Met495delins(40)] and c.1743C>A [p.Cys581]) in the first individual, a homozygous variant (c.1320_1326del [p.Arg440Serfs3]) in the second individual, and a homozygous splice site variant (c.1630+1G>A) in two siblings from the third family. We show that ACLP enhances collagen polymerization and binds to several fibrillar collagens via its discoidin domain. These studies support the conclusion that biallelic pathogenic variants in AEBP1 are the cause of this autosomal-recessive EDS subtype.

Bi-allelic Alterations in AEBP1 Lead to Defective Collagen Assembly and Connective Tissue Structure Resulting in a Variant of Ehlers-Danlos Syndrome

KAUST Repository

Blackburn, Patrick R.

2018-03-29

AEBP1 encodes the aortic carboxypeptidase-like protein (ACLP) that associates with collagens in the extracellular matrix (ECM) and has several roles in development, tissue repair, and fibrosis. ACLP is expressed in bone, the vasculature, and dermal tissues and is involved in fibroblast proliferation and mesenchymal stem cell differentiation into collagen-producing cells. Aebp1 mice have abnormal, delayed wound repair correlating with defects in fibroblast proliferation. In this study, we describe four individuals from three unrelated families that presented with a unique constellation of clinical findings including joint laxity, redundant and hyperextensible skin, poor wound healing with abnormal scarring, osteoporosis, and other features reminiscent of Ehlers-Danlos syndrome (EDS). Analysis of skin biopsies revealed decreased dermal collagen with abnormal collagen fibrils that were ragged in appearance. Exome sequencing revealed compound heterozygous variants in AEBP1 (c.1470delC [p.Asn490_Met495delins(40)] and c.1743C>A [p.Cys581]) in the first individual, a homozygous variant (c.1320_1326del [p.Arg440Serfs3]) in the second individual, and a homozygous splice site variant (c.1630+1G>A) in two siblings from the third family. We show that ACLP enhances collagen polymerization and binds to several fibrillar collagens via its discoidin domain. These studies support the conclusion that biallelic pathogenic variants in AEBP1 are the cause of this autosomal-recessive EDS subtype.

Osteogenesis imperfecta due to mutations in non-collagenous genes: lessons in the biology of bone formation.

Science.gov (United States)

Marini, Joan C; Reich, Adi; Smith, Simone M

2014-08-01

Osteogenesis imperfecta or 'brittle bone disease' has mainly been considered a bone disorder caused by collagen mutations. Within the last decade, however, a surge of genetic discoveries has created a new paradigm for osteogenesis imperfecta as a collagen-related disorder, where most cases are due to autosomal dominant type I collagen defects, while rare, mostly recessive, forms are due to defects in genes whose protein products interact with collagen protein. This review is both timely and relevant in outlining the genesis, development, and future of this paradigm shift in the understanding of osteogenesis imperfecta. Bone-restricted interferon-induced transmembrane (IFITM)-like protein (BRIL) and pigment epithelium-derived factor (PEDF) defects cause types V and VI osteogenesis imperfecta via defective bone mineralization, while defects in cartilage-associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1), and cyclophilin B (CYPB) cause types VII-IX osteogenesis imperfecta via defective collagen post-translational modification. Heat shock protein 47 (HSP47) and FK506-binding protein-65 (FKBP65) defects cause types X and XI osteogenesis imperfecta via aberrant collagen crosslinking, folding, and chaperoning, while defects in SP7 transcription factor, wingless-type MMTV integration site family member 1 (WNT1), trimeric intracellular cation channel type b (TRIC-B), and old astrocyte specifically induced substance (OASIS) disrupt osteoblast development. Finally, absence of the type I collagen C-propeptidase bone morphogenetic protein 1 (BMP1) causes type XII osteogenesis imperfecta due to altered collagen maturation/processing. Identification of these multiple causative defects has provided crucial information for accurate genetic counseling, inspired a recently proposed functional grouping of osteogenesis imperfecta types by shared mechanism to simplify current nosology, and has prodded investigations into common pathways in osteogenesis imperfecta. Such

Collagen-Induced Arthritis: A model for Murine Autoimmune Arthritis

OpenAIRE

Pietrosimone, K. M.; Jin, M.; Poston, B.; Liu, P.

2015-01-01

Collagen-induced arthritis (CIA) is a common autoimmune animal model used to study rheumatoid arthritis (RA). The development of CIA involves infiltration of macrophages and neutrophils into the joint, as well as T and B cell responses to type II collagen. In murine CIA, genetically susceptible mice (DBA/1J) are immunized with a type II bovine collagen emulsion in complete Freund’s adjuvant (CFA), and receive a boost of type II bovine collagen in incomplete Freund’s adjuvant (IFA) 21 days aft...

Characterization of electron beam irradiated collagen-polyvinylpyrrolidone (PVP) and collagen-dextran (DEX) blends

International Nuclear Information System (INIS)

Dumitrascu, M.; Sima, E.; Minea, R.; Vancea, C.; Meltze, V.; Albu, M.G.

2011-01-01

Complete text of publication follows. The aim of the present study was to investigate the influence of electron beam irradiation on some blends of collagen-polyvinylpyrrolidone (PVP) and collagen-dextran (DEX). The blends were prepared by mixing different quantities of collagen, PVP and DEX in distilled water. After irradiation the obtained hydrogels were processed by controlled drying and freeze-drying. Both types of materials were characterized by FT-IR, FT-Raman, TG, DSC, water uptake and SEM. The intensity of the characteristic bands, in the range 2800-3600 cm -1 from FT-IR spectra, varied considerably as function of absorbed radiation dose. Raman spectra revealed the absence of the characteristic peak at 2700 cm -1 for irradiated blends at 30 kGy. Kinetic parameters were calculated from the TG, DTG and DSC data by means of isoconversion methods at different heating rates. Thereby a relation between absorbed radiation dose and activation energy was established. Water uptake studies were carried out in PBS solution (phosphate buffer saline) at 37 deg C and pH = 7.4 and the results revealed a decrease of the water uptake with increasing of absorbed radiation dose.

Collagen turnover after tibial fractures

DEFF Research Database (Denmark)

Joerring, S; Krogsgaard, M; Wilbek, H

1994-01-01

Collagen turnover after tibial fractures was examined in 16 patients with fracture of the tibial diaphysis and in 8 patients with fracture in the tibial condyle area by measuring sequential changes in serological markers of turnover of types I and III collagen for up to 26 weeks after fracture....... The markers were the carboxy-terminal extension peptide of type I procollagen (PICP), the amino-terminal extension peptide of type III procollagen (PIIINP), and the pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen (ICTP). The latter is a new serum marker of degradation of type I...... collagen. A group comparison showed characteristic sequential changes in the turnover of types I and III collagen in fractures of the tibial diaphysis and tibial condyles. The turnover of type III collagen reached a maximum after 2 weeks in both groups. The synthesis of type I collagen reached a maximum...

Collagens--structure, function, and biosynthesis.

Science.gov (United States)

Gelse, K; Pöschl, E; Aigner, T

2003-11-28

The extracellular matrix represents a complex alloy of variable members of diverse protein families defining structural integrity and various physiological functions. The most abundant family is the collagens with more than 20 different collagen types identified so far. Collagens are centrally involved in the formation of fibrillar and microfibrillar networks of the extracellular matrix, basement membranes as well as other structures of the extracellular matrix. This review focuses on the distribution and function of various collagen types in different tissues. It introduces their basic structural subunits and points out major steps in the biosynthesis and supramolecular processing of fibrillar collagens as prototypical members of this protein family. A final outlook indicates the importance of different collagen types not only for the understanding of collagen-related diseases, but also as a basis for the therapeutical use of members of this protein family discussed in other chapters of this issue.

Inelastic behaviour of collagen networks in cell–matrix interactions and mechanosensation

Science.gov (United States)

Mohammadi, Hamid; Arora, Pamma D.; Simmons, Craig A.; Janmey, Paul A.; McCulloch, Christopher A.

2015-01-01

The mechanical properties of extracellular matrix proteins strongly influence cell-induced tension in the matrix, which in turn influences cell function. Despite progress on the impact of elastic behaviour of matrix proteins on cell–matrix interactions, little is known about the influence of inelastic behaviour, especially at the large and slow deformations that characterize cell-induced matrix remodelling. We found that collagen matrices exhibit deformation rate-dependent behaviour, which leads to a transition from pronounced elastic behaviour at fast deformations to substantially inelastic behaviour at slow deformations (1 μm min−1, similar to cell-mediated deformation). With slow deformations, the inelastic behaviour of floating gels was sensitive to collagen concentration, whereas attached gels exhibited similar inelastic behaviour independent of collagen concentration. The presence of an underlying rigid support had a similar effect on cell–matrix interactions: cell-induced deformation and remodelling were similar on 1 or 3 mg ml−1 attached collagen gels while deformations were two- to fourfold smaller in floating gels of high compared with low collagen concentration. In cross-linked collagen matrices, which did not exhibit inelastic behaviour, cells did not respond to the presence of the underlying rigid foundation. These data indicate that at the slow rates of collagen compaction generated by fibroblasts, the inelastic responses of collagen gels, which are influenced by collagen concentration and the presence of an underlying rigid foundation, are important determinants of cell–matrix interactions and mechanosensation. PMID:25392399

Peripheral hepatic arterial embolization with cross-linked collagen fibers

International Nuclear Information System (INIS)

Daniels, J.R.; Kerlan, R.K. Jr.; Dodds, L.; McLaughlin, P.; La Berge, J.M.; Harrington, D.; Daniels, A.M.; Ring, E.J.

1986-01-01

Hepatic artery embolization with a nonimmunogenic, cross-linked collagen preparation (Angiostat, collagen for embolization, Target Therapeutics) was studied in mongrel dogs. Flow-directed technique was used to achieve complete distal arterial occlusion. Serial liver function evaluation demonstrated marked alterations at 48 to 72 hours, partial correction at 1 week, and resolution of abnormalities by 1 month. Restoration of large-vessel blood flow was angiographically demonstrable at 1 week. Recanalization, achieved by migration of endothelial cells around the collagen, resulted in complete restoration of normal hepatic vascular and tissue anatomy at 1 month. Repeated embolization at biweekly intervals was well tolerated

Corneal collagen crosslinking for keratoconus. A review

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M. M. Bikbov

2014-10-01

Full Text Available Photochemical crosslinking is widely applied in ophthalmology. Its biochemical effect is due to the release of singlet oxygen that promotes anaerobic photochemical reaction. Keratoconus is one of the most common corneal ectasia affecting 1 in 250 to 250 000 persons. Currently, the rate of iatrogenic ectasia following eximer laser refractive surgery increases due to biomechanical weakening of the cornea. Morphologically and biochemically, ectasia is characterized by corneal layers thinning, contact between the stroma and epithelium resulting from Bowman’s membrane rupture, chromatin fragmentation in keratocyte nuclei, phagocytosis, abnormal staining and arrangement of collagen fibers, enzyme system disorders, and keratocyte apoptosis. In corneal ectasia, altered enzymatic processes result in the synthesis of abnormal collagen. Collagen packing is determined by the activity of various extracellular matrix enzymes which bind amines and aldehydes of collagen fiber amino acids. In the late stage, morphological changes of Descemet’s membrane (i.e., rupture and detachment develop. Abnormal hexagonal-shaped keratocytes and their apoptosis are the signs of endothelial dystrophy. The lack of analogs in domestic ophthalmology encouraged the scientists of Ufa Eye Research Institute to develop a device for corneal collagen crosslinking. The parameters of ultraviolet (i.e., wavelength, exposure time, power to achieve the desired effect were identified. The specifics of some photosensitizers in the course of the procedure were studied. UFalink, a device for UV irradiation of cornea, and photosensitizer Dextralink were developed and adopted. Due to the high risk of endothelial damage, this treatment is contraindicated in severe keratoconus (CCT less than 400 microns. Major effects of corneal collagen crosslinking are the following: Young’s modulus (modulus of elasticity increase by 328.9 % (on average, temperature tolerance increase by 5�

Second-harmonic generation imaging of collagen in ancient bone

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B. Thomas

2017-12-01

Full Text Available Second-harmonic generation imaging (SHG captures triple helical collagen molecules near tissue surfaces. Biomedical research routinely utilizes various imaging software packages to quantify SHG signals for collagen content and distribution estimates in modern tissue samples including bone. For the first time using SHG, samples of modern, medieval, and ice age bones were imaged to test the applicability of SHG to ancient bone from a variety of ages, settings, and taxa. Four independent techniques including Raman spectroscopy, FTIR spectroscopy, radiocarbon dating protocols, and mass spectrometry-based protein sequencing, confirm the presence of protein, consistent with the hypothesis that SHG imaging detects ancient bone collagen. These results suggest that future studies have the potential to use SHG imaging to provide new insights into the composition of ancient bone, to characterize ancient bone disorders, to investigate collagen preservation within and between various taxa, and to monitor collagen decay regimes in different depositional environments.

Differential effects of collagen prolyl 3-hydroxylation on skeletal tissues.

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Erica P Homan

2014-01-01

Full Text Available Mutations in the genes encoding cartilage associated protein (CRTAP and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1 were the first identified causes of recessive Osteogenesis Imperfecta (OI. These proteins, together with cyclophilin B (encoded by PPIB, form a complex that 3-hydroxylates a single proline residue on the α1(I chain (Pro986 and has cis/trans isomerase (PPIase activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1(H662A . This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I and α1(II collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase

Osteogenesis Imperfecta due to Mutations in Non-Collagenous Genes-Lessons in the Biology of Bone Formation

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Marini, Joan C.; Reich, Adi; Smith, Simone M.

2014-01-01

Purpose of Review Osteogenesis imperfecta (OI), or “brittle bone disease”, has mainly been considered a bone disorder caused by collagen mutations. Within the last decade, however, a surge of genetic discoveries has created a new paradigm for OI as a collagen-related disorder, where autosomal dominant type I collagen defects cause most cases, while rare, mostly recessive forms are due to defects in genes whose protein products interact with collagen protein. This review is both timely and relevant in outlining the genesis, development and future of this paradigm shift in the understanding of OI. Recent Findings BRIL and PEDF defects cause types V and VI OI via defective bone mineralization, while defects in CRTAP, P3H1 and CyPB cause types VII-IX via defective collagen post-translational modification. Hsp47 and FKBP65 defects cause types X and XI OI via aberrant collagen crosslinking, folding and chaperoning, while defects in SP7, WNT1, TRIC-B and OASIS disrupt osteoblast development. Finally, absence of the type I collagen C-propeptidase BMP1 causes type XII OI due to altered collagen maturation/processing. Summary Identification of these multiple causative defects has provided crucial information for accurate genetic counseling, inspired a recently proposed functional grouping of OI types by shared mechanism to simplify current nosology, and should prod investigations into common pathways in OI. Such investigations could yield critical information on cellular and bone tissue mechanisms and translate to new mechanistic insight into clinical therapies for patients. PMID:25007323

Delphinidin prevents high glucose-induced cell proliferation and collagen synthesis by inhibition of NOX-1 and mitochondrial superoxide in mesangial cells

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Seung Eun Song

2016-04-01

Full Text Available This study examined the effect of delphinidin on high glucose-induced cell proliferation and collagen synthesis in mesangial cells. Glucose dose-dependently (5.6–25 mM increased cell proliferation and collagen I and IV mRNA levels, whereas pretreatment with delphinidin (50 μM prevented cell proliferation and the increased collagen mRNA levels induced by high glucose (25 mM. High glucose increased reactive oxygen species (ROS generation, and this was suppressed by pretreating delphinidin or the antioxidant N-acetyl cysteine. NADPH oxidase (NOX 1 was upregulated by high glucose, but pretreatment with delphinidin abrogated this upregulation. Increased mitochondrial superoxide by 25 mM glucose was also suppressed by delphinidin. The NOX inhibitor apocynin and mitochondria-targeted antioxidant Mito TEMPO inhibited ROS generation and cell proliferation induced by high glucose. Phosphorylation of extracellular signal regulated kinase (ERK1/2 was increased by high glucose, which was suppressed by delphinidin, apocynin or Mito TEMPO. Furthermore, PD98059 (an ERK1/2 inhibitor prevented the high glucose-induced cell proliferation and increased collagen mRNA levels. Transforming growth factor (TGF-β protein levels were elevated by high glucose, and pretreatment with delphinidin or PD98059 prevented this augmentation. These results suggest that delphinidin prevents high glucose-induced cell proliferation and collagen synthesis by inhibition of NOX-1 and mitochondrial superoxide in mesangial cells.

Bone Collagen: New Clues to its Mineralization Mechanism From Recessive Osteogenesis Imperfecta

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Eyre, David R.; Ann Weis, Mary

2013-01-01

Until 2006 the only mutations known to cause osteogenesis imperfecta (OI) were in the two genes coding for type I collagen chains. These dominant mutations affecting the expression or primary sequence of collagen α1(I) and α2(I) chains account for over 90% of OI cases. Since then a growing list of mutant genes causing the 5–10% of recessive cases has rapidly emerged. They include CRTAP, LEPRE1 and PPIB, which encode three proteins forming the prolyl 3-hydroxylase complex; PLOD2 and FKBP10, which encode respectively lysyl hydroxylase 2 and a foldase required for its activity in forming mature cross-links in bone collagen; SERPIN H1, which encodes the collagen chaperone HSP47; SERPIN F1, which encodes pigment epithelium-derived factor required for osteoid mineralization; and BMP1, which encodes the type I procollagen C-propeptidase. All cause fragile bone in infancy, which can include over-mineralization or under-mineralization defects as well as abnormal collagen post-translational modifications. Consistently both dominant and recessive variants lead to abnormal cross-linking chemistry in bone collagen. These recent discoveries strengthen the potential for a common pathogenic mechanism of misassembled collagen fibrils. Of the new genes identified, eight encode proteins required for collagen post-translational modification, chaperoning of newly synthesized collagen chains into native molecules or transport through the endoplasmic reticulum and Golgi for polymerization, cross-linking and mineralization. In reviewing these findings, we conclude that a common theme is emerging in the pathogenesis of brittle bone disease of mishandled collagen assembly with important insights on post-translational features of bone collagen that have evolved to optimize it as a biomineral template. PMID:23508630

Extraction and Characterization of Collagen from Sea Cucumber Flesh

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Alhana

2015-11-01

Full Text Available Sea cucumber (Stichopus variegatus is one of the Echinodermata phylum that grows along Indonesian coastal. Sea cucumber is potential source of collagen. The purposes of this research were to determine the optimal concentration of NaOH and CH3COOH solution in collagen production and analyze the physicochemical characteristics of collagen from S. variegatus. Yield of the collagen was 1.5% (based on wet weight basis, produced by pretreatment with NaOH 0,30%, hydrolysis with CH3COOH 0.10% and extracted using distilled water. Protein, moisture, and ash content of the collagen was 67.68%, 13.64%, and 4.15%, respectively. Collagen was extracted using distilled water at 45°C during 2h and still had triple helix structure ; pH 7.37 ; melting temperature 163.67°C and whiteness 69.25%. The major amino acid content of collagen were glycine, alanine, proline and glutamic acid.

Studies on collagen-tannic acid-collagenase ternary system: Inhibition of collagenase against collagenolytic degradation of extracellular matrix component of collagen.

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Krishnamoorthy, Ganesan; Sehgal, Praveen Kumar; Mandal, Asit Baran; Sadulla, Sayeed

2012-06-01

We report the detailed studies on the inhibitory effect of tannic acid (TA) on Clostridium histolyticum collagenase (ChC) activity against degradation of extracellular matrix component of collagen. The TA treated collagen exhibited 64% resistance against collagenolytic hydrolysis by ChC, whereas direct interaction of TA with ChC exhibited 99% inhibition against degradation of collagen and the inhibition was found to be concentration dependant. The kinetic inhibition of ChC has been deduced from the extent of hydrolysis of N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA). This data provides a selective competitive mode of inhibition on ChC activity seems to be influenced strongly by the nature and structure of TA. TA showed inhibitor activity against the ChC by molecular docking method. This result demonstrated that TA containing digalloyl radical possess the ability to inhibit the ChC. The inhibition of ChC in gaining new insight into the mechanism of stabilization of collagen by TA is discussed.

Human pro. cap alpha. 1)(I) collagen: cDNA sequence for the C-propeptide domain

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Maekelae, J K; Raassina, M; Virta, A; Vuorio, E

1988-01-11

The authors have previously constructed a cDNA clone pHCAL1, covering most of the C-terminal propeptide domain of human pro..cap alpha..1(I) collagen mRNA,by inserting a 678 bp EcoRI-XhoI fragment of cDNA into pBR322. Since the XhoI/SalI ligation prevented removal of the insert, they used the same strategy to obtain a similar clone in pUC8. RNA was isolated from fetal calvarial bones. The cDNA was digested with EcoRI and XhoI and fractionated on a 1 % agarose gel. Fragments of 650-700 bp were cloned in pUC8 at the polylinker site, which now permits easy removal of the insert. The new clone was named pHCAL1U since the RNA was isolated from another individual. The approach outlined is useful for studies on individual variation which is important to recognize when searching for disease-related mutations in type I collagen.

Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane type IV collagens and form a novel family widely distributed in invertebrates.

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Aouacheria, Abdel; Geourjon, Christophe; Aghajari, Nushin; Navratil, Vincent; Deléage, Gilbert; Lethias, Claire; Exposito, Jean-Yves

2006-12-01

Collagens are thought to represent one of the most important molecular innovations in the metazoan line. Basement membrane type IV collagen is present in all Eumetazoa and was found in Homoscleromorpha, a sponge group with a well-organized epithelium, which may represent the first stage of tissue differentiation during animal evolution. In contrast, spongin seems to be a demosponge-specific collagenous protein, which can totally substitute an inorganic skeleton, such as in the well-known bath sponge. In the freshwater sponge Ephydatia mülleri, we previously characterized a family of short-chain collagens that are likely to be main components of spongins. Using a combination of sequence- and structure-based methods, we present evidence of remote homology between the carboxyl-terminal noncollagenous NC1 domain of spongin short-chain collagens and type IV collagen. Unexpectedly, spongin short-chain collagen-related proteins were retrieved in nonsponge animals, suggesting that a family related to spongin constitutes an evolutionary sister to the type IV collagen family. Formation of the ancestral NC1 domain and divergence of the spongin short-chain collagen-related and type IV collagen families may have occurred before the parazoan-eumetazoan split, the earliest divergence among extant animal phyla. Molecular phylogenetics based on NC1 domain sequences suggest distinct evolutionary histories for spongin short-chain collagen-related and type IV collagen families that include spongin short-chain collagen-related gene loss in the ancestors of Ecdyzosoa and of vertebrates. The fact that a majority of invertebrates encodes spongin short-chain collagen-related proteins raises the important question to the possible function of its members. Considering the importance of collagens for animal structure and substratum attachment, both families may have played crucial roles in animal diversification.

Doxazosin stimulates galectin-3 expression and collagen synthesis in HL-1 cardiomyocytes independent of protein kinase C pathway

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Xiaoqian Qian

2016-12-01

Full Text Available Doxazosin, a drug commonly prescribed for hypertension and prostate disease, increases heart failure risk. However, the underlying mechanism remains unclear. Galectin-3 is an important mediator that plays a pathogenic role in cardiac hypertrophy and heart failure. In the present study, we investigated whether doxazosin could stimulate galectin-3 expression and collagen synthesis in cultured HL-1 cardiomyocytes. We found that doxazosin dose-dependently induced galectin-3 protein expression, with a statistically significant increase in expression with a dose as low as 0.01 μM. Doxazosin upregulated collagen I and α-smooth muscle actin (α-SMA protein levels and also induced apoptotic protein caspase-3 in HL-1 cardiomyocytes. Although we previously reported that activation of protein kinase C (PKC stimulates galectin-3 expression, blocking the PKC pathway with the PKC inhibitor chelerythrine did not prevent doxazosin-induced galectin-3 and collagen expression. Consistently, doxazosin treatment did not alter total and phosphorylated PKC. These results suggest that doxazosin-stimulated galectin-3 is independent of PKC pathway. To determine if the α1-adrenergic pathway is involved, we pretreated the cells with the irreversible α-adrenergic receptor blocker phenoxybenzamine and found that doxazosin-stimulated galectin-3 and collagen expression was similar to controls, suggesting that doxazosin acts independently of α1-adrenergic receptor blockade. Collectively, we show a novel effect of doxazosin on cardiomycytes by stimulating heart fibrosis factor galectin-3 expression. The mechanism of action of doxazosin is not mediated through either activation of the PKC pathway or antagonism of α1-adrenergic receptors.

High resolution imaging of collagen organisation and synthesis using a versatile collagen specific probe

NARCIS (Netherlands)

Boerboom, R.A.; Krahn - Nash, K.; Megens, R.T.A.; Zandvoort, van M.; Merkx, M.; Bouten, C.V.C.

2007-01-01

Collagen is the protein primarily responsible for the load-bearing properties of tissues and collagen architecture is one of the main determinants of the mechanical properties of tissues. Visualisation of changes in collagen three-dimensional structure is essential in order to improve our

Collagen mRNA levels changes during colorectal cancer carcinogenesis

DEFF Research Database (Denmark)

Skovbjerg, Hanne; Anthonsen, Dorit; Lothe, Inger M B

2009-01-01

BACKGROUND: Invasive growth of epithelial cancers is a complex multi-step process which involves dissolution of the basement membrane. Type IV collagen is a major component in most basement membranes. Type VII collagen is related to anchoring fibrils and is found primarily in the basement membrane...... zone of stratified epithelia. Immunohistochemical studies have previously reported changes in steady-state levels of different alpha(IV) chains in several epithelial cancer types. In the present study we aimed to quantitatively determine the mRNA levels of type IV collagen (alpha1/alpha 4/alpha 6......) and type VII collagen (alpha1) during colorectal cancer carcinogenesis. METHODS: Using quantitative RT-PCR, we have determined the mRNA levels for alpha1(IV), alpha 4(IV), alpha 6(IV), and alpha1(VII) in colorectal cancer tissue (n = 33), adenomas (n = 29) and in normal tissue from the same individuals...

Changes in collagen synthesis and degradation during skeletal muscle growth

International Nuclear Information System (INIS)

Laurent, G.J.; McAnulty, R.J.; Gibson, J.

1985-01-01

The changes in collagen metabolism during skeletal muscle growth were investigated by measuring rates of synthesis and degradation during stretch-induced hypertrophy of the anterior latissimus dorsi muscle of the adult chicken (Gallus domesticus). Synthesis rates were obtained from the uptake of tritiated proline injected intravenously with a flooding dose of unlabeled proline. Degradation of newly synthesized and ''mature'' collagen was estimated from the amount of hydroxyproline in the free pool as small molecular weight moieties. In normal muscle, the synthesis rate was 1.1 +/- 0.3%/day, with 49 +/- 7% of the newly produced collagen degraded rapidly after synthesis. During hypertrophy there was an increase of about fivefold in the rate of synthesis (P less than 0.01), a 60% decrease in the rate of degradation of newly synthesized collagen (P less than 0.02), and an increase of about fourfold in the amount of degradation of mature collagen (P less than 0.01). These results suggest an important role for degradative as well as synthetic processes in the regulation of collagen mass. They indicate that enhanced degradation of mature collagen is required for muscle growth and suggest a physiological role for the pathway whereby in normal muscle, a large proportion of newly produced collagen is rapidly degraded

Epicutaneous Immunization with Type II Collagen Inhibits both Onset and Progression of Chronic Collagen-Induced Arthritis

OpenAIRE

Strid, Jessica; Tan, Lee Aun; Strobel, Stephan; Londei, Marco; Callard, Robin

2007-01-01

Epicutaneous immunization is a potential non-invasive technique for antigen-specific immune-modulation. Topical application of protein antigens to barrier-disrupted skin induces potent antigen-specific immunity with a strong Th2-bias. In this study, we investigate whether the autoimmune inflammatory response of chronic collagen-induced arthritis (CCIA) in DBA/1-TCR-beta Tg mice can be modified by epicutaneous immunization. We show that epicutaneous immunization with type II collagen (CII) inh...

MT1-MMP promotes cell growth and ERK activation through c-Src and paxillin in three-dimensional collagen matrix

International Nuclear Information System (INIS)

Takino, Takahisa; Tsuge, Hisashi; Ozawa, Terumasa; Sato, Hiroshi

2010-01-01

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is essential for tumor invasion and growth. We show here that MT1-MMP induces extracellular signal-regulated kinase (ERK) activation in cancer cells cultured in collagen gel, which is indispensable for their proliferation. Inhibition of MT1-MMP by MMP inhibitor or small interfering RNA suppressed activation of focal adhesion kinase (FAK) and ERK in MT1-MMP-expressing cancer cells, which resulted in up-regulation of p21 WAF1 and suppression of cell growth in collagen gel. Cell proliferation was also abrogated by the inhibitor against ERK pathway without affecting FAK phosphorylation. MT1-MMP and integrin α v β 3 were shown to be involved in c-Src activation, which induced FAK and ERK activation in collagen gel. These MT1-MMP-mediated signal transductions were paxillin dependent, as knockdown of paxillin reduced cell growth and ERK activation, and co-expression of MT1-MMP with paxillin induced ERK activation. The results suggest that MT1-MMP contributes to proliferation of cancer cells in the extracellular matrix by activating ERK through c-Src and paxillin.

Lack of collagen XVIII/endostatin exacerbates immune-mediated glomerulonephritis.

Science.gov (United States)

Hamano, Yuki; Okude, Takashi; Shirai, Ryota; Sato, Ikumi; Kimura, Ryota; Ogawa, Makoto; Ueda, Yoshihiko; Yokosuka, Osamu; Kalluri, Raghu; Ueda, Shiro

2010-09-01

Collagen XVIII is a component of the highly specialized extracellular matrix associated with basement membranes of epithelia and endothelia. In the normal kidney, collagen XVIII is distributed throughout glomerular and tubular basement membranes, mesangial matrix, and Bowman's capsule. Proteolytic cleavage within its C-terminal domain releases the fragment endostatin, which has antiangiogenic properties. Because damage to the glomerular basement membrane (GBM) accompanies immune-mediated renal injury, we investigated the role of collagen XVIII/endostatin in this disorder. We induced anti-GBM glomerulonephritis in collagen XVIII alpha1-null and wild-type mice and compared the resulting matrix accumulation, inflammation, and capillary rarefaction. Anti-GBM disease upregulated collagen XVIII/endostatin expression within the GBM and Bowman's capsule of wild-type mice. Collagen XVIII/endostatin-deficient mice developed more severe glomerular and tubulointerstitial injury than wild-type mice. Collagen XVIII/endostatin deficiency altered matrix remodeling, enhanced the inflammatory response, and promoted capillary rarefaction and vascular endothelial cell damage, but did not affect endothelial proliferation. Supplementing collagen XVIII-deficient mice with exogenous endostatin did not affect the progression of anti-GBM disease. Taken together, these results suggest that collagen XVIII/endostatin preserves the integrity of the extracellular matrix and capillaries in the kidney, protecting against progressive glomerulonephritis.

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

Science.gov (United States)

Saito, M; Takenouchi, Y; Kunisaki, N; Kimura, S

2001-05-01

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.

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

Science.gov (United States)

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

Measurement of the quadratic hyperpolarizability of the collagen triple helix and application to second harmonic imaging of natural and biomimetic collagenous tissues

Science.gov (United States)

Deniset-Besseau, A.; Strupler, M.; Duboisset, J.; De Sa Peixoto, P.; Benichou, E.; Fligny, C.; Tharaux, P.-L.; Mosser, G.; Brevet, P.-F.; Schanne-Klein, M.-C.

2009-09-01

Collagen is a major protein of the extracellular matrix that is characterized by triple helical domains. It plays a central role in the formation of fibrillar and microfibrillar networks, basement membranes, as well as other structures of the connective tissue. Remarkably, fibrillar collagen exhibits efficient Second Harmonic Generation (SHG) so that SHG microscopy proved to be a sensitive tool to probe the three-dimensional architecture of fibrillar collagen and to assess the progression of fibrotic pathologies. We obtained sensitive and reproducible measurements of the fibrosis extent, but we needed quantitative data at the molecular level to further process SHG images. We therefore performed Hyper- Rayleigh Scattering (HRS) experiments and measured a second order hyperpolarisability of 1.25 10-27 esu for rat-tail type I collagen. This value is surprisingly large considering that collagen presents no strong harmonophore in its aminoacid sequence. In order to get insight into the physical origin of this nonlinear process, we performed HRS measurements after denaturation of the collagen triple helix and for a collagen-like short model peptide [(Pro-Pro- Gly)10]3. It showed that the collagen large nonlinear response originates in the tight alignment of a large number of weakly efficient harmonophores, presumably the peptide bonds, resulting in a coherent amplification of the nonlinear signal along the triple helix. To illustrate this mechanism, we successfully recorded SHG images in collagenous biomimetic matrices.

Isolation and Characterization of Collagen and Antioxidant Collagen Peptides from Scales of Croceine Croaker (Pseudosciaena crocea

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Bin Wang

2013-11-01

Full Text Available Acid soluble collagen (ASC from scales of croceine croaker (ASC-C was successfully isolated with the yield of 0.37% ± 0.08% (dry weight basis, and characterized as type I collagen on the basis of amino acid analysis and electrophoretic pattern. The antioxidant hydrolysate of ASC-C (ACH was prepared through a two-stage in vitro digestion (4-h trypsin followed by 4-h pepsin, and three antioxidant peptides (ACH-P1, ACH-P2, and ACH-P3 were further isolated from ACH using ultrafiltration, gel chromatography, and RP-HPLC, and their amino acid sequences were identified as GFRGTIGLVG (ACH-P1, GPAGPAG (ACH-P2, and GFPSG (ACH-P3. ACH-P1, ACH-P2, and ACH-P3 showed good scavenging activities on hydroxyl radical (IC50 0.293, 0.240, and 0.107 mg/mL, respectively, DPPH radical (IC50 1.271, 0.675, and 0.283 mg/mL, respectively, superoxide radical (IC50 0.463, 0.099, and 0.151 mg/mL, respectively, and ABTS radical (IC50 0.421, 0.309, and 0.210 mg/mL, respectively. ACH-P3 was also effectively against lipid peroxidation in the model system. The antioxidant activities of three collagen peptides were due to the presence of hydrophobic amino acid residues within the peptide sequences. The collagen peptides might be used as antioxidant for the therapy of diseases associated with oxidative stress, or reducing oxidative changes during storage.

Heat Shock Protein 47: A Novel Biomarker of Phenotypically Altered Collagen-Producing Cells

International Nuclear Information System (INIS)

Taguchi, Takashi; Nazneen, Arifa; Al-Shihri, Abdulmonem A.; Turkistani, Khadijah A.; Razzaque, Mohammed S.

2011-01-01

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that helps the molecular maturation of various types of collagens. A close association between increased expression of HSP47 and the excessive accumulation of collagens is found in various human and experimental fibrotic diseases. Increased levels of HSP47 in fibrotic diseases are thought to assist in the increased assembly of procollagen, and thereby contribute to the excessive deposition of collagens in fibrotic areas. Currently, there is not a good universal histological marker to identify collagen-producing cells. Identifying phenotypically altered collagen-producing cells is essential for the development of cell-based therapies to reduce the progression of fibrotic diseases. Since HSP47 has a single substrate, which is collagen, the HSP47 cellular expression provides a novel universal biomarker to identify phenotypically altered collagen-producing cells during wound healing and fibrosis. In this brief article, we explained why HSP47 could be used as a universal marker for identifying phenotypically altered collagen-producing cells

Periurethral injection of collagen in the treatment of urinary stress incontinence: ultrasonographic appearance

Energy Technology Data Exchange (ETDEWEB)

Leonhardt, C.; Krysl, J.; Arenson, A.M.; Herschorn, S. [Toronto Univ., ON (Canada). Faculty of Medicine

1995-06-01

Transvesical and transvaginal ultrasonography (US) was performed 26 times in 23 patients, 3 to 36 months after periurethral injection of collagen to treat symptomatic urinary stress incontinence. The appearance, location and volume of the collagen were recorded. In all the patients the injected collagen had the appearance of a well-circumscribed mass of variable size, located at the bladder base. Transvesical US demonstrated the collagen in only 17 of the patients, and allowed only limited visualization of the collagen in five of these 17 patients. However, transvaginal US demonstrated the collagen in all of them. The collagen collections showed various levels of echogenicity with both techniques. However, in patients with more than one deposit of collagen, the collections had similar echogenicity. The study indicated that US provides a rapid, noninvasive method of assessing collagen after periurethral injection, and that transvaginal US was the best method of visualizing such collections. 10 refs., 5 figs.

Assembly of collagen into microribbons: effects of pH and electrolytes.

Science.gov (United States)

Jiang, Fengzhi; Hörber, Heinrich; Howard, Jonathon; Müller, Daniel J

2004-12-01

Collagen represents the major structural protein of the extracellular matrix. Elucidating the mechanism of its assembly is important for understanding many cell biological and medical processes as well as for tissue engineering and biotechnological approaches. In this work, conditions for the self-assembly of collagen type I molecules on a supporting surface were characterized. By applying hydrodynamic flow, collagen assembled into ultrathin ( approximately 3 nm) highly anisotropic ribbon-like structures coating the entire support. We call these novel collagen structures microribbons. High-resolution atomic force microscopy topographs show that subunits of these microribbons are built by fibrillar structures. The smallest units of these fibrillar structures have cross-sections of approximately 3 x 5nm, consistent with current models of collagen microfibril formation. By varying the pH and electrolyte of the buffer solution during the self-assembly process, the microfibril density and contacts formed within this network could be controlled. Under certain electrolyte compositions the microribbons and microfibers display the characteristic D-periodicity of approximately 65 nm observed for much thicker collagen fibrils. In addition to providing insight into the mechanism of collagen assembly, the ultraflat collagen matrices may also offer novel ways to bio-functionalize surfaces.

Suprachoroidal drainage with collagen sheet implant- a novel technique for non-penetrating glaucoma surgery.

Science.gov (United States)

Szurman, Peter; Januschowski, Kai; Boden, Karl Thomas; Seuthe, Anna-Maria

2018-02-01

Our purpose was both to introduce the new technique of suprachoroidal drainage with collagen sheet implantation as a novel technique of non-penetrating glaucoma surgery and to present first results of a prospective pilot study. A superficial rectangular sclera flap of half-scleral thickness sized 4 × 4.5 mm is dissected anteriorly until clear cornea. Then, a second scleral flap is created underneath the first one sized 3.5x4mm and is cut down full-thickness to the choroid exposing the suprachoroidal space. The flap is then bluntly prepared until the scleral spur is reached. Sharp dissection above the sclera spur exposes Schlemm's canal, which is located directly anteriorly. Schlemm's canal is unroofed, juxtacanalicular meshwork is peeled and the deep flap is cut off at its base. An absorbable collagen sheet (Ologen®, Dahlhausen, Cologne, Germany) is placed into the suprachoroidal space at the level of the ciliary body, and the superficial sclera flap is sutured tightly to prevent leakage. We prospectively analyzed 65 eyes that underwent suprachoroidal drainage with collagen sheet implantation. Mean reduction of intraocular pressure after 3 months was 35.1% (from 21.0 ± 4.3 mmHg to 13.5 ± 3.4 mmHg)(p < 0.01) and after 12 months 35.6% (from 21.0 ± 4.3 mmHg to 13.5 ± 3.0  mmHg)(p < 0.01). The number of topical IOP-reducing medication decreased significantly from 3.5 ± 0.7 to 0.6 ± 0.9 and to 0.9 ± 1.1 after 3 and 12 months, respectively (p < 0.01). No serious complications occurred. Suprachoroidal drainage with collagen sheet implantation seems to be a safe and effective surgical technique for non-penetrating glaucoma surgery that yields the opportunity of a sufficient IOP reduction for eyes unsuitable for canaloplasty.

Enhancing amine terminals in an amine-deprived collagen matrix.

LENUS (Irish Health Repository)

Tiong, William H C

2008-10-21

Collagen, though widely used as a core biomaterial in many clinical applications, is often limited by its rapid degradability which prevents full exploitation of its potential in vivo. Polyamidoamine (PAMAM) dendrimer, a highly branched macromolecule, possesses versatile multiterminal amine surface groups that enable them to be tethered to collagen molecules and enhance their potential. In this study, we hypothesized that incorporation of PAMAM dendrimer in a collagen matrix through cross-linking will result in a durable, cross-linked collagen biomaterial with free -NH 2 groups available for further multi-biomolecular tethering. The aim of this study was to assess the physicochemical properties of a G1 PAMAM cross-linked collagen matrix and its cellular sustainability in vitro. Different amounts of G1 PAMAM dendrimer (5 or 10 mg) were integrated into bovine-derived collagen matrices through a cross-linking process, mediated by 5 or 25 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in 5 mM N-hydroxysuccinimide (NHS) and 50 mM 2-morpholinoethane sulfonic acid buffer at pH 5.5. The physicochemical properties of resultant matrices were investigated with scanning electron microscopy (SEM), collagenase degradation assay, differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectra, and ninhydrin assay. Cellular sustainability of the matrices was assessed with Alamar Blue assay and SEM. There was no significant difference in cellular behavior between the treated and nontreated groups. However, the benefit of incorporating PAMAM in the cross-linking reaction was limited when higher concentrations of either agent were used. These results confirm the hypothesis that PAMAM dendrimer can be incorporated in the collagen cross-linking process in order to modulate the properties of the resulting cross-linked collagen biomaterial with free -NH 2 groups available for multi-biomolecular tethering.

PHAGOCYTOSIS AND REMODELING OF COLLAGEN MATRICES

OpenAIRE

Abraham, Leah C.; Dice, J Fred.; Lee, Kyongbum; Kaplan, David L.

2007-01-01

The biodegradation of collagen and the deposition of new collagen-based extracellular matrices are of central importance in tissue remodeling and function. Similarly, for collagen-based biomaterials used in tissue engineering, the degradation of collagen scaffolds with accompanying cellular infiltration and generation of new extracellular matrix is critical for integration of in vitro grown tissues in vivo. In earlier studies we observed significant impact of collagen structure on primary lun...

Platelet adhesion and plasma protein adsorption control of collagen surfaces by He+ ion implantation

International Nuclear Information System (INIS)

Kurotobi, K.; Suzuki, Y.; Nakajima, H.; Suzuki, H.; Iwaki, M.

2003-01-01

He + ion implanted collagen-coated tubes with a fluence of 1 x 10 14 ions/cm 2 were exhibited antithrombogenicity. To investigate the mechanisms of antithrombogenicity of these samples, plasma protein adsorption assay and platelet adhesion experiments were performed. The adsorption of fibrinogen (Fg) and von Willebrand factor (vWf) was minimum on the He + ion implanted collagen with a fluence of 1 x 10 14 ions/cm 2 . Platelet adhesion (using platelet rich plasma) was inhibited on the He + ion implanted collagen with a fluence of 1 x 10 14 ions/cm 2 and was accelerated on the untreated collagen and ion implanted collagen with fluences of 1 x 10 13 , 1 x 10 15 and 1 x 10 16 ions/cm 2 . Platelet activation with washed platelets was observed on untreated collagen and He + ion implanted collagen with a fluence of 1 x 10 14 ions/cm 2 and was inhibited with fluences of 1 x 10 13 , 1 x 10 15 and 1 x 10 16 ions/cm 2 . Generally, platelets can react with a specific ligand inside the collagen (GFOGER sequence). The results of platelets adhesion experiments using washed platelets indicated that there were no ligands such as GFOGER on the He + ion implanted collagen over a fluence of 1 x 10 13 ions/cm 2 . On the 1 x 10 14 ions/cm 2 implanted collagen, no platelet activation was observed due to the influence of plasma proteins. >From the above, it is concluded that the decrease of adsorbed Fg and vWf caused the antithrombogenicity of He + ion implanted collagen with a fluence of 1 x 10 14 ions/cm 2 and that plasma protein adsorption took an important role repairing the graft surface

Effect of Bizhongxiao decoction and its dismantled formulae on IL-1 and TNF levels in collagen-induced arthritis in rat synovial joints

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Guo Ya-jing

2012-11-01

Full Text Available Abstract Background Rheumatoid arthritis (RA, a chronic autoimmune disease, affects sufferers in many different ways. Treatment of this chronic condition is particularly challenging. Traditional Chinese Medicine (TCM provides alternatives. Bizhongxiao decoction (BZX is a TCM complex, which has been used clinically for many years to treat RA. The purpose of this study is to compare the effects of BZX decoction and its dismantled formulae on IL-1 and TNF-1 levels in rats with RA, and to elucidate its mechanism of action. Methods Ninety healthy normal female SD rats were randomly divided into six groups: normal (control, model, BZX decoction, and the three dismantled formulae (I: heat-clearing and detoxication, II: dissipating dampness, and III: blood circulation promotion. Apart from the normal (control group, the rats in each group were injected subcutaneously with bovine type II collagen and complete Freund adjuvant to establish a collagen-induced arthritis model, so that inhibition of foot swelling in the rats by BZX decoction and its dismantled formulae could be observed. Immunohistochemistry was used to assess the levels of the inflammatory cytokines IL-1 and TNF in synovial joints at various time points. Results Twenty-one days after the model was established, the levels of TNF and IL-1 were significantly higher in the model group, BZX decoction group and dismantled formula groups I, II and III than in the normal controls (P  Conclusions BZX decoction and the three dismantled formulae examined down-regulated the inflammatory factors IL-1 and TNF in collagen-induced arthritis rat models, but BZX exerted the strongest effect.

Chitosan: collagen sponges. In vitro mineralization; Mineralizacao in vitro de esponjas de quitosana: colageno

Energy Technology Data Exchange (ETDEWEB)

Martins, Virginia da C.A.; Silva, Gustavo M.; Plepis, Ana Maria G., E-mail: virginia@iqsc.usp.br [Instituto de Quimica de Sao Carlos- IQSC, Universidade de Sao Paulo, Sao Carlos, SP (Brazil)

2011-07-01

The regeneration of bone tissue is a problem that affects many people and scaffolds for bone tissue growth has been widely studied. The aim of this study was the in vitro mineralization of chitosan, chitosan:native collagen and chitosan:anionic collagen sponges. The sponges were obtained by lyophilization and mineralization was made by soaking the sponges in alternating solutions containing Ca{sup 2+} and PO{sub 4}{sup 3-}. The mineralization was confirmed by infrared spectroscopy, energy dispersive X-ray and X-ray diffraction observing the formation of phosphate salts, possibly a carbonated hydroxyapatite since Ca/P=1.80. The degree of mineralization was obtained by thermogravimetry calculating the amount of residue at 750 deg C. The chitosan:anionic collagen sponge showed the highest degree of mineralization probably due to the fact that anionic collagen provides additional sites for interaction with the inorganic phase. (author)

Elevated expression of NEU1 sialidase in idiopathic pulmonary fibrosis provokes pulmonary collagen deposition, lymphocytosis, and fibrosis.

Science.gov (United States)

Luzina, Irina G; Lockatell, Virginia; Hyun, Sang W; Kopach, Pavel; Kang, Phillip H; Noor, Zahid; Liu, Anguo; Lillehoj, Erik P; Lee, Chunsik; Miranda-Ribera, Alba; Todd, Nevins W; Goldblum, Simeon E; Atamas, Sergei P

2016-05-15

Idiopathic pulmonary fibrosis (IPF) poses challenges to understanding its underlying cellular and molecular mechanisms and the development of better therapies. Previous studies suggest a pathophysiological role for neuraminidase 1 (NEU1), an enzyme that removes terminal sialic acid from glycoproteins. We observed increased NEU1 expression in epithelial and endothelial cells, as well as fibroblasts, in the lungs of patients with IPF compared with healthy control lungs. Recombinant adenovirus-mediated gene delivery of NEU1 to cultured primary human cells elicited profound changes in cellular phenotypes. Small airway epithelial cell migration was impaired in wounding assays, whereas, in pulmonary microvascular endothelial cells, NEU1 overexpression strongly impacted global gene expression, increased T cell adhesion to endothelial monolayers, and disrupted endothelial capillary-like tube formation. NEU1 overexpression in fibroblasts provoked increased levels of collagen types I and III, substantial changes in global gene expression, and accelerated degradation of matrix metalloproteinase-14. Intratracheal instillation of NEU1 encoding, but not control adenovirus, induced lymphocyte accumulation in bronchoalveolar lavage samples and lung tissues and elevations of pulmonary transforming growth factor-β and collagen. The lymphocytes were predominantly T cells, with CD8(+) cells exceeding CD4(+) cells by nearly twofold. These combined data indicate that elevated NEU1 expression alters functional activities of distinct lung cell types in vitro and recapitulates lymphocytic infiltration and collagen accumulation in vivo, consistent with mechanisms implicated in lung fibrosis.

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

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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

LARP6 Meets Collagen mRNA: Specific Regulation of Type I Collagen Expression

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Yujie Zhang

2016-03-01

Full Text Available Type I collagen is the most abundant structural protein in all vertebrates, but its constitutive rate of synthesis is low due to long half-life of the protein (60–70 days. However, several hundred fold increased production of type I collagen is often seen in reparative or reactive fibrosis. The mechanism which is responsible for this dramatic upregulation is complex, including multiple levels of regulation. However, posttranscriptional regulation evidently plays a predominant role. Posttranscriptional regulation comprises processing, transport, stabilization and translation of mRNAs and is executed by RNA binding proteins. There are about 800 RNA binding proteins, but only one, La ribonucleoprotein domain family member 6 (LARP6, is specifically involved in type I collagen regulation. In the 5′untranslated region (5’UTR of mRNAs encoding for type I and type III collagens there is an evolutionally conserved stem-loop (SL structure; this structure is not found in any other mRNA, including any other collagen mRNA. LARP6 binds to the 5′SL in sequence specific manner to regulate stability of collagen mRNAs and their translatability. Here, we will review current understanding of how is LARP6 involved in posttranscriptional regulation of collagen mRNAs. We will also discuss how other proteins recruited by LARP6, including nonmuscle myosin, vimentin, serine threonine kinase receptor associated protein (STRAP, 25 kD FK506 binding protein (FKBP25 and RNA helicase A (RHA, contribute to this process.

The transmembrane collagen COL-99 guides longitudinally extending axons in C. elegans.

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Taylor, Jesse; Unsoeld, Thomas; Hutter, Harald

2018-06-01

We have identified the transmembrane collagen, COL-99, in a genetic screen for novel genes involved in axon guidance in the nematode C. elegans. COL-99 is similar to transmembrane collagens type XIII, XXIII and XXV in vertebrates. col-99 mutants exhibit guidance defects in axons extending along the major longitudinal axon tracts, most prominently the left ventral nerve cord (VNC). COL-99 is expressed in the hypodermis during the time of axon outgrowth. We provide evidence that a furin cleavage site in COL-99 is essential for function, suggesting that COL-99 is released from the cells producing it. Vertebrate homologs of COL-99 have been shown to be expressed in mammalian nervous systems and linked to various neurological disease but have not been associated with guidance of extending neurons. col-99 acts genetically with the discoidin domain receptors ddr-1 and ddr-2, which are expressed by neurons affected in col-99 mutants. Discoidin domain receptors are activated by collagens in vertebrates. DDR-1 and DDR-2 may function as receptors for COL-99. Our results establish a novel role for a transmembrane collagen in axonal guidance and asymmetry establishment of the VNC. Copyright © 2018 Elsevier Inc. All rights reserved.

Bi-allelic Alterations in AEBP1 Lead to Defective Collagen Assembly and Connective Tissue Structure Resulting in a Variant of Ehlers-Danlos Syndrome.

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Blackburn, Patrick R; Xu, Zhi; Tumelty, Kathleen E; Zhao, Rose W; Monis, William J; Harris, Kimberly G; Gass, Jennifer M; Cousin, Margot A; Boczek, Nicole J; Mitkov, Mario V; Cappel, Mark A; Francomano, Clair A; Parisi, Joseph E; Klee, Eric W; Faqeih, Eissa; Alkuraya, Fowzan S; Layne, Matthew D; McDonnell, Nazli B; Atwal, Paldeep S

2018-04-05

AEBP1 encodes the aortic carboxypeptidase-like protein (ACLP) that associates with collagens in the extracellular matrix (ECM) and has several roles in development, tissue repair, and fibrosis. ACLP is expressed in bone, the vasculature, and dermal tissues and is involved in fibroblast proliferation and mesenchymal stem cell differentiation into collagen-producing cells. Aebp1 -/- mice have abnormal, delayed wound repair correlating with defects in fibroblast proliferation. In this study, we describe four individuals from three unrelated families that presented with a unique constellation of clinical findings including joint laxity, redundant and hyperextensible skin, poor wound healing with abnormal scarring, osteoporosis, and other features reminiscent of Ehlers-Danlos syndrome (EDS). Analysis of skin biopsies revealed decreased dermal collagen with abnormal collagen fibrils that were ragged in appearance. Exome sequencing revealed compound heterozygous variants in AEBP1 (c.1470delC [p.Asn490_Met495delins(40)] and c.1743C>A [p.Cys581 ∗ ]) in the first individual, a homozygous variant (c.1320_1326del [p.Arg440Serfs ∗ 3]) in the second individual, and a homozygous splice site variant (c.1630+1G>A) in two siblings from the third family. We show that ACLP enhances collagen polymerization and binds to several fibrillar collagens via its discoidin domain. These studies support the conclusion that bi-allelic pathogenic variants in AEBP1 are the cause of this autosomal-recessive EDS subtype. Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Enhanced migration of murine fibroblast-like 3T3-L1 preadipocytes on type I collagen-coated dish is reversed by silibinin treatment.

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Liu, Xiaoling; Xu, Qian; Liu, Weiwei; Yao, Guodong; Zhao, Yeli; Xu, Fanxing; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Yamato, Masayuki; Ikejima, Takashi

2018-04-01

Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, whereas excessive migration is often responsible for impaired adipose tissue related with obesity and fibrotic diseases. Type I collagen (collagen I) is the most abundant component of extracellular matrix and has been shown to regulate fibroblast migration in vitro, but its role in adipose tissue is not known. Silibinin is a bioactive natural flavonoid with antioxidant and antimetastasis activities. In this study, we found that type I collagen coating promoted the proliferation and migration of murine 3T3-L1 preadipocytes in a dose-dependent manner, implying that collagen I could be an extracellular signal. Regarding the mechanisms of collagen I-stimulated 3T3-L1 migration, we found that NF-κB p65 is activated, including the increased nuclear translocation of NF-κB p65 as well as the upregulation of NF-κB p65 phosphorylation and acetylation, accompanied by the increased expressions of proinflammatory factors and the generation of reactive oxygen species (ROS). Reduction of collagen I-enhanced migration of cells by treatment with silibinin was associated with suppression of NF-κB p65 activity and ROS generation, and negatively correlated with the increasing sirt1 expression. Taken together, the enhanced migration of 3T3-L1 cells induced on collagen I-coated dish is mediated by the activation of NF-κB p65 function and ROS generation that can be alleviated with silibinin by upregulation of sirt1, leading to the repression of NF-κB p65 function and ROS generation.

Rheology of Heterotypic Collagen Networks

NARCIS (Netherlands)

Piechocka, I.K.; van Oosten, A.S.G.; Breuls, R.G.M.; Koenderink, G.H.

2011-01-01

Collagen fibrils are the main structural element of connective tissues. In many tissues, these fibrils contain two fibrillar collagens (types I and V) in a ratio that changes during tissue development, regeneration, and various diseases. Here we investigate the influence of collagen composition on

Effect of Vaginal or Systemic Estrogen on Dynamics of Collagen Assembly in the Rat Vaginal Wall1

Science.gov (United States)

Montoya, T. Ignacio; Maldonado, P. Antonio; Acevedo, Jesus F.; Word, R. Ann

2014-01-01

ABSTRACT The objective of this study was to compare the effects of systemic and local estrogen treatment on collagen assembly and biomechanical properties of the vaginal wall. Ovariectomized nulliparous rats were treated with estradiol or conjugated equine estrogens (CEEs) either systemically, vaginal CEE, or vaginal placebo cream for 4 wk. Low-dose local CEE treatment resulted in increased vaginal epithelial thickness and significant vaginal growth without uterine hyperplasia. Furthermore, vaginal wall distensibility increased without compromise of maximal force at failure. Systemic estradiol resulted in modest increases in collagen type I with no change in collagen type III mRNA. Low-dose vaginal treatment, however, resulted in dramatic increases in both collagen subtypes whereas moderate and high dose local therapies were less effective. Consistent with the mRNA results, low-dose vaginal estrogen resulted in increased total and cross-linked collagen content. The inverse relationship between vaginal dose and collagen expression may be explained in part by progressive downregulation of estrogen receptor-alpha mRNA with increasing estrogen dose. We conclude that, in this menopausal rat model, local estrogen treatment increased total and cross-linked collagen content and markedly stimulated collagen mRNA expression in an inverse dose-effect relationship. High-dose vaginal estrogen resulted in downregulation of estrogen receptor-alpha and loss of estrogen-induced increases in vaginal collagen. These results may have important clinical implications regarding the use of local vaginal estrogen therapy and its role as an adjunctive treatment in women with loss of vaginal support. PMID:25537371

Effect of internal structure of collagen/hydroxyapatite scaffold on the osteogenic differentiation of mesenchymal stem cells.

Science.gov (United States)

Chen, Guobao; Lv, Yonggang; Dong, Chanjuan; Yang, Li

2015-01-01

Consisting of seed cells and scaffold, regenerative medicine provides a new way for the repair and regeneration of tissue and organ. Collagen/hydroxyapatite (HA) biocomposite scaffold is highlighted due to its advantageous features of two major components of bone matrix: collagen and HA. The aim of this study is to investigate the effect of internal structure of collagen/HA scaffold on the fate of rat mesenchymal stem cells (MSCs). The internal structure of collagen/HA scaffold was characterized by micro-CT. It is found that the porosity decreased while average compressive modulus increased with the increase of collagen proportion. Within the collagen proportion of 0.35%, 0.5% and 0.7%, the porosities were 89.08%, 78.37% and 75.36%, the pore sizes were 140.6±75.5 μm, 133.9±48.4 μm and 160.7±119.6 μm, and the average compressive moduli were 6.74±1.16 kPa, 8.82±2.12 kPa and 23.61±8.06 kPa, respectively. Among these three kinds of scaffolds, MSCs on the Col 0.35/HA 22 scaffold have the highest viability and the best cell proliferation. On the contrary, the Col 0.7/HA 22 scaffold has the best ability to stimulate MSCs to differentiate into osteoblasts in a relatively short period of time. In vivo research also demonstrated that the internal structure of collagen/HA scaffold has significant effect on the cell infiltration. Therefore, precise control of the internal structure of collagen/HA scaffold can provide a more efficient carrier to the repair of bone defects.

Greener synthesis of electrospun collagen/hydroxyapatite composite fibers with an excellent microstructure for bone tissue engineering

Science.gov (United States)

Zhou, Yuanyuan; Yao, Hongchang; Wang, Jianshe; Wang, Dalu; Liu, Qian; Li, Zhongjun

2015-01-01

In bone tissue engineering, collagen/hydroxyapatite (HAP) fibrous composite obtained via electrospinning method has been demonstrated to support the cells’ adhesion and bone regeneration. However, electrospinning of natural collagen often requires the use of cytotoxic organic solvents, and the HAP crystals were usually aggregated and randomly distributed within a fibrous matrix of collagen, limiting their clinical potential. Here, an effective and greener method for the preparation of collagen/HAP composite fibers was developed for the first time, and this green product not only had 40 times higher mechanical properties than that previously reported, but also had an excellent microstructure similar to that of natural bone. By dissolving type I collagen in environmentally friendly phosphate buffered saline/ethanol solution instead of the frequently-used cytotoxic organic solvents, followed with the key step of desalination, co-electrospinning the collagen solution with the HAP sol, generates a collagen/HAP composite with a uniform and continuous fibrous morphology. Interestingly, the nano-HAP needles were found to preferentially orient along the longitudinal direction of the collagen fibers, which mimicked the nanostructure of natural bones. Based on the characterization of the related products, the formation mechanism for this novel phenomenon was proposed. After cross-linking with 1-ethyl-3-(3-dimethyl-aminopropyl)-1-carbodiimide hydrochloride/N-hydroxysuccinimide, the obtained composite exhibited a significant enhancement in mechanical properties. In addition, the biocompatibility of the obtained composite fibers was evaluated by in vitro culture of the human myeloma cells (U2-OS). Taken together, the process outlined herein provides an effective, non-toxic approach for the fabrication of collagen/HAP composite nanofibers that could be good candidates for bone tissue engineering. PMID:25995630

Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells.

Science.gov (United States)

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

2015-10-15

Collagen is a widely used naturally occurring biomaterial for scaffolding, whereas mesenchymal stem cells (MSCs) represent a promising cell source in tissue engineering and regenerative medicine. It is generally known that cells are able to remodel their environment by simultaneous degradation of the scaffolds and deposition of newly synthesized extracellular matrix. Nevertheless, the interactions between MSCs and collagen biomaterials are poorly known, and the strategies enhancing the extracellular matrix deposition are yet to be defined. In this study, we aim to investigate the fate of collagen when it is in contact with MSCs and hypothesize that protease inhibition will enhance their extracellular deposition of collagen fibrils. Specifically, human MSCs (hMSCs) were exposed to fluorescence-labeled collagen with and without intracellular or extracellular protease inhibitors (or both) before tracing the collagen at both intracellular and extracellular spaces. Collagen were internalized by hMSCs and degraded intracellularly in lysosomes. In the presence of protease inhibitors, both intracellular collagen fibril growth and extracellular deposition of collagen fibrils were enhanced. Moreover, protease inhibitors work synergistically with ascorbic acid, a well-known matrix deposition-enhancing reagent, in further enhancing collagen fibril deposition at the extracellular space. These findings provide a better understanding of the interactions between hMSCs and collagen biomaterials and suggest a method to manipulate matrix remodeling and deposition of hMSCs, contributing to better scaffolding for tissue engineering and regenerative medicine.

Glycation Contributes to Interaction Between Human Bone Alkaline Phosphatase and Collagen Type I.

Science.gov (United States)

Halling Linder, Cecilia; Enander, Karin; Magnusson, Per

2016-03-01

Bone is a biological composite material comprised primarily of collagen type I and mineral crystals of calcium and phosphate in the form of hydroxyapatite (HA), which together provide its mechanical properties. Bone alkaline phosphatase (ALP), produced by osteoblasts, plays a pivotal role in the mineralization process. Affinity contacts between collagen, mainly type II, and the crown domain of various ALP isozymes were reported in a few in vitro studies in the 1980s and 1990s, but have not attracted much attention since, although such interactions may have important implications for the bone mineralization process. The objective of this study was to investigate the binding properties of human collagen type I to human bone ALP, including the two bone ALP isoforms B1 and B2. ALP from human liver, human placenta and E. coli were also studied. A surface plasmon resonance-based analysis, supported by electrophoresis and blotting, showed that bone ALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. Further, the B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform. Human bone and liver ALP (with identical amino acid composition) displayed pronounced differences in binding, revealing that post-translational glycosylation properties govern these interactions to a large extent. In conclusion, this study presents the first evidence that glycosylation differences in human ALPs are of crucial importance for protein-protein interactions with collagen type I, although the presence of the ALP crown domain may also be necessary. Different binding affinities among the bone ALP isoforms may influence the mineral-collagen interface, mineralization kinetics, and degree of bone matrix mineralization, which are important factors determining the material properties of bone.

COLLAGENOUS SPHERULES OF THE BREAST: A DIAGNOSTIC ENIGMA

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Amrit Kaur

2016-05-01

Full Text Available INTRODUCTION Collagenous spherule (CS is an enigmatic finding in a breast lesion involving the lobular acini and ductules and is defined with the presence of eosinophilic intraluminal collagen rich spherules measuring 20-100 microns in diameter, surrounded by flattened myoepithelial cells. 1 It is an uncommon incidental finding in less than 1-2% of biopsies associated with various benign and malignant diseases occurring in isolation or multifocally. 2 A major growing concern surrounding collagenous spherules is that it might be misinterpreted as atypical ductal hyperplasia (ADH, cribriform ductal carcinoma in situ (DCIS, cribriform carcinoma or adenoid cystic carcinoma of breast. We present a case of mobile cystic mass of the breast reported as fibrocystic disease of the breast with focal areas showing adenosis and hyperplastic changes with multiple ducts displayed a peculiar change with the presence of extracellular concentric hyaline material present within the intraluminal space, diagnostic of collagenous spherules.

Cyclophilin B Deficiency Causes Abnormal Dentin Collagen Matrix.

Science.gov (United States)

Terajima, Masahiko; Taga, Yuki; Cabral, Wayne A; Nagasawa, Masako; Sumida, Noriko; Hattori, Shunji; Marini, Joan C; Yamauchi, Mitsuo

2017-08-04

Cyclophilin B (CypB) is an endoplasmic reticulum-resident protein that regulates collagen folding, and also contributes to prolyl 3-hydroxylation (P3H) and lysine (Lys) hydroxylation of collagen. In this study, we characterized dentin type I collagen in CypB null (KO) mice, a model of recessive osteogenesis imperfecta type IX, and compared to those of wild-type (WT) and heterozygous (Het) mice. Mass spectrometric analysis demonstrated that the extent of P3H in KO collagen was significantly diminished compared to WT/Het. Lys hydroxylation in KO was significantly diminished at the helical cross-linking sites, α1/α2(I) Lys-87 and α1(I) Lys-930, leading to a significant increase in the under-hydroxylated cross-links and a decrease in fully hydroxylated cross-links. The extent of glycosylation of hydroxylysine residues was, except α1(I) Lys-87, generally higher in KO than WT/Het. Some of these molecular phenotypes were distinct from other KO tissues reported previously, indicating the dentin-specific control mechanism through CypB. Histological analysis revealed that the width of predentin was greater and irregular, and collagen fibrils were sparse and significantly smaller in KO than WT/Het. These results indicate a critical role of CypB in dentin matrix formation, suggesting a possible association between recessive osteogenesis imperfecta and dentin defects that have not been clinically detected.

Mechanical enhancement and in vitro biocompatibility of nanofibrous collagen-chitosan scaffolds for tissue engineering.

Science.gov (United States)

Zou, Fengjuan; Li, Runrun; Jiang, Jianjun; Mo, Xiumei; Gu, Guofeng; Guo, Zhongwu; Chen, Zonggang

2017-12-01

The collagen-chitosan complex with a three-dimensional nanofiber structure was fabricated to mimic native ECM for tissue repair and biomedical applications. Though the three-dimensional hierarchical fibrous structures of collagen-chitosan composites could provide more adequate stimulus to facilitate cell adhesion, migrate and proliferation, and thus have the potential as tissue engineering scaffolding, there are still limitations in their applications due to the insufficient mechanical properties of natural materials. Because poly (vinyl alcohol) (PVA) and thermoplastic polyurethane (TPU) as biocompatible synthetic polymers can offer excellent mechanical properties, they were introduced into the collagen-chitosan composites to fabricate the mixed collagen/chitosan/PVA fibers and a sandwich structure (collagen/chitosan-TPU-collagen/chitosan) of nanofiber in order to enhance the mechanical properties of the nanofibrous collagen-chitosan scaffold. The results showed that the tensile behavior of materials was enhanced to different degrees with the difference of collagen content in the fibers. Besides the Young's modulus had no obvious changes, both the break strength and the break elongation of materials were heightened after reinforced by PVA. For the collagen-chitosan nanofiber reinforced by TPU, both the break strength and the Young's modulus of materials were heightened in different degrees with the variety of collagen content in the fibers despite the decrease of the break elongation of materials to some extent. In vitro cell test demonstrated that the materials could provide adequate environment for cell adhesion and proliferation. All these indicated that the reinforced collagen-chitosan nanofiber could be as potential scaffold for tissue engineering according to the different mechanical requirements in clinic.

Snail1 induced in breast cancer cells in 3D collagen I gel environment suppresses cortactin and impairs effective invadopodia formation.

Science.gov (United States)

Lee, Mi-Sook; Kim, Sudong; Kim, Baek Gil; Won, Cheolhee; Nam, Seo Hee; Kang, Suki; Kim, Hye-Jin; Kang, Minkyung; Ryu, Jihye; Song, Haeng Eun; Lee, Doohyung; Ye, Sang-Kyu; Jeon, Noo Li; Kim, Tai Young; Cho, Nam Hoon; Lee, Jung Weon

2014-09-01

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents. Copyright © 2014 Elsevier B.V. All rights reserved.

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

International Nuclear Information System (INIS)

Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G.

2012-01-01

Highlights: ► We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. ► YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. ► There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. ► The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. ► The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929–933 sequence of the β1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

Distinct characteristics of mandibular bone collagen relative to long bone collagen: relevance to clinical dentistry.

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Matsuura, Takashi; Tokutomi, Kentaro; Sasaki, Michiko; Katafuchi, Michitsuna; Mizumachi, Emiri; Sato, Hironobu

2014-01-01

Bone undergoes constant remodeling throughout life. The cellular and biochemical mechanisms of bone remodeling vary in a region-specific manner. There are a number of notable differences between the mandible and long bones, including developmental origin, osteogenic potential of mesenchymal stem cells, and the rate of bone turnover. Collagen, the most abundant matrix protein in bone, is responsible for determining the relative strength of particular bones. Posttranslational modifications of collagen, such as intermolecular crosslinking and lysine hydroxylation, are the most essential determinants of bone strength, although the amount of collagen is also important. In comparison to long bones, the mandible has greater collagen content, a lower amount of mature crosslinks, and a lower extent of lysine hydroxylation. The great abundance of immature crosslinks in mandibular collagen suggests that there is a lower rate of cross-link maturation. This means that mandibular collagen is relatively immature and thus more readily undergoes degradation and turnover. The greater rate of remodeling in mandibular collagen likely renders more flexibility to the bone and leaves it more suited to constant exercise. As reviewed here, it is important in clinical dentistry to understand the distinctive features of the bones of the jaw.

Distinct Characteristics of Mandibular Bone Collagen Relative to Long Bone Collagen: Relevance to Clinical Dentistry

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Takashi Matsuura

2014-01-01

Full Text Available Bone undergoes constant remodeling throughout life. The cellular and biochemical mechanisms of bone remodeling vary in a region-specific manner. There are a number of notable differences between the mandible and long bones, including developmental origin, osteogenic potential of mesenchymal stem cells, and the rate of bone turnover. Collagen, the most abundant matrix protein in bone, is responsible for determining the relative strength of particular bones. Posttranslational modifications of collagen, such as intermolecular crosslinking and lysine hydroxylation, are the most essential determinants of bone strength, although the amount of collagen is also important. In comparison to long bones, the mandible has greater collagen content, a lower amount of mature crosslinks, and a lower extent of lysine hydroxylation. The great abundance of immature crosslinks in mandibular collagen suggests that there is a lower rate of cross-link maturation. This means that mandibular collagen is relatively immature and thus more readily undergoes degradation and turnover. The greater rate of remodeling in mandibular collagen likely renders more flexibility to the bone and leaves it more suited to constant exercise. As reviewed here, it is important in clinical dentistry to understand the distinctive features of the bones of the jaw.

Demineralized dentin matrix composite collagen material for bone tissue regeneration.

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Li, Jianan; Yang, Juan; Zhong, Xiaozhong; He, Fengrong; Wu, Xiongwen; Shen, Guanxin

2013-01-01

Demineralized dentin matrix (DDM) had been successfully used in clinics as bone repair biomaterial for many years. However, particle morphology of DDM limited it further applications. In this study, DDM and collagen were prepared to DDM composite collagen material. The surface morphology of the material was studied by scanning electron microscope (SEM). MC3T3-E1 cells responses in vitro and tissue responses in vivo by implantation of DDM composite collagen material in bone defect of rabbits were also investigated. SEM analysis showed that DDM composite collagen material evenly distributed and formed a porous scaffold. Cell culture and animal models results indicated that DDM composite collagen material was biocompatible and could support cell proliferation and differentiation. Histological evaluation showed that DDM composite collagen material exhibited good biocompatibility, biodegradability and osteoconductivity with host bone in vivo. The results suggested that DDM composite collagen material might have a significant clinical advantage and potential to be applied in bone and orthopedic surgery.

Glutaraldehyde-induced remineralization improves the mechanical properties and biostability of dentin collagen

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Chen, Chaoqun; Mao, Caiyun; Sun, Jian; Chen, Yi; Wang, Wei [Department of Stomatology, The First Affiliated Hospital, College of Medicine, Zhejiang University (China); Pan, Haihua; Tang, Ruikang [Centre for Biopathways and Biomaterials, Department of Chemistry, Zhejiang University (China); Gu, Xinhua [Department of Stomatology, The First Affiliated Hospital, College of Medicine, Zhejiang University (China)

2016-10-01

The purpose of this study was to induce a biomimetic remineralization process by using glutaraldehyde (GA) to reconstruct the mechanical properties and biostability of demineralized collagen. Demineralized dentin disks (35% phosphoric acid, 10 s) were pretreated with a 5% GA solution for 3 min and then cultivated in a calcium phosphate remineralization solution. The remineralization kinetics and superstructure of the remineralization layer were evaluated by Raman spectroscopy, transmission electron microscopy, scanning electron microscopy and nanoindentation tests. The biostability was examined by enzymatic degradation experiments. A significant difference was found in dentin remineralization process between dentin with and without GA pretreating. GA showed a specific affinity to dentin collagen resulting in the formation of a cross-linking superstructure. GA pretreating could remarkably shorten remineralization time from 7 days to 2 days. The GA-induced remineralized collagen fibrils were well encapsulated by newly formed hydroxyapatite mineral nanocrystals. With the nano-hydroxyapatite coating, both the mechanical properties (elastic modulus and hardness) and the biostability against enzymatic degradation of the collagen were significantly enhanced, matching those of natural dentin. The results indicated that GA cross-linking of dentin collagen could promote dentin biomimetic remineralization, resulting in an improved mechanical properties and biostability. It may provide a promising tissue-engineering technology for dentin repair. - Highlights: • GA cross-linking can promote the remineralization kinetics of dentin collagen. • GA-induced remineralization can reshape the demineralized dentin collagen layer. • The GA-induced remineralization enhances the degradation resistance of collagen. • GA-induced remineralization provides a new approach to improve bonding durability.

Glutaraldehyde-induced remineralization improves the mechanical properties and biostability of dentin collagen

International Nuclear Information System (INIS)

Chen, Chaoqun; Mao, Caiyun; Sun, Jian; Chen, Yi; Wang, Wei; Pan, Haihua; Tang, Ruikang; Gu, Xinhua

2016-01-01

The purpose of this study was to induce a biomimetic remineralization process by using glutaraldehyde (GA) to reconstruct the mechanical properties and biostability of demineralized collagen. Demineralized dentin disks (35% phosphoric acid, 10 s) were pretreated with a 5% GA solution for 3 min and then cultivated in a calcium phosphate remineralization solution. The remineralization kinetics and superstructure of the remineralization layer were evaluated by Raman spectroscopy, transmission electron microscopy, scanning electron microscopy and nanoindentation tests. The biostability was examined by enzymatic degradation experiments. A significant difference was found in dentin remineralization process between dentin with and without GA pretreating. GA showed a specific affinity to dentin collagen resulting in the formation of a cross-linking superstructure. GA pretreating could remarkably shorten remineralization time from 7 days to 2 days. The GA-induced remineralized collagen fibrils were well encapsulated by newly formed hydroxyapatite mineral nanocrystals. With the nano-hydroxyapatite coating, both the mechanical properties (elastic modulus and hardness) and the biostability against enzymatic degradation of the collagen were significantly enhanced, matching those of natural dentin. The results indicated that GA cross-linking of dentin collagen could promote dentin biomimetic remineralization, resulting in an improved mechanical properties and biostability. It may provide a promising tissue-engineering technology for dentin repair. - Highlights: • GA cross-linking can promote the remineralization kinetics of dentin collagen. • GA-induced remineralization can reshape the demineralized dentin collagen layer. • The GA-induced remineralization enhances the degradation resistance of collagen. • GA-induced remineralization provides a new approach to improve bonding durability.

Combined MSC and GLP-1 Therapy Modulates Collagen Remodeling and Apoptosis following Myocardial Infarction

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Elizabeth J. Wright

2016-01-01

Full Text Available Background. Mesenchymal stem cells (MSCs and glucagon-like peptide-1 (GLP-1 are being tested as treatment strategies for myocardial infarction (MI; however, their mechanisms in the heart are not fully understood. Methods. We examined the effects of MSCs, either native, or engineered to secrete a GLP-1 fusion protein (MSCs ± GLP-1, on human cardiomyocyte apoptosis in vitro. The effect on cardiac remodeling when encapsulated in alginate beads (CellBeads-MSC and CellBeads-MSC + GLP-1 was also evaluated in a pig MI model, whereby pigs were treated with Empty Beads, CellBeads-MSC, or CellBeads-MSC + GLP-1 and sacrificed at one or four weeks following MI. Results. MSC + GLP-1 conditioned media demonstrated antiapoptotic effects on ischaemic human cardiomyocytes in vitro. In vivo, qRT-PCR revealed large changes in the expression of several genes involved in extracellular matrix remodeling, which were altered following MSC ± GLP treatment. After four weeks, infarcted areas were imaged using atomic force microscopy, demonstrating significant alterations between groups in the structure of collagen fibrils and resulting scar. Conclusions. These data demonstrate that MSCs ± GLP-1 exhibit modulatory effects on healing post-MI, affecting both apoptosis and collagen scar formation. These data support the premise that both MSCs and GLP-1 could be beneficial in MI treatment.

Nanoscale Structure of Type I Collagen Fibrils: Quantitative Measurement of D-spacing

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Erickson, Blake; Fang, Ming; Wallace, Joseph M.; Orr, Bradford G.; Les, Clifford M.; Holl, Mark M. Banaszak

2012-01-01

This paper details a quantitative method to measure the D-periodic spacing of Type I collagen fibrils using Atomic Force Microscopy coupled with analysis using a 2D Fast Fourier Transform approach. Instrument calibration, data sampling and data analysis are all discussed and comparisons of the data to the complementary methods of electron microscopy and X-ray scattering are made. Examples of the application of this new approach to the analysis of Type I collagen morphology in disease models of estrogen depletion and Osteogenesis Imperfecta are provided. We demonstrate that it is the D-spacing distribution, not the D-spacing mean, that showed statistically significant differences in estrogen depletion associated with early stage Osteoporosis and Osteogenesis Imperfecta. The ability to quantitatively characterize nanoscale morphological features of Type I collagen fibrils will provide important structural information regarding Type I collagen in many research areas, including tissue aging and disease, tissue engineering, and gene knock out studies. Furthermore, we also envision potential clinical applications including evaluation of tissue collagen integrity under the impact of diseases or drug treatments. PMID:23027700

Ridge preservation of extraction sockets with chronic pathology using Bio-Oss® Collagen with or without collagen membrane: an experimental study in dogs.

Science.gov (United States)

Kim, Jung-Ju; Schwarz, Frank; Song, Hyun Young; Choi, YoonMi; Kang, Kyung-Rim; Koo, Ki-Tae

2017-06-01

This study aimed to evaluate the dynamics of newly bone formation and dimensional change in diseased extraction sockets using Bio-Oss ® Collagen with or without a collagen membrane. In six beagle dogs, right and left 3rd and 4th mandibular premolars were hemisected and the distal roots were removed. Combined endodontic-periodontic lesions were induced in all sites using black silk, collagen sponge, endodontic files, and application of Porphyromonas gingivalis. After 4 months, among 4 premolars, three teeth were randomly selected per dog and allocated to the following experimental groups: Control group (no treatment but debridement), Test 1 group (only Bio-Oss ® Collagen graft), and Test 2 group (Bio-Oss ® Collagen graft with a collagen membrane). After 7 months from the baseline, the beagle dogs were sacrificed for histomorphometric and Micro-CT analysis. The vertical distance between buccal and lingual crests in the Control group (2.22 ± 0.26 mm) and Test 2 group (1.80 ± 0.16 mm) was significantly different. The socket of the Test 2 group (27.04 ± 5.25%) was occupied by a greater quantity of bone graft compared to the Test 1 group (18.49 ± 2.11%). Ridge preservation in diseased extraction sockets could compensate for buccal bone resorption by contact osteogenesis surrounding the bone graft particles at the bucco-coronal area during socket healing, and the application of a collagen membrane at the entrance of the socket is useful for preserving graft material at the coronal part of the socket. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Characterization of Acid Soluble Collagen from Redbelly Yellowtail Fusilier Fish Skin (Caesio cuning

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Ika Astiana

2016-04-01

Full Text Available Fish skin can be used as raw material for producing collagen. The collagen can be extracted by chemical or combination of chemical and enzymatic processes. Extraction of collagen chemically can do with the acid process that produces acid soluble collagen (ASC. This study aimed to determine the optimum concentration and time of pretreatment and extraction, also to determine the characteristics of the acid soluble collagen from the skin of yellow tail fish. Extraction of collagen done by pretreatment using NaOH at the concentration of 0.05; 0.1; and 0.15 M and extraction using acetic acid at the concentration of 0.3; 0.5; and 0.7 M. Pretreatment NaOH with concentration 0.05 M and soaking time of 8 hours is the best combination for eliminating non collagen protein. Combination treatment of acetic acid at the concentration of 0.3 M for 3 days obtained the best solubility. The yield of collagen ASC was 18.4±1.49% (db and 5.79±0.47% (wb. Amino acid composition that is dominant in the ASC collagen was glycine (25.09±0.003%, alanine (13.71±0.075%, and proline (12.15±0.132%. Collagen from yellow tail fish skin has α1, α2, β and γ protein structure with the molecular weight of 125, 113, 170-181, and 208 KDa. The transition and melting temperatures of collagen were 67.69oC and 144.4oC. The surface structure of collagen by analysis of SEM has fibers on the surface.

Edaravone suppresses degradation of type II collagen.

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Huang, Chen; Liao, Guangjun; Han, Jian; Zhang, Guofeng; Zou, Benguo

2016-05-13

Osteoarthritis (OA) is a degenerative joint disease affecting millions of people. The degradation and loss of type II collagen induced by proinflammatory cytokines secreted by chondrocytes, such as factor-α (TNF-α) is an important pathological mechanism to the progression of OA. Edaravone is a potent free radical scavenger, which has been clinically used to treat the neuronal damage following acute ischemic stroke. However, whether Edaravone has a protective effect in articular cartilage hasn't been reported before. In this study, we investigated the chondrocyte protective effects of Edaravone on TNF-α induced degradation of type Ⅱ collagen. And our results indicated that TNF-α treatment resulted in degradation of type Ⅱ collagen, which can be ameliorated by treatment with Edaravone in a dose dependent manner. Notably, it was found that the inhibitory effects of Edaravone on TNF-α-induced reduction of type Ⅱ collagen were mediated by MMP-3 and MMP-13. Mechanistically, we found that Edaravone alleviated TNF-α induced activation of STAT1 and expression of IRF-1. These findings suggest a potential protective effect of Edaravone in OA. Copyright © 2016. Published by Elsevier Inc.

Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding.

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Barnes, Aileen M; Carter, Erin M; Cabral, Wayne A; Weis, MaryAnn; Chang, Weizhong; Makareeva, Elena; Leikin, Sergey; Rotimi, Charles N; Eyre, David R; Raggio, Cathleen L; Marini, Joan C

2010-02-11

Osteogenesis imperfecta is a heritable disorder that causes bone fragility. Mutations in type I collagen result in autosomal dominant osteogenesis imperfecta, whereas mutations in either of two components of the collagen prolyl 3-hydroxylation complex (cartilage-associated protein [CRTAP] and prolyl 3-hydroxylase 1 [P3H1]) cause autosomal recessive osteogenesis imperfecta with rhizomelia (shortening of proximal segments of upper and lower limbs) and delayed collagen folding. We identified two siblings who had recessive osteogenesis imperfecta without rhizomelia. They had a homozygous start-codon mutation in the peptidyl-prolyl isomerase B gene (PPIB), which results in a lack of cyclophilin B (CyPB), the third component of the complex. The proband's collagen had normal collagen folding and normal prolyl 3-hydroxylation, suggesting that CyPB is not the exclusive peptidyl-prolyl cis-trans isomerase that catalyzes the rate-limiting step in collagen folding, as is currently thought. 2010 Massachusetts Medical Society

Tendon collagen synthesis declines with immobilization in elderly humans

DEFF Research Database (Denmark)

Dideriksen, Kasper; Boesen, Anders P; Reitelseder, Søren

2017-01-01

-80 yr) were randomly assigned to NSAIDs (ibuprofen 1,200 mg/day; Ibu) or placebo (Plc). One lower limb was immobilized in a cast for 2 wk and retrained for 6 wk. Tendon collagen protein synthesis, mechanical properties, size, expression of genes related to collagen turnover and remodeling, and signal...... intensity (from magnetic resonance imaging) were investigated. Tendon collagen synthesis decreased (P ... immobilization in both groups, whereas scleraxis mRNA decreased with inactivity in the Plc group only (P collagen protein synthesis decreased after 2 wk of immobilization, whereas tendon stiffness and modulus were only marginally reduced, and NSAIDs had no influence upon this...

Cartilage collagen damage in hip osteoarthritis similar to that seen in knee osteoarthritis; a case-control study of relationship between collagen, glycosaminoglycan and cartilage swelling.

Science.gov (United States)

Hosseininia, Shahrzad; Lindberg, Lisbeth R; Dahlberg, Leif E

2013-01-09

It remains to be shown whether OA shares molecular similarities between different joints in humans. This study provides evidence for similarities in cartilage molecular damage in osteoarthritic (OA) joints. Articular cartilage from osteoarthritic hip joints were analysed and compared to non-OA controls regarding collagen, glycosaminoglycan and water content. Femoral heads from 16 osteoarthritic (OA) and 20 reference patients were obtained from hip replacement surgery due to OA and femoral neck fracture, respectively. Cartilage histological changes were assessed by Mankin grading and denatured collagen type II immunostaining and cartilage was extracted by α-chymotrypsin. Hydroxyproline and Alcian blue binding assays were used to measure collagen and glycosaminoglycan (GAG) content, respectively. Mankin and immunohistology scores were significantly higher in hip OA samples than in reference samples. Cartilage water content was 6% higher in OA samples than in references. 2.5 times more collagen was extracted from OA than from reference samples. There was a positive association between water content and percentage of extractable collagen pool (ECP) in both groups. The amounts of collagen per wet and dry weights did not differ statistically between OA and reference cartilage. % Extractable collagen was not related to collagen per dry weight in either group. However when collagen was expressed by wet weight there was a negative correlation between % extractable and collagen in OA cartilage. The amount of GAG per wet weight was similar in both groups but the amount of GAG per dry weight was higher in OA samples compared to reference samples, which suggests a capacity for GAG biosynthesis in hip OA cartilage. Neither of the studied parameters was related to age in either group. Increased collagen extractability and water content in human hip cartilage is associated with OA pathology and can be observed at early stages of the degenerative hip OA process. Our results

Reinforcement of a porous collagen scaffold with surface-activated PLA fibers.

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Liu, Xi; Huang, Changbin; Feng, Yujie; Liang, Jie; Fan, Yujiang; Gu, Zhongwei; Zhang, Xingdong

2010-01-01

A hybrid porous collagen scaffold mechanically reinforced with surface-activated poly(lactic acid) (PLA) fiber was prepared. PLA fibers, 20 mum in diameter and 1 mm in length, were aminolyzed with hexanediamine to introduce free amino groups on the surfaces. After the amino groups were transferred to aldehyde groups by treatment with glutaraldehyde, different amounts (1.5, 3, 5 and 8 mg) of surface-activated PLA fibers were homogeneously mixed with 2 ml type-I collagen solution (pH 2.8, 0.6 wt%). This mixture solution was then freeze-dried and cross-linked to obtain collagen sponges with surface-activated PLA fiber. Scanning electron microscopy observation indicated that the collagen sponges had a highly interconnected porous structure with an average pore size of 170 mum, irrespective of PLA fiber incorporation. The dispersion of surface-activated PLA fibers was homogeneous in collagen sponge, in contrast to unactivated PLA fibers. The compression modulus test results showed that, compared with unactivated PLA fibers, the surface-activated PLA fibers enhanced the resistance of collagen sponge to compression more significantly. Cytotoxicity assay by MTT test showed no cytotoxicity of these collagen sponges. L929 mouse fibroblast cell-culture studies in vitro revealed that the number of L929 cells attached to the collagen sponge with surface-activated PLA fibers, both 6 h and 24 h after seeding, was higher than that in pure collagen sponge and sponge with unactivated PLA fibers. In addition, a better distribution of cells infiltrated in collagen sponge with surface-activated PLA fibers was observed by histological staining. These results indicated that the collagen sponge reinforced with surface-activated PLA fibers is a promising biocompatible scaffold for tissue engineering.

Structure to function: Spider silk and human collagen

Science.gov (United States)

Rabotyagova, Olena S.

Nature has the ability to assemble a variety of simple molecules into complex functional structures with diverse properties. Collagens, silks and muscles fibers are some examples of fibrous proteins with self-assembling properties. One of the great challenges facing Science is to mimic these designs in Nature to find a way to construct molecules that are capable of organizing into functional supra-structures by self-assembly. In order to do so, a construction kit consisting of molecular building blocks along with a complete understanding on how to form functional materials is required. In this current research, the focus is on spider silk and collagen as fibrous protein-based biopolymers that can shed light on how to generate nanostructures through the complex process of self-assembly. Spider silk in fiber form offers a unique combination of high elasticity, toughness, and mechanical strength, along with biological compatibility and biodegrability. Spider silk is an example of a natural block copolymer, in which hydrophobic and hydrophilic blocks are linked together generating polymers that organize into functional materials with extraordinary properties. Since silks resemble synthetic block copolymer systems, we adopted the principles of block copolymer design from the synthetic polymer literature to build block copolymers based on spider silk sequences. Moreover, we consider spider silk to be an important model with which to study the relationships between structure and properties in our system. Thus, the first part of this work was dedicated to a novel family of spider silk block copolymers, where we generated a new family of functional spider silk-like block copolymers through recombinant DNA technology. To provide fundamental insight into relationships between peptide primary sequence, block composition, and block length and observed morphological and structural features, we used these bioengineered spider silk block copolymers to study secondary structure

Association of collagen architecture with glioblastoma patient survival.

Science.gov (United States)

Pointer, Kelli B; Clark, Paul A; Schroeder, Alexandra B; Salamat, M Shahriar; Eliceiri, Kevin W; Kuo, John S

2017-06-01

OBJECTIVE Glioblastoma (GBM) is the most malignant primary brain tumor. Collagen is present in low amounts in normal brain, but in GBMs, collagen gene expression is reportedly upregulated. However, to the authors' knowledge, direct visualization of collagen architecture has not been reported. The authors sought to perform the first direct visualization of GBM collagen architecture, identify clinically relevant collagen signatures, and link them to differential patient survival. METHODS Second-harmonic generation microscopy was used to detect collagen in a GBM patient tissue microarray. Focal and invasive GBM mouse xenografts were stained with Picrosirius red. Quantitation of collagen fibers was performed using custom software. Multivariate survival analysis was done to determine if collagen is a survival marker for patients. RESULTS In focal xenografts, collagen was observed at tumor brain boundaries. For invasive xenografts, collagen was intercalated with tumor cells. Quantitative analysis showed significant differences in collagen fibers for focal and invasive xenografts. The authors also found that GBM patients with more organized collagen had a longer median survival than those with less organized collagen. CONCLUSIONS Collagen architecture can be directly visualized and is different in focal versus invasive GBMs. The authors also demonstrate that collagen signature is associated with patient survival. These findings suggest that there are collagen differences in focal versus invasive GBMs and that collagen is a survival marker for GBM.

Low‑dose halofuginone inhibits the synthesis of type I collagen without influencing type II collagen in the extracellular matrix of chondrocytes.

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Li, Zeng; Fei, Hao; Wang, Zhen; Zhu, Tianyi

2017-09-01

Full‑thickness and large area defects of articular cartilage are unable to completely repair themselves and require surgical intervention, including microfracture, autologous or allogeneic osteochondral grafts, and autologous chondrocyte implantation. A large proportion of regenerative cartilage exists as fibrocartilage, which is unable to withstand impacts in the same way as native hyaline cartilage, owing to excess synthesis of type I collagen in the matrix. The present study demonstrated that low‑dose halofuginone (HF), a plant alkaloid isolated from Dichroa febrifuga, may inhibit the synthesis of type I collagen without influencing type II collagen in the extracellular matrix of chondrocytes. In addition, HF was revealed to inhibit the phosphorylation of mothers against decapentaplegic homolog (Smad)2/3 and promoted Smad7 expression, as well as decrease the synthesis of type I collagen synthesis. Results from the present study indicated that HF treatment suppressed the synthesis of type I collagen by inhibiting the transforming growth factor‑β signaling pathway in chondrocytes. These results may provide an alternative solution to the problems associated with fibrocartilage, and convert fibrocartilage into hyaline cartilage at the mid‑early stages of cartilage regeneration. HF may additionally be used to improve monolayer expansion or 3D cultures of seed cells for the tissue engineering of cartilage.

Comparison of thermal properties of fish collagen and bovine collagen in the temperature range 298-670K.

Science.gov (United States)

Gauza-Włodarczyk, Marlena; Kubisz, Leszek; Mielcarek, Sławomir; Włodarczyk, Dariusz

2017-11-01

The increased interest in fish collagen is a consequence of the risk of exposure to Creutzfeld-Jacob disease (CJD) and the bovine spongiform encephalopathy (BSE), whose occurrence is associated with prions carried by bovine collagen. Collagen is the main biopolymer in living organisms and the main component of the skin and bones. Until the discovery of the BSE, bovine collagen had been widely used. The BSE epidemic increased the interest in new sources of collagen such as fish skin collagen (FSC) and its properties. Although the thermal properties of collagen originating from mammals have been well described, less attention has been paid to the thermal properties of FSC. Denaturation temperature is a particularly important parameter, depending on the collagen origin and hydration level. In the reported experiment, the free water and bound water release processes along with thermal denaturation process were studied by means of the differential scanning calorimetry (DSC). Measurements were carried out using a DSC 7 instrument (Elmer-Perkin), in the temperature range 298-670K. The study material was FSC derived by acidic hydration method. The bovine Achilles tendon (BAT) collagen type I was used as the control material. The thermograms recorded revealed both, exothermic and endothermic peaks. For both materials, the peaks in the temperature range of 330-360K were assigned to the release of free water and bound water. The denaturation temperatures of FSC and BAT collagen were determined as 420K and 493K, respectively. Thermal decomposition process was observed at about 500K for FSC and at about 510K for BAT collagen. These results show that FSC is less resistant to high temperature than BAT collagen. Copyright © 2017 Elsevier B.V. All rights reserved.

Collagen Induced Arthritis in DBA/1J Mice Associates with Oxylipin Changes in Plasma

Directory of Open Access Journals (Sweden)

Min He

2015-01-01

Full Text Available Oxylipins play important roles in various biological processes and are considered as mediators of inflammation for a wide range of diseases such as rheumatoid arthritis (RA. The purpose of this research was to study differences in oxylipin levels between a widely used collagen induced arthritis (CIA mice model and healthy control (Ctrl mice. DBA/1J male mice (age: 6-7 weeks were selected and randomly divided into two groups, namely, a CIA and a Ctrl group. The CIA mice were injected intraperitoneally (i.p. with the joint cartilage component collagen type II (CII and an adjuvant injection of lipopolysaccharide (LPS. Oxylipin metabolites were extracted from plasma for each individual sample using solid phase extraction (SPE and were detected with high performance liquid chromatography/tandem mass spectrometry (HPLC-ESI-MS/MS, using dynamic multiple reaction monitoring (dMRM. Both univariate and multivariate statistical analyses were applied. The results in univariate Student’s t-test revealed 10 significantly up- or downregulated oxylipins in CIA mice, which were supplemented by another 6 additional oxylipins, contributing to group clustering upon multivariate analysis. The dysregulation of these oxylipins revealed the presence of ROS-generated oxylipins and an increase of inflammation in CIA mice. The results also suggested that the collagen induced arthritis might associate with dysregulation of apoptosis, possibly inhibited by activated NF-κB because of insufficient PPAR-γ ligands.

Structure of collagen-glycosaminoglycan matrix and the influence to its integrity and stability.

Science.gov (United States)

Bi, Yuying; Patra, Prabir; Faezipour, Miad

2014-01-01

Glycosaminoglycan (GAG) is a chain-like disaccharide that is linked to polypeptide core to connect two collagen fibrils/fibers and provide the intermolecular force in Collagen-GAG matrix (C-G matrix). Thus, the distribution of GAG in C-G matrix contributes to the integrity and mechanical properties of the matrix and related tissue. This paper analyzes the transverse isotropic distribution of GAG in C-G matrix. The angle of GAGs related to collagen fibrils is used as parameters to qualify the GAGs isotropic characteristic in both 3D and 2D rendering. Statistical results included that over one third of GAGs were perpendicular directed to collagen fibril with symmetrical distribution for both 3D matrix and 2D plane cross through collagen fibrils. The three factors tested in this paper: collagen radius, collagen distribution, and GAGs density, were not statistically significant for the strength of Collagen-GAG matrix in 3D rendering. However in 2D rendering, a significant factor found was the radius of collagen in matrix for the GAGs directed to orthogonal plane of Collagen-GAG matrix. Between two cross-section selected from Collagen-GAG matrix model, the plane cross through collagen fibrils was symmetrically distributed but the total percentage of perpendicular directed GAG was deducted by decreasing collagen radius. There were some symmetry features of GAGs angle distribution in selected 2D plane that passed through space between collagen fibrils, but most models showed multiple peaks in GAGs angle distribution. With less GAGs directed to perpendicular of collagen fibril, strength in collagen cross-section weakened. Collagen distribution was also a factor that influences GAGs angle distribution in 2D rendering. True hexagonal collagen packaging is reported in this paper to have less strength at collagen cross-section compared to quasi-hexagonal collagen arrangement. In this work focus is on GAGs matrix within the collagen and its relevance to anisotropy.

Regulation of collagen biosynthesis in cultured bovine aortic smooth muscle cells

International Nuclear Information System (INIS)

Stepp, M.A.

1986-01-01

Aortic smooth muscles cells have been implicated in the etiology of lesions which occur in atherosclerosis and hypertension. Both diseases involve proliferation of smooth muscle cells and accumulation of excessive amounts of extracellular matrix proteins, including collagen type I and type III produced by the smooth muscle cells. To better understand the sites of regulation of collagen biosynthesis and to correlate these with the growth rate of the cells, cultured bovine aortic smooth muscle cells were studied as a function of the number of days (3 to 14) in second passage. Cells grew rapidly up to day 6 when confluence was reached. The total incorporation of [ 3 H]-proline into proteins was highest at day 3 and decreased to a constant level after the cultures reached confluence. In contrast, collagen protein production was lowest before confluence and continued to increase over the entire time course of the experiments. cDNA clones for the α1 and α2 chains of type I and the α1 chain of type III collagen were used to quantitate the steady state level of collagen mRNAs. RNA was tested in a cell-free translation system. Changes in the translational activity of collagen mRNAs parallelled the observed increases in collagen protein production. Thus, at later time points, collagen mRNAs are more active in directing synthesis of preprocollagens, even though less collagen mRNA is present. The conclusion is that the site of regulation of the expression of collagen genes is a function of the growth rate of cultured smooth muscle cells

Skin collagen glycation, glycoxidation, and crosslinking are lower in subjects with long-term intensive versus conventional therapy of type 1 diabetes - Relevance of glycated collagen products versus HbA(1c) as markers of diabetic complications

NARCIS (Netherlands)

Monnier, VM; Bautista, O; Kenny, D; Sell, DR; Fogarty, J; Dahms, W; Cleary, PA; Lachin, J; Genuth, S

The relationships between long-term intensive control of glycemia and indicators of skin collagen glycation (furosine), glycoxidation (pentosidine and N-epsilon-[carboxymethyl]-lysine [CML]), and crosslinking (acid and pepsin solubility) were examined in 216 patients with type 1 diabetes from the

Collagenous colitis: histopathology and clinical course.

Science.gov (United States)

Goff, J S; Barnett, J L; Pelke, T; Appelman, H D

1997-01-01

Collagenous colitis is a chronic diarrheal disease characterized by a normal or near-normal mucosa endoscopically and microscopic inflammation in the lamina propria, surface epithelial injury and a thick subepithelial collagen layer. The symptoms of collagenous colitis vary in duration and intensity, and long periods of remission have been described, but long-term follow-up data are limited. Our goal was to determine the natural clinical history of collagenous colitis and to determine whether there was a relationship between histopathologic changes and course of disease. Cases were identified at the University of Michigan Hospitals using surgical pathology records before 1992. All charts, including medical records from other hospitals, were reviewed, and a telephone interview was conducted with each locatable patient (pt). Biopsy specimens were reviewed by two pathologists for degree of collagen layer thickness, epithelial damage, and inflammation. There were 31 patients (26 F, 5 M) with a mean age of 66 yr (range 33-83) and a mean duration of symptoms of 5.4 yr at the time of diagnosis. Of the 31 patients, 18 (56%) had some form of arthritis, and 22 (71%) were using NSAIDS regularly at the time of diagnosis. Follow-up interviews were conducted at least 2 yr after diagnosis (mean 3.5 yr, range 2-5 yr) with 27 of 31 patients (3 could not be located, 1 died). Two definable groups of patients were identified: (1) those with either spontaneous or treatment-related symptom resolution (63%), and (2) those with ongoing or intermittent symptoms requiring at least intermittent therapy (37%). There was no significant difference between the two groups with regard to sex, age, associated diseases, and use of medications. Patients with symptom resolution (mean duration 3.1 yr) had been treated with antidiarrheals (6), sulfasalazine (3), discontinuation of NSAIDS (3), reversal of jejunoilial bypass (1), or nothing (4). Those with ongoing symptoms experienced a wide range of

Preparation and characterization of collagen/hydroxypropyl methylcellulose (HPMC) blend film.

Science.gov (United States)

Ding, Cuicui; Zhang, Min; Li, Guoying

2015-03-30

This study aimed to prepare and characterize the collagen/HPMC blend film (1/1). Thermogravimetric analysis and differential scanning calorimetry were used to investigate the thermal properties of the film. Both thermal decomposition temperature and denaturation temperature of the blend film were higher than those of the collagen film due to the intermolecular hydrogen bonding interaction between collagen and HPMC, which was demonstrated by Fourier transform infrared spectroscopy. Additionally, the morphologies, mechanical properties and hydrophilicity of films were examined. The blend film exhibited a more homogeneous and compact structure compared with that of the collagen film, as observed from scanning electron microscopy and atomic force microscopy. The tensile strength, ultimate elongation and hydrophilicity of the blend film were superior to those of the pure collagen film. Furthermore, the introduction of polyethylene glycol 1500 had almost no influence on the thermal properties of the blend film but obviously improved its stretch-ability and smoothness. Copyright © 2014 Elsevier Ltd. All rights reserved.

On the role of type IX collagen in the extracellular matrix of cartilage: type IX collagen is localized to intersections of collagen fibrils

OpenAIRE

1986-01-01

The tissue distribution of type II and type IX collagen in 17-d-old chicken embryo was studied by immunofluorescence using polyclonal antibodies against type II collagen and a peptic fragment of type IX collagen (HMW), respectively. Both proteins were found only in cartilage where they were co-distributed. They occurred uniformly throughout the extracellular matrix, i.e., without distinction between pericellular, territorial, and interterritorial matrices. Tissues that undergo endochondral bo...

Endogenous collagen peptide activation of CD1d-restricted NKT cells ameliorates tissue-specific inflammation in mice.

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Liu, Yawei; Teige, Anna; Mondoc, Emma; Ibrahim, Saleh; Holmdahl, Rikard; Issazadeh-Navikas, Shohreh

2011-01-01

NKT cells in the mouse recognize antigen in the context of the MHC class I-like molecule CD1d and play an important role in peripheral tolerance and protection against autoimmune and other diseases. NKT cells are usually activated by CD1d-presented lipid antigens. However, peptide recognition in the context of CD1 has also been documented, although no self-peptide ligands have been reported to date. Here, we have identified an endogenous peptide that is presented by CD1d to activate mouse NKT cells. This peptide, the immunodominant epitope from mouse collagen type II (mCII707-721), was not associated with either MHC class I or II. Activation of CD1d-restricted mCII707-721-specific NKT cells was induced via TCR signaling and classical costimulation. In addition, mCII707-721-specific NKT cells induced T cell death through Fas/FasL, in an IL-17A-independent fashion. Moreover, mCII707-721-specific NKT cells suppressed a range of in vivo inflammatory conditions, including delayed-type hypersensitivity, antigen-induced airway inflammation, collagen-induced arthritis, and EAE, which were all ameliorated by mCII707-721 vaccination. The findings presented here offer new insight into the intrinsic roles of NKT cells in health and disease. Given the results, endogenous collagen peptide activators of NKT cells may offer promise as novel therapeutics in tissue-specific autoimmune and inflammatory diseases.

ISOCT study of collagen crosslinking of collagen in cancer models (Conference Presentation)

Science.gov (United States)

Spicer, Graham; Young, Scott T.; Yi, Ji; Shea, Lonnie D.; Backman, Vadim

2016-03-01

The role of extracellular matrix modification and signaling in cancer progression is an increasingly recognized avenue for the progression of the disease. Previous study of field effect carcinogenesis with Inverse Spectroscopic Optical Coherence Tomography (ISOCT) has revealed pronounced changes in the nanoscale-sensitive mass fractal dimension D measured from field effect tissue when compared to healthy tissue. However, the origin of this difference in tissue ultrastructure in field effect carcinogenesis has remained poorly understood. Here, we present findings supporting the idea that enzymatic crosslinking of the extracellular matrix is an effect that presents at the earliest stages of carcinogenesis. We use a model of collagen gel with crosslinking induced by lysyl oxidase (LOXL4) to recapitulate the difference in D previously reported from healthy and cancerous tissue biopsies. Furthermore, STORM imaging of this collagen gel model verifies the morphologic effects of enzymatic crosslinking at length scales as small as 40 nm, close to the previously reported lower length scale sensitivity threshold of 35 nm for ISOCT. Analysis of the autocorrelation function from STORM images of collagen gels and subsequent fitting to the Whittle-Matérn correlation function shows a similar effect of LOXL4 on D from collagen measured with ISOCT and STORM. We extend this to mass spectrometric study of tissue to directly measure concentrations of collagen crosslink residues. The validation of ISOCT as a viable tool for non-invasive rapid quantification of collagen ultrastructure lends it to study other physiological phenomena involving ECM restructuring such as atherosclerotic plaque screening or cervical ripening during pregnancy.

GH receptor blocker administration and muscle-tendon collagen synthesis in humans

DEFF Research Database (Denmark)

Nielsen, Rie Harboe; Doessing, Simon; Goto, Kazushige

2011-01-01

Collagen is the predominant structural protein in tendons and ligaments, and can be controlled by hormonal changes. In animals, injections of insulin-like growth factor I (IGF-I) has been shown to increase collagen synthesis in tendons and ligaments and to improve structural tissue healing......, but the effect of local IGF-I administration on tendon collagen synthesis in human has not been studied. The purpose of this study was to study whether local injections of IGF-I would have a stimulating effect on tendon collagen synthesis. Twelve healthy nonsmoking men [age 62 ± 1 years (mean ± SEM), BMI 27 ± 1......] participated. Two injections of either human recombinant IGF-I (0.1 mL Increlex©) or saline (control) into each patellar tendon were performed 24-h apart, respectively. Tendon collagen fractional synthesis rate (FSR) was measured by stable isotope technique in the hours after the second injection...

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

International Nuclear Information System (INIS)

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

2010-01-01

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.

Characterization of Acid Soluble Collagen from Redbelly Yellowtail Fusilier Fish Skin (Caesio cuning

Directory of Open Access Journals (Sweden)

Ika Astiana

2016-04-01

Full Text Available Fish skin can be used as raw material for producing collagen. The collagen can be extracted by chemicalor combination of chemical and enzymatic processes. Extraction of collagen chemically can do with theacid process that produces acid soluble collagen (ASC. This study aimed to determine the optimumconcentration and time of pretreatment and extraction, also to determine the characteristics of the acidsoluble collagen from the skin of yellow tail fish. Extraction of collagen done by pretreatment using NaOH atthe concentration of 0.05; 0.1; and 0.15 M and extraction using acetic acid at the concentration of 0.3; 0.5; and0.7 M. Pretreatment NaOH with concentration 0.05 M and soaking time of 8 hours is the best combinationfor eliminating non collagen protein. Combination treatment of acetic acid at the concentration of 0.3 Mfor 3 days obtained the best solubility. The yield of collagen ASC was 18.4±1.49% (db and 5.79±0.47%(wb. Amino acid composition that is dominant in the ASC collagen was glycine (25.09±0.003%, alanine(13.71±0.075%, and proline (12.15±0.132%. Collagen from yellow tail fish skin has α1, α2, β and γprotein structure with the molecular weight of 125, 113, 170-181, and 208 KDa. The transition and meltingtemperatures of collagen were 67.69oC and 144.4oC. The surface structure of collagen by analysis of SEM hasfibers on the surface.Keywords: cholesterol, fatty acids, meat tissue, proximate, red snapper (L. argentimaculatus

uPARAP/Endo180 is essential for cellular uptake of collagen and promotes fibroblast collagen adhesion

DEFF Research Database (Denmark)

Engelholm, Lars H; List, Karin; Netzel-Arnett, Sarah

2003-01-01

The uptake and lysosomal degradation of collagen by fibroblasts constitute a major pathway in the turnover of connective tissue. However, the molecular mechanisms governing this pathway are poorly understood. Here, we show that the urokinase plasminogen activator receptor-associated protein (u......, these cells had diminished initial adhesion to a range of different collagens, as well as impaired migration on fibrillar collagen. These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions....

Quantitative Characterization of Collagen in the Fibrotic Capsule Surrounding Implanted Polymeric Microparticles through Second Harmonic Generation Imaging.

Science.gov (United States)

Akilbekova, Dana; Bratlie, Kaitlin M

2015-01-01

The collagenous capsule formed around an implant will ultimately determine the nature of its in vivo fate. To provide a better understanding of how surface modifications can alter the collagen orientation and composition in the fibrotic capsule, we used second harmonic generation (SHG) microscopy to evaluate collagen organization and structure generated in mice subcutaneously injected with chemically functionalized polystyrene particles. SHG is sensitive to the orientation of a molecule, making it a powerful tool for measuring the alignment of collagen fibers. Additionally, SHG arises from the second order susceptibility of the interrogated molecule in response to the electric field. Variation in these tensor components distinguishes different molecular sources of SHG, providing collagen type specificity. Here, we demonstrated the ability of SHG to differentiate collagen type I and type III quantitatively and used this method to examine fibrous capsules of implanted polystyrene particles. Data presented in this work shows a wide range of collagen fiber orientations and collagen compositions in response to surface functionalized polystyrene particles. Dimethylamino functionalized particles were able to form a thin collagenous matrix resembling healthy skin. These findings have the potential to improve the fundamental understanding of how material properties influence collagen organization and composition quantitatively.

Collagen like peptide bioconjugates for targeted drug delivery applications

Science.gov (United States)

Luo, Tianzhi

the coil/globule conformational transition of the PDEGMEMA building block above its LCST with stabilization of the nanostructures by the hydrophilic CLP. To the best of our knowledge, this is the first report on such assembled nanostructures from collagen-like peptide containing copolymers. Due to the strong propensity for CLPs to bind to natural collagen via strand invasion processes, these nanosized vesicles may be used as drug carriers for targeted delivery. In addition to synthetic polymers, the collagen like peptide is then conjugated with a thermoresponsive elastin-like peptide (ELP). The resulting ELP-CLP diblock conjugates show a remarkable reduction in the inverse transition temperature of the ELP domain, attributed to the anchoring effect of the CLP triple helix. The lower transition temperature of the conjugate enables facile formation of well-defined vesicles at physiological temperature and the unexpected resolubilization of the vesicles at elevated temperatures upon unfolding of the CLP domain. Given the ability of CLPs to modify collagens, this work provides not only a simple and versatile avenue for controlling the inverse transition behavior of elastin-like peptides, but also suggest future opportunities for these thermoresponsive nanostructures in biologically relevant environments. In the last section, the potential of using the ELP-CLP nanoparticles as drug delivery vehicles for targeting collagen containing matrices is evaluated. A sustained release of clinically relevant amount of encapsulated modelled drug is achieved within three weeks, followed by a thermally controlled burst release. As expected, the ELP-CLP nanoparticles show strong retention on collagen substrate, via specific binding through collagen triple helix hybridization. Additionally, cell viability and proliferation studies using fibroblasts and chondrocytes suggest the nanoparticles are non-cytotoxic. Additionally, almost no TNF-alpha expression from macrophages is observed

Urotensin II contributes to collagen synthesis and up-regulates Egr-1 expression in cultured pulmonary arterial smooth muscle cells through the ERK1/2 pathway

Energy Technology Data Exchange (ETDEWEB)

Li, Wei [Biomedical Engineering Institute, School of Control Science and Engineering, Shandong University, Jinan 250061 (China); Cai, Zhifeng; Liu, Mengmeng [Department of Pediatrics, Qilu Hospital, Shandong University, Jinan 250012 (China); Zhao, Cuifen, E-mail: zhaocuifen@sdu.edu.cn [Department of Pediatrics, Qilu Hospital, Shandong University, Jinan 250012 (China); Li, Dong [Research Room of Hypothermia Medicine, Qilu Hospital, Shandong University, Jinan 250012 (China); Lv, Chenguang; Wang, Yuping; Xu, Tengfei [Biomedical Engineering Institute, School of Control Science and Engineering, Shandong University, Jinan 250061 (China)

2015-11-27

Aim: The objective of this study was to investigate the effects of urotensin II (UII) treatment on the proliferation and collagen synthesis of cultured rat pulmonary arterial smooth muscle cells (PASMCs) and to explore whether these effects are mediated by mitogen-activated protein kinase (MAPK) signaling pathways and early growth response 1 (Egr-1). Methods: The proliferation of cultured PASMCs stimulated with different doses of UII was detected by BrdU incorporation. The mRNA expression levels of procollagen I (procol I), procollagen III (procol III), extracellular regulated protein kinase 1/2 (ERK1/2), stress-stimulated protein kinase (Sapk), p38 MAPK (p38), and Egr-1 mRNA in cultured PASMCs after treatment with UII, the UII-specific antagonist urantide, and the ERK1/2 inhibitor PD98059 were detected by real-time polymerase chain reaction (PCR), and the protein expression levels of procol I, procol III, phosphorylated (p)-ERK1/2, p-Sapk, p-p38, and Egr-1 were detected by Western blotting. Results: Treatment with UII increased the proliferation of cultured PASMCs in a dose-dependent manner (P < 0.05). However, treatment with urantide and PD98059 inhibited the promoting effect of UII on PASMC proliferation (P < 0.05). Real-time PCR analysis showed that UII up-regulated the expression of procol I, procol III, ERK1/2, Sapk, and Egr-1 mRNA (P < 0.05), but not p38 mRNA. However, the up-regulating effect of UII was inhibited by PD98059 and urantide. Western blotting analysis showed that UII increased the synthesis of collagen I, collagen III, p-ERK1/2, p-Sapk, and Egr-1, and these effects also were inhibited by PD98059 and urantide (P < 0.05). Conclusions: Egr-1 participates in the UII-mediated proliferation and collagen synthesis of cultured rat PASMCs via activation of the ERK1/2 signaling pathway.

In vivo evaluation of hybrid patches composed of PLA based copolymers and collagen/chondroitin sulfate for ligament tissue regeneration.

Science.gov (United States)

Pinese, Coline; Gagnieu, Christian; Nottelet, Benjamin; Rondot-Couzin, Capucine; Hunger, Sylvie; Coudane, Jean; Garric, Xavier

2017-10-01

Biomaterials for soft tissues regeneration should exhibit sufficient mechanical strength, demonstrating a mechanical behavior similar to natural tissues and should also promote tissues ingrowth. This study was aimed at developing new hybrid patches for ligament tissue regeneration by synergistic incorporation of a knitted structure of degradable polymer fibers to provide mechanical strength and of a biomimetic matrix to help injured tissues regeneration. PLA- Pluronic ® (PLA-P) and PLA-Tetronic ® (PLA-T) new copolymers were shaped as knitted patches and were associated with collagen I (Coll) and collagen I/chondroitine-sulfate (Coll CS) 3-dimensional matrices. In vitro study using ligamentocytes showed the beneficial effects of CS on ligamentocytes proliferation. Hybrid patches were then subcutaneously implanted in rats for 4 and 12 weeks. Despite degradation, patches retained strength to answer the mechanical physiological needs. Tissue integration capacity was assessed with histological studies. We showed that copolymers, associated with collagen and chondroitin sulfate sponge, exhibited very good tissue integration and allowed neotissue synthesis after 12 weeks in vivo. To conclude, PLA-P/CollCS and PLA-T/CollCS hybrid patches in terms of structure and composition give good hopes for tendon and ligament regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1778-1788, 2017. © 2016 Wiley Periodicals, Inc.

Synergistic intrafibrillar/extrafibrillar mineralization of collagen scaffolds based on a biomimetic strategy to promote the regeneration of bone defects

Directory of Open Access Journals (Sweden)

Wang Y

2016-05-01

Full Text Available Yao Wang,1 Ngo Van Manh,1,2 Haorong Wang,1 Xue Zhong,1 Xu Zhang,1 Changyi Li1 1School of Dentistry, Hospital of Stomatology, Tianjin Medical University, Tianjin, People’s Republic of China; 2Thaibinh University of Medicine and Pharmacy, Thaibinh, Vietnam Abstract: The mineralization of collagen scaffolds can improve their mechanical properties and biocompatibility, thereby providing an appropriate microenvironment for bone regeneration. The primary purpose of the present study is to fabricate a synergistically intra- and extrafibrillar mineralized collagen scaffold, which has many advantages in terms of biocompatibility, biomechanical properties, and further osteogenic potential. In this study, mineralized collagen scaffolds were fabricated using a traditional mineralization method (ie, immersed in simulated body fluid as a control group and using a biomimetic method based on the polymer-induced liquid precursor process as an experimental group. In the polymer-induced liquid precursor process, a negatively charged polymer, carboxymethyl chitosan (CMC, was used to stabilize amorphous calcium phosphate (ACP to form nanocomplexes of CMC/ACP. Collagen scaffolds mineralized based on the polymer-induced liquid precursor process were in gel form such that nanocomplexes of CMC/ACP can easily be drawn into the interstices of the collagen fibrils. Scanning electron microscopy and transmission electron microscopy were used to examine the porous micromorphology and synergistic mineralization pattern of the collagen scaffolds. Compared with simulated body fluid, nanocomplexes of CMC/ACP significantly increased the modulus of the collagen scaffolds. The results of in vitro experiments showed that the cell count and differentiated degrees in the experimental group were higher than those in the control group. Histological staining and micro-computed tomography showed that the amount of new bone regenerated in the experimental group was larger than that in the

Prominent Vascularization Capacity of Mesenchymal Stem Cells in Collagen-Gold Nanocomposites.

Science.gov (United States)

Hsieh, Shu-Chen; Chen, Hui-Jye; Hsu, Shan-Hui; Yang, Yi-Chin; Tang, Cheng-Ming; Chu, Mei-Yun; Lin, Pei-Ying; Fu, Ru-Huei; Kung, Mei-Lang; Chen, Yun-Wen; Yeh, Bi-Wen; Hung, Huey-Shan

2016-10-26

The ideal characteristics of surface modification on the vascular graft for clinical application would be with excellent hemocompatibility, endothelialization capacity, and antirestenosis ability. Here, Fourier transform infrared spectroscopy (FTIR), surface enhanced Raman spectroscopy (SERS), atomic force microscopy (AFM), contact angle (θ) measurement, and thermogravimetric analysis (TGA) were used to evaluate the chemical and mechanical properties of collagen-gold nanocomposites (collagen+Au) with 17.4, 43.5, and 174 ppm of Au and suggested that the collagen+Au with 43.5 ppm of Au had better biomechanical properties and thermal stability than pure collagen. Besides, stromal-derived factor-1α (SDF-1α) at 50 ng/mL promoted the migration of mesenchymal stem cells (MSCs) on collagen+Au material through the α5β3 integrin/endothelial oxide synthase (eNOS)/metalloproteinase (MMP) signaling pathway which can be abolished by the knockdown of vascular endothelial growth factor (VEGF). The potentiality of collagen+Au with MSCs for vascular regeneration was evaluated by our in vivo rat model system. Artery tissues isolated from an implanted collagen+Au-coated catheter with MSCs expressed substantial CD-31 and α-SMA, displayed higher antifibrotic ability, antithrombotic activity, as well as anti-inflammatory response than all other materials. Our results indicated that the implantation of collagen+Au-coated catheters with MSCs could be a promising strategy for vascular regeneration.

In vivo determination of arterial collagen synthesis in atherosclerotic rabbits

International Nuclear Information System (INIS)

Opsahl, W.P.; DeLuca, D.J.; Ehrhart, L.A.

1986-01-01

Collagen and non-collagen protein synthesis rates were determined in vivo in tissues from rabbits fed a control or atherogenic diet supplemented with 2% peanut oil and 0.25% cholesterol for 4 months. Rabbits received a bolus intravenous injection of L-[ 3 H]-proline (1.0 mCi/kg) and unlabeled L-proline (7 mmoles/kg) in 0.9% NaCl. Plasma proline specific activity decreased only 20% over 5 hr and was similar to the specific activity of free proline in tissues. Thoracic aortas from atherosclerotic rabbits exhibited raised plaques covering at least 75% of the surface. Thoracic intima plus a portion of the media (TIM) was separated from the remaining media plus adventitia (TMA). Dry delipidated weight, total collagen content, and collagen as a percent of dry weight were increased significantly in the TIM of atherosclerotic rabbits. Collagen synthesis rates and collagen synthesis as a percent of total protein synthesis were likewise increased both in the TIM and in the abdominal aortas. No differences from controls either in collagen content or collagen synthesis rates were observed in the TMA, lung or skin. These results demonstrate for the first time in vivo that formation of atherosclerotic plaques is associated with increased rates of collagen synthesis. Furthermore, as previously observed with incubations in vitro, collagen synthesis was elevated to a greater extent than noncollagen protein synthesis in atherosclerotic aortas from rabbits fed cholesterol plus peanut oil

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, So Young [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Jang, Hwan-Hee [Functional Food and Nutrition Division, Department of Agrofood Resources, Rural Development Administration, Suwon 441-853 (Korea, Republic of); Ryu, Sung Ho [Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, Beom Joon [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Taehoon G., E-mail: taehoon@novacelltech.com [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of)

2012-11-23

Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate

A novel combined polyphenol-aldehyde crosslinking of collagen film-Applications in biomedical materials.

Science.gov (United States)

Liu, Ting; Shi, Lu; Gu, Zhipeng; Dan, Weihua; Dan, Nianhua

2017-08-01

Despite its crucial role in directing cell fate in healthy and diseased tissues, improvements in physical-chemical properties and biocompatibility of type-I collagen are still needed. In this report, we described combined and facile method to modify collagen. The collagen film was first modified by procyanidins solution, in which, then subjected to further crosslinked by dialdehyde alginate, resulting in collagen-procyanidins-dialdehyde alginate film. The properties of the crosslinked collagen films were investigated and the results were discussed. Results from differential scanning calorimetry and thermo gravimetric analysis suggested that the thermal stabilities of the collagen-procyanidins-dialdehyde alginate film were significantly improved. The mechanical properties of collagen-procyanidins-dialdehyde alginate film in terms of elongation at break and tensile strength increased approximately 2-fold and 3-fold, respectively compare to pure collagen film. In addition, the resistance to collagenase degradation of collagen-procyanidins-dialdehyde alginate film was remarkably promoted. The results from methyltetrazolium assay and confocal laser scanning microscopy showed that no cytotoxicity of collagen film was introduced by the combined crosslinking method. Thus, the novel combined by procyanidins-dialdehyde alginate crosslinking method shown in this study provided a non-toxic and efficient crosslinking method that improved various properties of collagen film, which has great potential applications in biomedical materials. Copyright © 2017 Elsevier B.V. All rights reserved.

Electrochemical deposition of mineralized BSA/collagen coating

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Zhuang, Junjun [School of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Zhejiang University, Hangzhou 310027 (China); Lin, Jun; Li, Juan; Wang, Huiming [The First Affiliated Hospital of Medical College, Zhejiang University, Hangzhou 310003 (China); Cheng, Kui [School of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Zhejiang University, Hangzhou 310027 (China); Weng, Wenjian, E-mail: wengwj@zju.edu.cn [School of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Zhejiang University, Hangzhou 310027 (China); The Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050 (China)

2016-09-01

In this work, mineralized collagen coatings with different loading quantity of bovine serum albumin (BSA) were prepared via in situ electrochemical deposition on titanium substrate. The microstructure and BSA loading quantity of the coatings could be controlled by the electrochemical deposition parameters, such as deposition potential, BSA concentration and its adding sequence in the electrolyte. The BSA loading quantity in the coatings was obtained in the range of 0.0170–0.173 mg/cm{sup 2}, enhancing the cell adhesion and proliferation of the coatings with the simultaneous release. The distinct release behaviors of BSA were attributed to their gradient distribution with different mineralization degrees, which could be adjusted by the deposition process. These results suggest that in situ electrochemical deposition is a promising way to incorporate functional molecules into the mineralized collagen coatings and the mineralized BSA/collagen coatings are highly promising for improving the rhBMP-2 loading capability (1.8-fold). - Highlights: • BSA is incorporated into mineralized collagen coating by electrochemical deposition. • The loading amount of BSA in coatings can be adjusted in the range of 0-173 ng. • The BSA/collagen coating shows good cytocompatibility with free-albumin culture. • The incorporation process is put forward for some other molecules deposition.

Double thermal transitions of type I collagen in acidic solution.

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Liu, Yan; Liu, Lingrong; Chen, Mingmao; Zhang, Qiqing

2013-01-01

Contributed equally to this work. To further understand the origin of the double thermal transitions of collagen in acidic solution induced by heating, the denaturation of acidic soluble collagen was investigated by micro-differential scanning calorimeter (micro-DSC), circular dichroism (CD), dynamic laser light scattering (DLLS), transmission electron microscopy (TEM), and two-dimensional (2D) synchronous fluorescence spectrum. Micro-DSC experiments revealed that the collagen exhibited double thermal transitions, which were located within 31-37 °C (minor thermal transition, T(s) ∼ 33 °C) and 37-55 °C (major thermal transition, T(m) ∼ 40 °C), respectively. The CD spectra suggested that the thermal denaturation of collagen resulted in transition from polyproline II type structure to unordered structure. The DLLS results showed that there were mainly two kinds of collagen fibrillar aggregates with different sizes in acidic solution and the larger fibrillar aggregates (T(p2) = 40 °C) had better heat resistance than the smaller one (T(p1) = 33 °C). TEM revealed that the depolymerization of collagen fibrils occurred and the periodic cross-striations of collagen gradually disappeared with increasing temperature. The 2D fluorescence correlation spectra were also applied to investigate the thermal responses of tyrosine and phenylalanine residues at the molecular level. Finally, we could draw the conclusion that (1) the minor thermal transition was mainly due to the defibrillation of the smaller collagen fibrillar aggregates and the unfolding of a little part of triple helices; (2) the major thermal transition primarily arose from the defibrillation of the larger collagen fibrillar aggregates and the complete denaturation of the majority part of triple helices.

Effect of starch on the mechanical and in vitro properties of collagen-hydroxyapatite sponges for applications in dentistry.

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Castro-Ceseña, Ana B; Camacho-Villegas, Tanya A; Lugo-Fabres, Pavel H; Novitskaya, Ekaterina E; McKittrick, Joanna; Licea-Navarro, Alexei

2016-09-05

This study sought to improve the mechanical and blood-absorbing properties of collagen sponges, while keeping them compressible, by incorporating blended hydroxyapatite (HA)-starch. Results were compared with CollaPlug(®) (pure collagen). The elastic modulus increased from 1.5±0.2kPa for CollaPlug(®) to 49±8kPa for sponges with composition 1:4:10 (collagen:HA:starch, by weight). The modified microstructure and surface area provided by the starch granules on the sponges improved cell viability. Sponges with composition 1:4:10 maintained their blood-clotting capability with almost no change from 5 to 15min after contact with blood, while CollaPlug(®) diminished to about half its capacity to absorb blood and form clots. Incorporation of HA-starch into the sponges with composition of 1:4:10, increased the elastic modulus of the collagen-HA sponges, making them more structurally robust. The viability of cells and the blood-clotting capability increased with starch incorporation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Complete Histological Resolution of Collagenous Sprue

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Hugh J Freeman

2004-01-01

Full Text Available A 65-year-old woman developed a watery diarrhea syndrome with collagenous colitis. Later, weight loss and hypoalbuminemia were documented. This prompted small bowel biopsies that showed pathological changes of collagenous sprue. An apparent treatment response to a gluten-free diet and prednisone resulted in reduced diarrhea, weight gain and normalization of serum albumin. Later repeated biopsies from multiple small and large bowel sites over a period of over three years, however, showed reversion to normal small intestinal mucosa but persistent collagenous colitis. These results indicate that collagenous inflammatory disease may be a far more extensive process in the gastrointestinal tract than is currently appreciated. Moreover, collagenous colitis may be a clinical signal that occult small intestinal disease is present. Finally, collagenous sprue may, in some instances, be a completely reversible small intestinal disorder.

Effect of photoactivated riboflavin on the biodegradation-resistance of root-dentin collagen.

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Priyadarshini, Balasankar Meera; Lu, Thong Beng; Fawzy, Amr S

2017-12-01

This study was conducted to evaluate the effect of UVA-activated 1% riboflavin solution on structural integrity; mechanical properties and stability; and collagenase-mediated collagen solubilisation resistance of demineralized root dentin collagen matrix. Root dentin specimens demineralized with 17% EDTA for 7days were treated with 1% RF for 1min followed by UVA photo-activation at intensity 7mW/cm 2 for 1min. Control specimens were completely devoid of riboflavin and/or UVA treatments. Specimens were challenged with bacterial collagenase type-I solution for different time-periods at 37°C. Collagen solubilisation resistance was evaluated in terms of hydroxyproline (HYP) liberation. Mechanical characterization of dentin specimens before and after 24h of exposure to collagenase solution was done in terms of apparent-elastic modulus (E appr ) and ultimate tensile strength (UTS). Variations in dentin collagen-network structure with exposure time in collagenase were visualized by TEM. Crosslinking dentin with UVA-activated riboflavin significantly decreased HYP release and increased E appr and UTS compared to control specimens with storage time in collagenase. Moreover, crosslinked specimens showed higher structural resistance to collagenase effect reflected from dense, well-formed collagen fibrils-network with characteristic collagen cross-banding. UVA-activated riboflavin treatment increased collagenase-mediated collagen degradation resistance and enhanced mechanical stability against collagenase challenges of root dentin after EDTA demineralization. Copyright © 2017 Elsevier B.V. All rights reserved.

Increasing extracellular matrix collagen level and MMP activity induces cyst development in polycystic kidney disease.

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Liu, Bin; Li, Chenghai; Liu, Zijuan; Dai, Zonghan; Tao, Yunxia

2012-09-11

Polycystic Kidney Disease (PKD) kidneys exhibit increased extracellular matrix (ECM) collagen expression and metalloproteinases (MMPs) activity. We investigated the role of these increases on cystic disease progression in PKD kidneys. We examined the role of type I collagen (collagen I) and membrane bound type 1 MMP (MT1-MMP) on cyst development using both in vitro 3 dimensional (3D) collagen gel culture and in vivo PCK rat model of PKD. We found that collagen concentration is critical in controlling the morphogenesis of MDCK cells cultured in 3D gels. MDCK cells did not form 3D structures at collagen I concentrations lower than 1 mg/ml but began forming tubules when the concentration reaches 1 mg/ml. Significantly, these cells began to form cyst when collagen I concentration reached to 1.2 mg/ml, and the ratios of cyst to tubule structures increased as the collagen I concentration increased. These cells exclusively formed cyst structures at a collagen I concentration of 1.8 mg/ml or higher. Overexpression of MT1-MMP in MDCK cells significantly induced cyst growth in 3D collagen gel culture. Conversely, inhibition of MMPs activity with doxycycline, a FDA approved pan-MMPs inhibitor, dramatically slowed cyst growth. More importantly, the treatment of PCK rats with doxycycline significantly decreased renal tubule cell proliferation and markedly inhibited the cystic disease progression. Our data suggest that increased collagen expression and MMP activity in PKD kidneys may induce cyst formation and expansion. Our findings also suggest that MMPs may serve as a therapeutic target for the treatment of human PKD.

Increasing extracellular matrix collagen level and MMP activity induces cyst development in polycystic kidney disease

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Liu Bin

2012-09-01

Full Text Available Abstract Background Polycystic Kidney Disease (PKD kidneys exhibit increased extracellular matrix (ECM collagen expression and metalloproteinases (MMPs activity. We investigated the role of these increases on cystic disease progression in PKD kidneys. Methods We examined the role of type I collagen (collagen I and membrane bound type 1 MMP (MT1-MMP on cyst development using both in vitro 3 dimensional (3D collagen gel culture and in vivo PCK rat model of PKD. Results We found that collagen concentration is critical in controlling the morphogenesis of MDCK cells cultured in 3D gels. MDCK cells did not form 3D structures at collagen I concentrations lower than 1 mg/ml but began forming tubules when the concentration reaches 1 mg/ml. Significantly, these cells began to form cyst when collagen I concentration reached to 1.2 mg/ml, and the ratios of cyst to tubule structures increased as the collagen I concentration increased. These cells exclusively formed cyst structures at a collagen I concentration of 1.8 mg/ml or higher. Overexpression of MT1-MMP in MDCK cells significantly induced cyst growth in 3D collagen gel culture. Conversely, inhibition of MMPs activity with doxycycline, a FDA approved pan-MMPs inhibitor, dramatically slowed cyst growth. More importantly, the treatment of PCK rats with doxycycline significantly decreased renal tubule cell proliferation and markedly inhibited the cystic disease progression. Conclusions Our data suggest that increased collagen expression and MMP activity in PKD kidneys may induce cyst formation and expansion. Our findings also suggest that MMPs may serve as a therapeutic target for the treatment of human PKD.

Protective Effect of Pyruvate Against Radiation-Induced Damage in Collagenized Tissues

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Griko, Y. V.; Yan, Xiaoli

2016-01-01

Exposure to high doses of ionizing radiation produces both acute and late effects on the collagenized tissues and have profound effects on wound healing. Because of the crucial practical importance for new radioprotective agents, our study has been focused on evaluation of the efficacy of non-toxic naturally occurring compounds to protect tissue integrity against high-dose gamma radiation. Here, we demonstrate that molecular integrity of collagen may serve as a sensitive biological marker for quantitative evaluation of molecular damage to collagenized tissue and efficacy of radioprotective agents. Increasing doses of gamma radiation (0-50kGy) result in progressive destruction of the native collagen fibrils, which provide a structural framework, strength, and proper milieu for the regenerating tissue. The strategy used in this study involved the thermodynamic specification of all structural changes in collagenized matrix of skin, aortic heart valve, and bone tissue induced by different doses and conditions of g-irradiation. This study describes a simple biophysical approach utilizing the Differential Scanning Calorimetry (DSC) to characterize the structural resistance of the aortic valve matrix exposed to different doses of g-irradiation. It allows us to identify the specific response of each constituent as well as to determine the influence of the different treatments on the characteristic parameters of protein structure. We found that pyruvate, a substance that naturally occurs in the body, provide significant protection (up to 80%) from biochemical and biomechanical damage to the collagenized tissue through the effective targeting of reactive oxygen species. The recently discovered role of pyruvate in the cell antioxidant defense to O2 oxidation, and its essential constituency in the daily human diet, indicate that the administration of pyruvate-based radioprotective formulations may provide safe and effective protection from deleterious effects of ionizing

Cartilage collagen damage in hip osteoarthritis similar to that seen in knee osteoarthritis; a case–control study of relationship between collagen, glycosaminoglycan and cartilage swelling

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Hosseininia Shahrzad

2013-01-01

Full Text Available Abstract Background It remains to be shown whether OA shares molecular similarities between different joints in humans. This study provides evidence for similarities in cartilage molecular damage in osteoarthritic (OA joints. Methods Articular cartilage from osteoarthritic hip joints were analysed and compared to non-OA controls regarding collagen, glycosaminoglycan and water content. Femoral heads from 16 osteoarthritic (OA and 20 reference patients were obtained from hip replacement surgery due to OA and femoral neck fracture, respectively. Cartilage histological changes were assessed by Mankin grading and denatured collagen type II immunostaining and cartilage was extracted by α-chymotrypsin. Hydroxyproline and Alcian blue binding assays were used to measure collagen and glycosaminoglycan (GAG content, respectively. Results Mankin and immunohistology scores were significantly higher in hip OA samples than in reference samples. Cartilage water content was 6% higher in OA samples than in references. 2.5 times more collagen was extracted from OA than from reference samples. There was a positive association between water content and percentage of extractable collagen pool (ECP in both groups. The amounts of collagen per wet and dry weights did not differ statistically between OA and reference cartilage. % Extractable collagen was not related to collagen per dry weight in either group. However when collagen was expressed by wet weight there was a negative correlation between % extractable and collagen in OA cartilage. The amount of GAG per wet weight was similar in both groups but the amount of GAG per dry weight was higher in OA samples compared to reference samples, which suggests a capacity for GAG biosynthesis in hip OA cartilage. Neither of the studied parameters was related to age in either group. Conclusions Increased collagen extractability and water content in human hip cartilage is associated with OA pathology and can be observed at

Computational study of packing a collagen-like molecule: quasi-hexagonal vs "Smith" collagen microfibril model.

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Lee, J; Scheraga, H A; Rackovsky, S

1996-01-01

The lateral packing of a collagen-like molecule, CH3CO-(Gly-L-Pro-L-Pro)4-NHCH3, has been examined by energy minimization with the ECEPP/3 force field. Two current packing models, the Smith collagen microfibril twisted equilateral pentagonal model and the quasi-hexagonal packing model, have been extensively investigated. In treating the Smith microfibril model, energy minimization was carried out on various conformations including those with the symmetry of equivalent packing, i.e., in which the triple helices were arranged equivalently with respect to each other. Both models are based on the experimental observation of the characteristic axial periodicity, D = 67 nm, of light and dark bands, indicating that, if any superstructure exists, it should consist of five triple helices. The quasi-hexagonal packing structure is found to be energetically more favorable than the Smith microfibril model by as much as 31.2 kcal/mol of five triple helices. This is because the quasi-hexagonal packing geometry provides more nonbonded interaction possibilities between triple helices than does the Smith microfibril geometry. Our results are consistent with recent x-ray studies with synthetic collagen-like molecules and rat tail tendon, in which the data were interpreted as being consistent with either a quasi-hexagonal or a square-triangular structure.

SHP-1, a novel peptide isolated from seahorse inhibits collagen release through the suppression of collagenases 1 and 3, nitric oxide products regulated by NF-kappaB/p38 kinase.

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Ryu, BoMi; Qian, Zhong-Ji; Kim, Se-Kwon

2010-01-01

Considerable efforts have been taken to identify natural peptides as potential bioactive substances. In this study, novel peptide (SHP-1) derived from seahorse (Hippocampus, Syngnathidae) hydrolysate was explored for its inhibitory effects on collagen release in arthritis with the investigation of its underlying mechanism of action. The efficacy of SHP-1 was determined on cartilage protective effects such as inhibition of collagen and GAG release. SHP-1 was able to suppress not only the expression of collagenases 1 and 3, but also the production of NO via down-regulation of iNOS. However, it presented an irrelevant effect on the level of GAG release in chondrocytic and osteoblastic cells. Inhibition of collagen release by SHP-1 is associated with restraining the phosphorylation of NF-kappaB and p38 kinase cascade. Therefore, it could be suggested that SHP-1 has a potential to be used in arthritis treatment.

Susceptibility tensor imaging and tractography of collagen fibrils in the articular cartilage.

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Wei, Hongjiang; Gibbs, Eric; Zhao, Peida; Wang, Nian; Cofer, Gary P; Zhang, Yuyao; Johnson, G Allan; Liu, Chunlei

2017-11-01

To investigate the B 0 orientation-dependent magnetic susceptibility of collagen fibrils within the articular cartilage and to determine whether susceptibility tensor imaging (STI) can detect the 3D collagen network within cartilage. Multiecho gradient echo datasets (100-μm isotropic resolution) were acquired from fixed porcine articular cartilage specimens at 9.4 T. The susceptibility tensor was calculated using phase images acquired at 12 or 15 different orientations relative to B 0 . The susceptibility anisotropy of the collagen fibril was quantified and diffusion tensor imaging (DTI) was compared against STI. 3D tractography was performed to visualize and track the collagen fibrils with DTI and STI. STI experiments showed the distinct and significant anisotropic magnetic susceptibility of collagen fibrils within the articular cartilage. STI can be used to measure and quantify susceptibility anisotropy maps. Furthermore, STI provides orientation information of the underlying collagen network via 3D tractography. The findings of this study demonstrate that STI can characterize the orientation variation of collagen fibrils where diffusion anisotropy fails. We believe that STI could serve as a sensitive and noninvasive marker to study the collagen fibrils microstructure. Magn Reson Med 78:1683-1690, 2017. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

Dwarfism in Mice Lacking Collagen-binding Integrins α2β1 and α11β1 Is Caused by Severely Diminished IGF-1 Levels*

Science.gov (United States)

Blumbach, Katrin; Niehoff, Anja; Belgardt, Bengt F.; Ehlen, Harald W. A.; Schmitz, Markus; Hallinger, Ralf; Schulz, Jan-Niklas; Brüning, Jens C.; Krieg, Thomas; Schubert, Markus; Gullberg, Donald; Eckes, Beate

2012-01-01

Mice with a combined deficiency in the α2β1 and α11β1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggesting a systemic cause for the overall size reduction. In accordance with a critical role of insulin-like growth factor (IGF)-1 in growth control and bone mineralization, circulating IGF-1 levels in the sera of mice lacking either α2β1 or α11β1 or both integrins were sharply reduced by 39%, 64%, or 81% of normal levels, respectively. Low hepatic IGF-1 production resulted from diminished growth hormone-releasing hormone expression in the hypothalamus and, subsequently, reduced growth hormone expression in the pituitary glands of these mice. These findings point out a novel role of collagen-binding integrin receptors in the control of growth hormone/IGF-1-dependent biological activities. Thus, coupling hormone secretion to extracellular matrix signaling via integrins represents a novel concept in the control of endocrine homeostasis. PMID:22210772

Dwarfism in mice lacking collagen-binding integrins α2β1 and α11β1 is caused by severely diminished IGF-1 levels.

Science.gov (United States)

Blumbach, Katrin; Niehoff, Anja; Belgardt, Bengt F; Ehlen, Harald W A; Schmitz, Markus; Hallinger, Ralf; Schulz, Jan-Niklas; Brüning, Jens C; Krieg, Thomas; Schubert, Markus; Gullberg, Donald; Eckes, Beate

2012-02-24

Mice with a combined deficiency in the α2β1 and α11β1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggesting a systemic cause for the overall size reduction. In accordance with a critical role of insulin-like growth factor (IGF)-1 in growth control and bone mineralization, circulating IGF-1 levels in the sera of mice lacking either α2β1 or α11β1 or both integrins were sharply reduced by 39%, 64%, or 81% of normal levels, respectively. Low hepatic IGF-1 production resulted from diminished growth hormone-releasing hormone expression in the hypothalamus and, subsequently, reduced growth hormone expression in the pituitary glands of these mice. These findings point out a novel role of collagen-binding integrin receptors in the control of growth hormone/IGF-1-dependent biological activities. Thus, coupling hormone secretion to extracellular matrix signaling via integrins represents a novel concept in the control of endocrine homeostasis.

Neuroprotective effects of collagen matrix in rats after traumatic brain injury.

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Shin, Samuel S; Grandhi, Ramesh; Henchir, Jeremy; Yan, Hong Q; Badylak, Stephen F; Dixon, C Edward

2015-01-01

In previous studies, collagen based matrices have been implanted into the site of lesion in different models of brain injury. We hypothesized that semisynthetic collagen matrix can have neuroprotective function in the setting of traumatic brain injury. Rats were subjected to sham injury or controlled cortical impact. They either received extracellular matrix graft (DuraGen) over the injury site or did not receive any graft and underwent beam balance/beam walking test at post injury days 1-5 and Morris water maze at post injury days 14-18. Animals were sacrificed at day 18 for tissue analysis. Collagen matrix implantation in injured rats did not affect motor function (beam balance test: p = 0.627, beam walking test: p = 0.921). However, injured group with collagen matrix had significantly better spatial memory acquisition (p < 0.05). There was a significant reduction in lesion volume, as well as neuronal loss in CA1 (p < 0.001) and CA3 (p < 0.05) regions of the hippocampus in injured group with collagen matrix (p < 0.05). Collagen matrix reduces contusional lesion volume, neuronal loss, and cognitive deficit after traumatic brain injury. Further studies are needed to demonstrate the mechanisms of neuroprotection by collagen matrix.

Collagen crosslinks in chondromalacia of the patella.

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Väätäinen, U; Kiviranta, I; Jaroma, H; Arokosi, J; Tammi, M; Kovanen, V

1998-02-01

The aim of the study was to determine collagen concentration and collagen crosslinks in cartilage samples from chondromalacia of the patella. To study the extracellular matrix alterations associated to chondromalacia, we determined the concentration of collagen (hydroxyproline) and its hydroxylysylpyridinoline and lysylpyridinoline crosslinks from chondromalacia foci of the patellae in 12 patients and 7 controls from apparently normal cadavers. The structure of the collagen network in 8 samples of grades II-IV chondromalacia was examined under polarized light microscopy. The full-thickness cartilage samples taken with a surgical knife from chondromalacia lesions did not show changes in collagen, hydroxylysylpyridinoline and lysylpyridinoline concentration as compared with the controls. Polarized light microscopy showed decreased birefringence in the superficial cartilage of chondromalacia lesions, indicating disorganization or disappearance of collagen fibers in this zone. It is concluded that the collagen network shows gradual disorganization with the severity of chondromalacia lesion of the patella without changes in the concentration or crosslinks of collagen.

Collagen triple helix repeat containing 1 is a new promigratory marker of arthritic pannus.

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Shekhani, Mohammed Talha; Forde, Toni S; Adilbayeva, Altynai; Ramez, Mohamed; Myngbay, Askhat; Bexeitov, Yergali; Lindner, Volkhard; Adarichev, Vyacheslav A

2016-07-19

The formation of destructive hypercellular pannus is critical to joint damage in rheumatoid arthritis (RA). The collagen triple helix repeat containing 1 (CTHRC1) protein expressed by activated stromal cells of diverse origin has previously been implicated in tissue remodeling and carcinogenesis. We recently discovered that the synovial Cthrc1 mRNA directly correlates with arthritis severity in mice. This study characterizes the role of CTHRC1 in arthritic pannus formation. Synovial joints of mice with collagen antibody-induced arthritis (CAIA) and human RA-fibroblast-like synoviocytes (FLS) were immunostained for CTHRC1, FLS and macrophage-specific markers. CTHRC1 levels in plasma from patients with RA were measured using sandwich ELISA. The migratory response of fibroblasts was studied with a transwell migration assay and time-lapse microscopy. Velocity and directness of cell migration was analyzed by recording the trajectories of cells treated with rhCTHRC1. Immunohistochemical analysis of normal and inflamed synovium revealed highly inducible expression of CTHRC1 in arthritis (10.9-fold). At the tissue level, CTHRC1-expressing cells occupied the same niche as large fibroblast-like cells positive for α-smooth muscle actin (α-SMA) and cadherin 11 (CDH11). CTHRC1 was produced by activated FLS predominantly located at the synovial intimal lining and at the bone-pannus interface. Cultured RA-FLS expressed CDH11, α-SMA, and CTHRC1. Upon treatment with exogenous rhCTHRC1, embryonic fibroblasts and RA-FLS significantly increased migration velocity, directness, and cell length along the front-tail axis (1.4-fold, p pannus.

Bioinspired coupled helical coils for soft tissue engineering of tubular structures - Improved mechanical behavior of tubular collagen type I templates.

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Janke, H P; Bohlin, J; Lomme, R M L M; Mihaila, S M; Hilborn, J; Feitz, W F J; Oosterwijk, E

2017-09-01

The design of constructs for tubular tissue engineering is challenging. Most biomaterials need to be reinforced with supporting structures such as knittings, meshes or electrospun material to comply with the mechanical demands of native tissues. In this study, coupled helical coils (CHCs) were manufactured to mimic collagen fiber orientation as found in nature. Monofilaments of different commercially available biodegradable polymers were wound and subsequently fused, resulting in right-handed and left-handed polymer helices fused together in joints where the filaments cross. CHCs of different polymer composition were tested to determine the tensile strength, strain recovery, hysteresis, compressive strength and degradation of CHCs of different composition. Subsequently, seamless and stable hybrid constructs consisting of PDSII® USP 2-0 CHCs embedded in porous collagen type I were produced. Compared to collagen alone, this hybrid showed superior strain recovery (93.5±0.9% vs 71.1±12.6% in longitudinal direction; 87.1±6.6% vs 57.2±4.6% in circumferential direction) and hysteresis (18.9±2.7% vs 51.1±12.0% in longitudinal direction; 11.5±4.6% vs 46.3±6.3% in circumferential direction). Furthermore, this hybrid construct showed an improved Young's modulus in both longitudinal (0.5±0.1MPavs 0.2±0.1MPa; 2.5-fold) and circumferential (1.65±0.07MPavs (2.9±0.3)×10 -2 MPa; 57-fold) direction, respectively, compared to templates created from collagen alone. Moreover, hybrid template characteristics could be modified by changing the CHC composition and CHCs were produced showing a mechanical behavior similar to the native ureter. CHC-enforced templates, which are easily tunable to meet different demands may be promising for tubular tissue engineering. Most tubular constructs lack sufficient strength and tunability to comply with the mechanical demands of native tissues. Therefore, we embedded coupled helical coils (CHCs) produced from biodegradable polymers - to

Slow Muscle Precursors Lay Down a Collagen XV Matrix Fingerprint to Guide Motor Axon Navigation.

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Guillon, Emilie; Bretaud, Sandrine; Ruggiero, Florence

2016-03-02

The extracellular matrix (ECM) provides local positional information to guide motoneuron axons toward their muscle target. Collagen XV is a basement membrane component mainly expressed in skeletal muscle. We have identified two zebrafish paralogs of the human COL15A1 gene, col15a1a and col15a1b, which display distinct expression patterns. Here we show that col15a1b is expressed and deposited in the motor path ECM by slow muscle precursors also called adaxial cells. We further demonstrate that collagen XV-B deposition is both temporally and spatially regulated before motor axon extension from the spinal cord in such a way that it remains in this region after the adaxial cells have migrated toward the periphery of the myotome. Loss- and gain-of-function experiments in zebrafish embryos demonstrate that col15a1b expression and subsequent collagen XV-B deposition and organization in the motor path ECM depend on a previously undescribed two-step mechanism involving Hedgehog/Gli and unplugged/MuSK signaling pathways. In silico analysis predicts a putative Gli binding site in the col15a1b proximal promoter. Using col15a1b promoter-reporter constructs, we demonstrate that col15a1b participates in the slow muscle genetic program as a direct target of Hedgehog/Gli signaling. Loss and gain of col15a1b function provoke pathfinding errors in primary and secondary motoneuron axons both at and beyond the choice point where axon pathway selection takes place. These defects result in muscle atrophy and compromised swimming behavior, a phenotype partially rescued by injection of a smyhc1:col15a1b construct. These reveal an unexpected and novel role for collagen XV in motor axon pathfinding and neuromuscular development. In addition to the archetypal axon guidance cues, the extracellular matrix provides local information that guides motor axons from the spinal cord to their muscle targets. Many of the proteins involved are unknown. Using the zebrafish model, we identified an

Quantitative Characterization of Collagen in the Fibrotic Capsule Surrounding Implanted Polymeric Microparticles through Second Harmonic Generation Imaging.

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Dana Akilbekova

Full Text Available The collagenous capsule formed around an implant will ultimately determine the nature of its in vivo fate. To provide a better understanding of how surface modifications can alter the collagen orientation and composition in the fibrotic capsule, we used second harmonic generation (SHG microscopy to evaluate collagen organization and structure generated in mice subcutaneously injected with chemically functionalized polystyrene particles. SHG is sensitive to the orientation of a molecule, making it a powerful tool for measuring the alignment of collagen fibers. Additionally, SHG arises from the second order susceptibility of the interrogated molecule in response to the electric field. Variation in these tensor components distinguishes different molecular sources of SHG, providing collagen type specificity. Here, we demonstrated the ability of SHG to differentiate collagen type I and type III quantitatively and used this method to examine fibrous capsules of implanted polystyrene particles. Data presented in this work shows a wide range of collagen fiber orientations and collagen compositions in response to surface functionalized polystyrene particles. Dimethylamino functionalized particles were able to form a thin collagenous matrix resembling healthy skin. These findings have the potential to improve the fundamental understanding of how material properties influence collagen organization and composition quantitatively.

Collagens - structure, function and biosynthesis.

OpenAIRE

Gelse, K; Poschl, E; Aigner, T

2003-01-01

The extracellular matrix represents a complex alloy of variable members of diverse protein families defining structural integrity and various physiological functions. The most abundant family is the collagens with more than 20 different collagen types identified so far. Collagens are centrally involved in the formation of fibrillar and microfibrillar networks of the extracellular matrix, basement membranes as well as other structures of the extracellular matrix. This review focuses on the dis...

Biomimetic soluble collagen purified from bones.

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Ferreira, Ana Marina; Gentile, Piergiorgio; Sartori, Susanna; Pagliano, Cristina; Cabrele, Chiara; Chiono, Valeria; Ciardelli, Gianluca

2012-11-01

Type I collagen has been extensively exploited as a biomaterial for biomedical applications and drug delivery; however, small molecular alterations occurring during the isolation procedure and its interaction with residual bone extracellular matrix molecules or proteins might affect the overall material biocompatibility and performance. The aim of the current work is to study the potential alterations in collagen properties and organization associated with the absence of proteoglycans, which mimic pathological conditions associated with age-related diseases. A new approach for evaluating the effect of proteoglycans on the properties of isolated type I collagen from the bone matrix is described. Additional treatment with guanidine hydrochloride was introduced to remove residual proteoglycans from the collagen matrix. The properties of the isolated collagen with/without guanidine hydrochloride treatment were investigated and compared with a commercial rabbit collagen as control. We demonstrate that the absence of proteoglycans in the isolated type I collagen affects its thermal properties, the extraction into its native structure, and its ability to hydrate and self-assemble into fibers. The fine control and tuning of all these features, linked to the absence of non-collagenous proteins as proteoglycans, offer the possibility of designing new strategies and biomaterials with advanced biomimetic properties aimed at regenerating bone tissue in the case of fragility and/or defects. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The spatial-temporal characteristics of type I collagen-based extracellular matrix.

Science.gov (United States)

Jones, Christopher Allen Rucksack; Liang, Long; Lin, Daniel; Jiao, Yang; Sun, Bo

2014-11-28

Type I collagen abounds in mammalian extracellular matrix (ECM) and is crucial to many biophysical processes. While previous studies have mostly focused on bulk averaged properties, here we provide a comprehensive and quantitative spatial-temporal characterization of the microstructure of type I collagen-based ECM as the gelation temperature varies. The structural characteristics including the density and nematic correlation functions are obtained by analyzing confocal images of collagen gels prepared at a wide range of gelation temperatures (from 16 °C to 36 °C). As temperature increases, the gel microstructure varies from a "bundled" network with strong orientational correlation between the fibers to an isotropic homogeneous network with no significant orientational correlation, as manifested by the decaying of length scales in the correlation functions. We develop a kinetic Monte-Carlo collagen growth model to better understand how ECM microstructure depends on various environmental or kinetic factors. We show that the nucleation rate, growth rate, and an effective hydrodynamic alignment of collagen fibers fully determines the spatiotemporal fluctuations of the density and orientational order of collagen gel microstructure. Also the temperature dependence of the growth rate and nucleation rate follow the prediction of classical nucleation theory.

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.

Science.gov (United States)

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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

DEFF Research Database (Denmark)

Nieswandt, B; Brakebusch, C; Bergmeier, W

2001-01-01

Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subs...

Dense tissue-like collagen matrices formed in cell-free conditions.

Science.gov (United States)

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.

Therapeutic effect of dioscin on collagen-induced arthritis through reduction of Th1/Th2.

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Guo, Yachun; Xing, Enhong; Song, Hongru; Feng, Guiying; Liang, Xiujun; An, Gao; Zhao, Xiaofei; Wang, Mi

2016-10-01

The aim of this study was to detect the therapeutic effect of dioscin on collagen-induced arthritis (CIA). Mice model of CIA was induced by chicken collagen II and arthritis index was assessed. After suspension of dioscin (100mg/kg/d) or triptolide was intragastrically administered, the left paw swelling and body weight of each mouse were measured. Then tissue samples were assayed by histopathological analysis. The levels of Th1 and Th2 were detected by flow cytometry. The expression of p-STAT1, p-STAT4 and p-STAT6 was demonstrated by western blot analysis, and T-bet and GATA-3 expression was detected by RT-PCR. The paw swelling and arthritis index were decreased and body weight was increased in the high dose of dioscin group compared to the model group (PTh1/Th2 in the dioscin group (0.82±0.24) and triptolide group (0.99±0.44) was lower than that in the model group (1.84±0.70, PTh1/Th2 cells, which could reduce the expression of p-STAT4, increase the expression of p-STAT6 and GATA3 in the synovial tissue. Copyright © 2016 Elsevier B.V. All rights reserved.

High-resolution study of the 3D collagen fibrillary matrix of Achilles tendons without tissue labelling and dehydrating.

Science.gov (United States)

Wu, Jian-Ping; Swift, Benjamin John; Becker, Thomas; Squelch, Andrew; Wang, Allan; Zheng, Yong-Chang; Zhao, Xuelin; Xu, Jiake; Xue, Wei; Zheng, Minghao; Lloyd, David; Kirk, Thomas Brett

2017-06-01

Knowledge of the collagen structure of an Achilles tendon is critical to comprehend the physiology, biomechanics, homeostasis and remodelling of the tissue. Despite intensive studies, there are still uncertainties regarding the microstructure. The majority of studies have examined the longitudinally arranged collagen fibrils as they are primarily attributed to the principal tensile strength of the tendon. Few studies have considered the structural integrity of the entire three-dimensional (3D) collagen meshwork, and how the longitudinal collagen fibrils are integrated as a strong unit in a 3D domain to provide the tendons with the essential tensile properties. Using second harmonic generation imaging, a 3D imaging technique was developed and used to study the 3D collagen matrix in the midportion of Achilles tendons without tissue labelling and dehydration. Therefore, the 3D collagen structure is presented in a condition closely representative of the in vivo status. Atomic force microscopy studies have confirmed that second harmonic generation reveals the internal collagen matrix of tendons in 3D at a fibril level. Achilles tendons primarily contain longitudinal collagen fibrils that braid spatially into a dense rope-like collagen meshwork and are encapsulated or wound tightly by the oblique collagen fibrils emanating from the epitenon region. The arrangement of the collagen fibrils provides the longitudinal fibrils with essential structural integrity and endows the tendon with the unique mechanical function for withstanding tensile stresses. A novel 3D microscopic method has been developed to examine the 3D collagen microstructure of tendons without tissue dehydrating and labelling. The study also provides new knowledge about the collagen microstructure in an Achilles tendon, which enables understanding of the function of the tissue. The knowledge may be important for applying surgical and tissue engineering techniques to tendon reconstruction. © 2017 The Authors

Alginate-Collagen Fibril Composite Hydrogel

Directory of Open Access Journals (Sweden)

Mahmoud Baniasadi

2015-02-01

Full Text Available We report on the synthesis and the mechanical characterization of an alginate-collagen fibril composite hydrogel. Native type I collagen fibrils were used to synthesize the fibrous composite hydrogel. We characterized the mechanical properties of the fabricated fibrous hydrogel using tensile testing; rheometry and atomic force microscope (AFM-based nanoindentation experiments. The results show that addition of type I collagen fibrils improves the rheological and indentation properties of the hydrogel.

Preparation and characterization of collagen/PLA, chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering.

Science.gov (United States)

Haaparanta, Anne-Marie; Järvinen, Elina; Cengiz, Ibrahim Fatih; Ellä, Ville; Kokkonen, Harri T; Kiviranta, Ilkka; Kellomäki, Minna

2014-04-01

In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.

A 48 kDa collagen-binding phosphoprotein isolated from bovine aortic endothelial cells interacts with the collagenous domain, but not the globular domain, of collagen type IV.

OpenAIRE

Yannariello-Brown, J; Madri, J A

1990-01-01

We have identified collagen-binding proteins in detergent extracts of metabolically labelled bovine aortic endothelial cells (BAEC) by collagen type IV-Sepharose affinity chromatography. The major collagen type IV-binding protein identified by SDS/PAGE had a molecular mass of 48 kDa, which we term the 'collagen-binding 48 kDa protein' (CB48). The pI of CB48 was 8.0-8.3 in a two-dimensional gel system, running non-equilibrium pH gel electrophoresis in the first dimension and SDS/PAGE in the se...

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

Science.gov (United States)

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.

IGF-1 decreases collagen degradation in diabetic NOD mesangial cells: implications for diabetic nephropathy.

Science.gov (United States)

Lupia, E; Elliot, S J; Lenz, O; Zheng, F; Hattori, M; Striker, G E; Striker, L J

1999-08-01

Nonobese diabetic (NOD) mice develop glomerulosclerosis shortly after the onset of diabetes. We showed that mesangial cells (MCs) from diabetic mice exhibited a stable phenotypic switch, consisting of both increased IGF-1 synthesis and proliferation (Elliot SJ, Striker LJ, Hattori M, Yang CW, He CJ, Peten EP, Striker GE: Mesangial cells from diabetic NOD mice constitutively secrete increased amounts of insulin-like growth factor-I. Endocrinology 133:1783-1788, 1993). Because the extracellular matrix (ECM) accumulation in diabetic glomerulosclerosis may be partly due to decreased degradation, we examined the effect of excess IGF-1 on collagen turnover and the activity of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) in diabetic and nondiabetic NOD-MC. Total collagen degradation was reduced by 58 +/- 18% in diabetic NOD-MCs, which correlated with a constitutive decrease in MMP-2 activity and mRNA levels, and nearly undetectable MMP-9 activity and mRNA. TIMP levels were slightly decreased in diabetic NOD-MC. The addition of recombinant IGF-1 to nondiabetic NOD-MC resulted in a decrease in MMP-2 and TIMP activity. Furthermore, treatment of diabetic NOD-MC with a neutralizing antibody against IGF-1 increased the latent form, and restored the active form, of MMP-2. In conclusion, the excessive production of IGF-1 contributes to the altered ECM turnover in diabetic NOD-MC, largely through a reduction of MMP-2 activity. These data suggest that IGF-1 could be a major contributor to the development of diabetic glomerulosclerosis.

A role for collagen IV in cardiovascular disease?

DEFF Research Database (Denmark)

Steffensen, Lasse Bach; Rasmussen, Lars M

2018-01-01

Over the past decade, studies have repeatedly found single nucleotide polymorphisms located in the COL4A1 and COL4A2 genes to be associated with cardiovascular disease (CVD), and the 13q34 locus harboring these genes is one of approximately 160 genome-wide significant risk loci for coronary artery...... disease. COL4A1 and COL4A2 encode the ⍺1- and ⍺2-chains of collagen IV, a major component of basement membranes in various tissues including arteries. In spite of the growing body of evidence indicating a role for collagen IV in CVD, remarkably few studies aim at directly investigating such a role....... The purpose of this review is to summarize the clinical reports linking 13q34 to coronary artery disease, atherosclerosis and artery stiffening and to assemble the scattered pieces of evidence from experimental studies based on vascular cells and -tissue collectively supporting a role for collagen IV...

Triple helix-forming oligonucleotide corresponding to the polypyrimidine sequence in the rat alpha 1(I) collagen promoter specifically inhibits factor binding and transcription.

Science.gov (United States)

Kovacs, A; Kandala, J C; Weber, K T; Guntaka, R V

1996-01-19

Type I and III fibrillar collagens are the major structural proteins of the extracellular matrix found in various organs including the myocardium. Abnormal and progressive accumulation of fibrillar type I collagen in the interstitial spaces compromises organ function and therefore, the study of transcriptional regulation of this gene and specific targeting of its expression is of major interest. Transient transfection of adult cardiac fibroblasts indicate that the polypurine-polypyrimidine sequence of alpha 1(I) collagen promoter between nucleotides - 200 and -140 represents an overall positive regulatory element. DNase I footprinting and electrophoretic mobility shift assays suggest that multiple factors bind to different elements of this promoter region. We further demonstrate that the unique polypyrimidine sequence between -172 and -138 of the promoter represents a suitable target for a single-stranded polypurine oligonucleotide (TFO) to form a triple helix DNA structure. Modified electrophoretic mobility shift assays show that this TFO specifically inhibits the protein-DNA interaction within the target region. In vitro transcription assays and transient transfection experiments demonstrate that the transcriptional activity of the promoter is inhibited by this oligonucleotide. We propose that TFOs represent a therapeutic potential to specifically influence the expression of alpha 1(I) collagen gene in various disease states where abnormal type I collagen accumulation is known to occur.

M2-like macrophages are responsible for collagen degradation through a mannose receptor–mediated pathway

Science.gov (United States)

Madsen, Daniel H.; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E.; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S.; Brenner, David A.; Burgdorf, Sven; Engelholm, Lars H.; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto

2013-01-01

Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase–dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor–associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process. PMID:24019537

The I kappa B kinase inhibitor ACHP strongly attenuates TGF beta 1-induced myofibroblast formation and collagen synthesis

NARCIS (Netherlands)

Mia, Masum M.; Bank, Ruud A.

2015-01-01

Excessive accumulation of a collagen-rich extracellular matrix (ECM) by myofibroblasts is a characteristic feature of fibrosis, a pathological state leading to serious organ dysfunction. Transforming growth factor beta1 (TGF beta 1) is a strong inducer of myofibroblast formation and subsequent

Identification of the NC1 domain of {alpha}3 chain as critical for {alpha}3{alpha}4{alpha}5 type IV collagen network assembly.

Science.gov (United States)

LeBleu, Valerie; Sund, Malin; Sugimoto, Hikaru; Birrane, Gabriel; Kanasaki, Keizo; Finan, Elizabeth; Miller, Caroline A; Gattone, Vincent H; McLaughlin, Heather; Shield, Charles F; Kalluri, Raghu

2010-12-31

The network organization of type IV collagen consisting of α3, α4, and α5 chains in the glomerular basement membrane (GBM) is speculated to involve interactions of the triple helical and NC1 domain of individual α-chains, but in vivo evidence is lacking. To specifically address the contribution of the NC1 domain in the GBM collagen network organization, we generated a mouse with specific loss of α3NC1 domain while keeping the triple helical α3 chain intact by connecting it to the human α5NC1 domain. The absence of α3NC1 domain leads to the complete loss of the α4 chain. The α3 collagenous domain is incapable of incorporating the α5 chain, resulting in the impaired organization of the α3α4α5 chain-containing network. Although the α5 chain can assemble with the α1, α2, and α6 chains, such assembly is incapable of functionally replacing the α3α4α5 protomer. This novel approach to explore the assembly type IV collagen in vivo offers novel insights in the specific role of the NC1 domain in the assembly and function of GBM during health and disease.

Type XII and XIV collagens mediate interactions between banded collagen fibers in vitro and may modulate extracellular matrix deformability.

Science.gov (United States)

Nishiyama, T; McDonough, A M; Bruns, R R; Burgeson, R E

1994-11-11

Type XII and XIV collagens are very large molecules containing three extended globular domains derived from the amino terminus of each alpha chain and an interrupted triple helix. Both collagens are genetically and immunologically unique and have distinct distributions in many tissues. These collagens localize near the surface of banded collagen fibrils. The function of the molecules is unknown. We have prepared a mixture of native type XII and XIV collagens that is free of contaminating proteins by electrophoretic criteria. In addition, we have purified the collagenase-resistant globular domains of type XII or XIV collagens (XII-NC-3 or XIV-NC-3). In this study, we have investigated the effect of intact type XII and XIV and XII-NC-3 or XIV-NC-3 on the interactions between fibroblasts and type I collagen fibrils. We find that both type XII and XIV collagens promote collagen gel contraction mediated by fibroblasts, even in the absence of serum. The activity is present in the NC-3 domains. The effect is dose-dependent and is inhibited by denaturation. The effect of type XII NC-3 is inhibited by the addition of anti-XII antiserum. To elucidate the mechanism underlying this phenomenon, we examined the effect of XII-NC-3 or XIV-NC-3 on deformability of collagen gels by centrifugal force. XII-NC-3 or XIV-NC-3 markedly promotes gel compression after centrifugation. The effect is also inhibited by denaturation, and the activity of type XII-NC3 is inhibited by the addition of anti-XII antiserum. The results indicate that the effect of XII-NC-3 or XIV-NC-3 on collagen gel contraction by fibroblasts is not due to activation of cellular events but rather results from the increase in mobility of hydrated collagen fibrils within the gel. These studies suggest that collagen types XII and XIV may modulate the biomechanical properties of tissues.

The Mineral–Collagen Interface in Bone

Science.gov (United States)

2015-01-01

The interface between collagen and the mineral reinforcement phase, carbonated hydroxyapatite (cAp), is essential for bone’s remarkable functionality as a biological composite material. The very small dimensions of the cAp phase and the disparate natures of the reinforcement and matrix are essential to the material’s performance but also complicate study of this interface. This article summarizes what is known about the cAp-collagen interface in bone and begins with descriptions of the matrix and reinforcement roles in composites, of the phases bounding the interface, of growth of cAp growing within the collagen matrix, and of the effect of intra- and extrafibrilar mineral on determinations of interfacial properties. Different observed interfacial interactions with cAp (collagen, water, non-collagenous proteins) are reviewed; experimental results on interface interactions during loading are reported as are their influence on macroscopic mechanical properties; conclusions of numerical modeling of interfacial interactions are also presented. The data suggest interfacial interlocking (bending of collagen molecules around cAp nanoplatelets) and water-mediated bonding between collagen and cAp are essential to load transfer. The review concludes with descriptions of areas where new research is needed to improve understanding of how the interface functions. PMID:25824581

Age Increases Monocyte Adhesion on Collagen

Science.gov (United States)

Khalaji, Samira; Zondler, Lisa; Kleinjan, Fenneke; Nolte, Ulla; Mulaw, Medhanie A.; Danzer, Karin M.; Weishaupt, Jochen H.; Gottschalk, Kay-E.

2017-05-01

Adhesion of monocytes to micro-injuries on arterial walls is an important early step in the occurrence and development of degenerative atherosclerotic lesions. At these injuries, collagen is exposed to the blood stream. We are interested whether age influences monocyte adhesion to collagen under flow, and hence influences the susceptibility to arteriosclerotic lesions. Therefore, we studied adhesion and rolling of human peripheral blood monocytes from old and young individuals on collagen type I coated surface under shear flow. We find that firm adhesion of monocytes to collagen type I is elevated in old individuals. Pre-stimulation by lipopolysaccharide increases the firm adhesion of monocytes homogeneously in older individuals, but heterogeneously in young individuals. Blocking integrin αx showed that adhesion of monocytes to collagen type I is specific to the main collagen binding integrin αxβ2. Surprisingly, we find no significant age-dependent difference in gene expression of integrin αx or integrin β2. However, if all integrins are activated from the outside, no differences exist between the age groups. Altered integrin activation therefore causes the increased adhesion. Our results show that the basal increase in integrin activation in monocytes from old individuals increases monocyte adhesion to collagen and therefore the risk for arteriosclerotic plaques.

Collagen films with stabilized liquid crystalline phases and concerns on osteoblast behaviors

Energy Technology Data Exchange (ETDEWEB)

Tang, Minjian; Ding, Shan; Min, Xiang; Jiao, Yanpeng, E-mail: tjiaoyp@jnu.edu.cn; Li, Lihua; Li, Hong; Zhou, Changren, E-mail: tcrz9@jnu.edu.cn

2016-01-01

To duplicate collagen's in vivo liquid crystalline (LC) phase and investigate the relationship between the morphology of LC collagen and osteoblast behavior, a self-assembly method was introduced for preparing collagen films with a stabilized LC phase. The LC texture and topological structure of the films before and after stabilization were observed with polarizing optical microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM). The relationship between the collagen films and osteoblast behavior was studied with the 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide method, proliferation index detection, alkaline phosphatase measurements, osteocalcin assay, inverted microscopy, SEM observation, AFM observation, and cytoskeleton fluorescence staining. The results showed that the LC collagen film had continuously twisting orientations in the cholesteric phase with a typical series of arced patterns. The collagen fibers assembled in a well-organized orientation in the LC film. Compared to the non-LC film, the LC collagen film can promote cell proliferation, and increase ALP and osteocalcin expression, revealing a contact guide effect on osteoblasts. - Highlights: • Collagen film with liquid crystalline (LC) phase was observed by POM, SEM and AFM. • The effect of LC collagen film on osteoblasts behaviors was studied in detail. • LC collagen film promoted osteoblast proliferation and osteogenesis activity.

Raman spectroscopy enables noninvasive biochemical identification of the collagen regeneration in cutaneous wound healing of diabetic mice treated with MSCs.

Science.gov (United States)

Yan, Wenxia; Liu, Hanping; Deng, Xiaoyuan; Jin, Ying; Sun, Huimin; Li, Caiyun; Wang, Ning; Chu, Jing

2017-07-01

Mesenchymal stem cells (MSCs) had been reported as a novel therapeutic strategy for non-healing diabetic cutaneous wound mainly by promoting the formation of extracellular matrix (ECM) and neovasculature. Collagen regeneration is one of the key processes of ECM remodeling in wound healing. Accordingly, rapid assessment of the collagen content in a noninvasive manner can promptly provide objective evaluation for MSC therapy of cutaneous wound healing and strength evidence to adjust therapeutic regimen. In the present study, noninvasive Raman microspectroscopy was used for tracing the regeneration status of collagen during diabetic wound healing with MSCs. Wound tissues of normal mice, diabetic mice, and MSC-treated diabetic mice were subjected to Masson trichrome staining assay and submitted to spectroscopic analysis by Raman microspectroscopy after wounding 7, 14, and 21 days. Masson trichrome staining demonstrated that there was more collagen deposition in diabetic + MSCs group relative to diabetic group. The relative intensity of Raman collagen peak positions at 937, 1004, 1321, 1452, and 1662 cm -1 increased in MSC-treated diabetic group compared to diabetic group, although normal mice group had the highest relative intensity of collagen peak bands. Correlation analysis suggested that the spectral bands had a high positive correlation with the collagen intensity detected by Masson trichrome staining in wound tissues of three groups. Our results demonstrate that Raman microspectroscopy has potential application in rapidly and quantitatively assessing diabetic wound healing with MSCs by monitoring collagen variation, which may provide a novel method for the study of skin regeneration.

Higher iron bioavailability of a human-like collagen iron complex.

Science.gov (United States)

Zhu, Chenhui; Yang, Fan; Fan, Daidi; Wang, Ya; Yu, Yuanyuan

2017-07-01

Iron deficiency remains a public health problem around the world due to low iron intake and/or bioavailability. FeSO 4 , ferrous succinate, and ferrous glycinate chelate are rich in iron but have poor bioavailability. To solve the problem of iron deficiency, following previous research studies, a thiolated human-like collagen-ironcomplex supplement with a high iron content was prepared in an anaerobic workstation. In addition, cell viability tests were evaluated after conducting an MTT assay, and a quantitative analysis of the thiolated human-like collagen-iron digesta samples was performed using the SDS-PAGE method coupled with gel filtration chromatography. The iron bioavailability was assessed using Caco-2 cell monolayers and iron-deficiency anemia mice models. The results showed that (1) one mole of thiolated human-like collagen-iron possessed approximately 35.34 moles of iron; (2) thiolated human-like collagen-iron did not exhibit cytotoxity and (3) thiolated human-like collagen- iron digesta samples had higher bioavailability than other iron supplements, including FeSO 4 , ferrous succinate, ferrous glycine chelate and thiolated human-like collagen-Fe iron. Finally, the iron bioavailability was significantly enhanced by vitamin C. These results indicated that thiolated human-like collagen-iron is a promising iron supplement for use in the future.

Adrenomedullin and adrenotensin regulate collagen synthesis and proliferation in pulmonary arterial smooth muscle cells

Energy Technology Data Exchange (ETDEWEB)

Li, W. [School of Control Science and Engineering, Biomedical Engineering Institute, Shandong University, Jinan, Shandong (China); Kong, Q.Y.; Zhao, C.F. [Department of Pediatrics, Qilu Hospital, Shandong University, Jinan, Shandong (China); Zhao, F. [Department of Medicine, Weill Medical College of Cornell University, New York, NY (United States); Li, F.H.; Xia, W. [Department of Pediatrics, Qilu Hospital, Shandong University, Jinan, Shandong (China); Wang, R. [Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan, Shandong (China); Hu, Y.M. [School of Control Science and Engineering, Biomedical Engineering Institute, Shandong University, Jinan, Shandong (China); Hua, M. [Shandong Institute of Scientific and Technical Information, Jinan, Shandong (China)

2013-12-10

To understand the pathophysiological mechanisms of pulmonary arterial smooth muscle cell (PASMC) proliferation and extracellular-matrix accumulation in the development of pulmonary hypertension and remodeling, this study determined the effects of different doses of adrenomedullin (ADM) and adrenotensin (ADT) on PASMC proliferation and collagen synthesis. The objective was to investigate whether extracellular signal-regulated kinase (ERK1/2) signaling was involved in ADM- and ADT-stimulated proliferation of PASMCs in 4-week-old male Wistar rats (body weight: 100-150 g, n=10). The proliferation of PASMCs was examined by 5-bromo-2-deoxyuridine incorporation. A cell growth curve was generated by the Cell Counting Kit-8 method. Expression of collagen I, collagen III, and phosphorylated ERK1/2 (p-ERK1/2) was evaluated by immunofluorescence. The effects of different concentrations of ADM and ADT on collagen I, collagen III, and p-ERK1/2 protein expression were determined by immunoblotting. We also investigated the effect of PD98059 inhibition on the expression of p-ERK1/2 protein by immunoblotting. ADM dose-dependently decreased cell proliferation, whereas ADT dose-dependently increased it; and ADM and ADT inhibited each other with respect to their effects on the proliferation of PASMCs. Consistent with these results, the expression of collagen I, collagen III, and p-ERK1/2 in rat PASMCs decreased after exposure to ADM but was upregulated after exposure to ADT. PD98059 significantly inhibited the downregulation by ADM and the upregulation by ADT of p-ERK1/2 expression. We conclude that ADM inhibited, and ADT stimulated, ERK1/2 signaling in rat PASMCs to regulate cell proliferation and collagen expression.

Layer-dependent role of collagen recruitment during loading of the rat bladder wall.

Science.gov (United States)

Cheng, Fangzhou; Birder, Lori A; Kullmann, F Aura; Hornsby, Jack; Watton, Paul N; Watkins, Simon; Thompson, Mark; Robertson, Anne M

2018-04-01

In this work, we re-evaluated long-standing conjectures as to the source of the exceptionally large compliance of the bladder wall. Whereas these conjectures were based on indirect measures of loading mechanisms, in this work we take advantage of advances in bioimaging to directly assess collagen fibers and wall architecture during biaxial loading. A custom biaxial mechanical testing system compatible with multiphoton microscopy was used to directly measure the layer-dependent collagen fiber recruitment in bladder tissue from 9 male Fischer rats (4 adult and 5 aged). As for other soft tissues, the bladder loading curve was exponential in shape and could be divided into toe, transition and high stress regimes. The relationship between collagen recruitment and loading curves was evaluated in the context of the inner (lamina propria) and outer (detrusor smooth muscle) layers. The large extensibility of the bladder was found to be possible due to folds in the wall (rugae) that provide a mechanism for low resistance flattening without any discernible recruitment of collagen fibers throughout the toe regime. For more extensible bladders, as the loading extended into the transition regime, a gradual coordinated recruitment of collagen fibers between the lamina propria layer and detrusor smooth muscle layer was found. A second important finding was that wall extensibility could be lost by premature recruitment of collagen in the outer wall that cut short the toe region. This change was correlated with age. This work provides, for the first time, a mechanistic understanding of the role of collagen recruitment in determining bladder extensibility and capacitance.

Factors regulating collagen synthesis and degradation during second-intention healing of wounds in the thoracic region and the distal aspect of the forelimb of horses.

Science.gov (United States)

Schwartz, Anne J; Wilson, David A; Keegan, Kevin G; Ganjam, Venkataseshu K; Sun, Yao; Weber, Karl T; Zhang, Jiakun

2002-11-01

To determine significant molecular and cellular factors responsible for differences in second-intention healing in thoracic and metacarpal wounds of horses. 6 adult mixed-breed horses. A full-thickness skin wound on the metacarpus and another such wound on the pectoral region were created, photographed, and measured, and tissue was harvested from these sites weekly for 4 weeks. Gene expression of type-I collagen, transforming growth factor (TGF)-beta1, matrix metalloproteinase (MMP)-1, and tissue inhibitor of metalloproteinase (TIMP)-1 were determined by quantitative in situ hybridization. Myofibroblasts were detected by immunohistochemical labeling with alpha-smooth muscle actin (alpha-SMA). Collagen accumulation was detected by use of picrosirius red staining. Tissue morphology was examined by use of H&E staining. Unlike thoracic wounds, forelimb wounds enlarged during the first 2 weeks. Myofibroblasts, detected by week 1, remained abundant with superior organization in thoracic wounds. Type-I collagen mRNA accumulated progressively in both wounds. More type-I collagen and TGF-beta1 mRNA were seen in forelimb wounds. Volume of MMP-1 mRNA decreased from day 0 in both wounds. By week 3, TIMP-1 mRNA concentration was greater in thoracic wounds. Greater collagen synthesis in metacarpal than thoracic wounds was documented by increased concentrations of myofibroblasts, type-I collagen mRNA,TGF-beta1 mRNA, and decreased collagen degradation (ie, MMP-1). Imbalanced collagen synthesis and degradation likely correlate with development of exuberant granulation tissue, delaying healing in wounds of the distal portions of the limbs. Factors that inhibit collagen synthesis or stimulate collagenase may provide treatment options for horses with exuberant granulation tissue.

Collagen metabolism in obesity

DEFF Research Database (Denmark)

Rasmussen, M H; Jensen, L T; Andersen, T

1995-01-01

OBJECTIVE: To investigate the impact of obesity, fat distribution and weight loss on collagen turnover using serum concentrations of the carboxyterminal propeptide of type I procollagen (S-PICP) and the aminoterminal propeptide of type III pro-collagen (S-PIIINP) as markers for collagen turnover...... (r = 0.37; P = 0.004), height (r = 0.27; P = 0.04), waist circumference (r = 0.35; P = 0.007), as well as with WHR (r = 0.33; P = 0.01) and was inversely correlated to age (r = -0.40; P = 0.002). Compared with randomly selected controls from a large pool of healthy volunteers, the obese patients had...... restriction (P obesity and associated with body fat distribution, suggesting...

Collagen as potential cell scaffolds for tissue engineering.

Science.gov (United States)

Annuar, N; Spier, R E

2004-05-01

Selections of collagen available commercially were tested for their biocompatibility as scaffold to promote cell growth in vitro via simple collagen fast test and cultivation of mammalian cells on the selected type of collagen. It was found that collagen type C9791 promotes the highest degree of aggregation as well as cells growth. This preliminary study also indicated potential use of collagen as scaffold in engineered tissue.

A novel functional role of collagen glycosylation

DEFF Research Database (Denmark)

Jürgensen, Henrik J; Madsen, Daniel H; Ingvarsen, Signe

2011-01-01

Collagens make up the most abundant component of interstitial extracellular matrices and basement membranes. Collagen remodeling is a crucial process in many normal physiological events and in several pathological conditions. Some collagen subtypes contain specific carbohydrate side chains......, the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose...... receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens...

Extraction of Collagen from Chicken Feet with Various Acidic Solutions and Soaking Time

Directory of Open Access Journals (Sweden)

Prayitno Prayitno

2007-05-01

Full Text Available The objective of this research was to know the ability of various acidic solutions on dissolving collagen  chicken feet, with different soaked time.  Each acid 5 percent (v/v, collagen extraction was done by washing chicken feet and then cutted into small pieces and finally grinded.  Every 100 gram treatment was soaked in acetic acid (a1, citric acid (a2, lactic acid (a3 and hydrochloric acid (a4, for 12, 24 and 36 hours.  Precipitated collagen in the filtrate was 5 percent NaOH to reach the neutral pH (pH 7.  Collagen precipitate was separated by filtration usingfilter paper and then  rendement was calculated, HPLC was used to determin amino acid composition, and SDS-PAGE was use determin the type of collagen.  This experiment use factorial completely randomized design (CRD 4 x 3 and three time replication.   Result showed that lactic acid has highest capability to dissolve collagen, while citric acid the lowest.  Combination of acid solution and soaking time had significant (P<0.01 effect on dissolving collagen of chicken feet.  Extracted collagen in all acid solution, hassame amino acid, composition but different in percentage of amino acid molecules.  Collagen type in treatment combination was the same, but for soaking time of 36 hours revealed some peptide band.  Lactic acid had highest capability of collagen extraction in chicken feet than citric acid, acetic acid and hydrochloric acid with soaking time of 12, 24 and 36 hours.  It was estimated that extracted collagen can be grouped to type I consisted of two chain of a1. (Animal Production 9(2: 99-104 (2007   Key Words : Chicken feet, acids, soaking time, collagen

Enough is as good as a feast - sufficiency as policy. Volume 1

International Nuclear Information System (INIS)

Darby, Sarah

2007-01-01

The concept of sufficiency has a long history, related as it is to the timeless issues of how best to distribute and use resources. Where energy is concerned, absolute reductions in demand are increasingly seen as necessary in response to climate change and energy security concerns. There is an acknowledgement that, collectively if not individually, humans have gone beyond safe limits in their use of fuels. The relatively wealthy and industrialised nations urgently need to move beyond a primary focus on efficiency to the more contentious issues surrounding demand reduction and sufficiency. The paper considers definitions of energy sufficiency, looks at a recent attempt to model future energy use in terms of efficiency and sufficiency, and discusses quantitative and qualitative aspects of sufficiency and how they might become institutionalised. There are many arguments in favour of sufficiency but they often founder in the face of political requirements for market growth and the employment generated by it. Some options for 'sufficiency policy' are selected, including a focus on energy in relation to livelihoods, energy implications of our use of time and making energy use more transparent

Enough is as good as a feast - sufficiency as policy. Volume 1

Energy Technology Data Exchange (ETDEWEB)

Darby, Sarah [Lower Carbon Futures, Environmental Change Inst., Oxford Univ. Centre for the Environment (United Kingdom)

2007-07-01

The concept of sufficiency has a long history, related as it is to the timeless issues of how best to distribute and use resources. Where energy is concerned, absolute reductions in demand are increasingly seen as necessary in response to climate change and energy security concerns. There is an acknowledgement that, collectively if not individually, humans have gone beyond safe limits in their use of fuels. The relatively wealthy and industrialised nations urgently need to move beyond a primary focus on efficiency to the more contentious issues surrounding demand reduction and sufficiency. The paper considers definitions of energy sufficiency, looks at a recent attempt to model future energy use in terms of efficiency and sufficiency, and discusses quantitative and qualitative aspects of sufficiency and how they might become institutionalised. There are many arguments in favour of sufficiency but they often founder in the face of political requirements for market growth and the employment generated by it. Some options for 'sufficiency policy' are selected, including a focus on energy in relation to livelihoods, energy implications of our use of time and making energy use more transparent.

[The genetics of collagen diseases].

Science.gov (United States)

Kaplan, J; Maroteaux, P; Frezal, J

1986-01-01

Heritable disorders of collagen include Ehler-Danlos syndromes (11 types are actually known), Larsen syndrome and osteogenesis imperfecta. Their clinical, genetic and biochemical features are reviewed. Marfan syndrome is closely related to heritable disorders of collagen.

Effect of collagen type IV, MMPs and TIMPs on remodeling of radiation pulmonary injury

International Nuclear Information System (INIS)

Diao Ruiying; Song Liangwen; Wang Shaoxia; Yin Jiye

2007-01-01

Objective: To explore the effect of collagen type IV, matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs(TIMPs) on early remodeling after radiation pulmonary injury. Methods: Right lungs of rats were irradiated by 60 Co γ-rays at a dose of 20 Gy to induce radiation pulmonary injury, and the lung specimens were taken at weeks 1, 2, 4 after irradiation. Quantitative analysis was performed on pulmonary collagen type IV, MMP-2, MMP-9, TIMP-2, TIMP-1 at the level of gene expression and protein synthesis using real-time PCR or immunohistochemistry. Results: Gene detection using real-time PCR: gene expression of collagen type IV increased at week 1 and decreased at week 2 after irradiation; MMP-2 reached peak at week 2 in which an opposed alteration trend was displayed; MMP-9 appeared a significant trend of elevation, then decrease and elevation again which was similar to those of collagen type IV; expression of TIMP-1 was lower, and there was no marked difference among all time points; TIMP-2 displayed a trend of slight elevation, then decrease and elevation again, which was opposed to MMP-2. Immunohistochemistry-image analysis: Pulmonary collagen type IV obviously increased at week 1, and began to decrease at week 2; MMP-2 decreased at week 2 and then increased; an opposed alteration trend to that of collagen type IV was displayed; alteration trend of MMP-9 was similar to that of collagen type IV but the extent was higher; gene expression of TIMP-1 slightly increased at 2 week and an opposed trend to of MMP-9 was displayed. Conclusions: Collagen type IV, MMP-2, MMP-9 and their tissue inhibitors were involved in ineffective remodeling in the early radiation pulmonary injury; MMP-2 and MMP-9 play an important role in degradation of collagen type IV; Disturbance of collagen type IV degradation might have relationship with the initiation of pulmonary fibrosis. (authors)

Confocal Raman mapping of collagen cross-link and crystallinity of human dentin-enamel junction

Science.gov (United States)

Slimani, Amel; Nouioua, Fares; Desoutter, Alban; Levallois, Bernard; Cuisinier, Frédéric J. G.; Tassery, Hervé; Terrer, Elodie; Salehi, Hamideh

2017-08-01

The separation zone between enamel and dentin [dentin-enamel junction (DEJ)] with different properties in biomechanical composition has an important role in preventing crack propagation from enamel to dentin. The understanding of the chemical structure (inorganic and organic components), physical properties, and chemical composition of the human DEJ could benefit biomimetic materials in dentistry. Spatial distribution of calcium phosphate crystallinity and the collagen crosslinks near DEJ were studied using confocal Raman microscopy and calculated by different methods. To obtain collagen crosslinking, the ratio of two peaks 1660 cm-1 over 1690 cm-1 (amide I bands) is calculated. For crystallinity, the inverse full-width at half maximum of phosphate peak at 960 cm-1, and the ratio of two Raman peaks of phosphate at 960/950 cm-1 is provided. In conclusion, the study of chemical and physical properties of DEJ provides many benefits in the biomaterial field to improve the synthesis of dental materials in respect to the natural properties of human teeth. Confocal Raman microscopy as a powerful tool provides the molecular structure to identify the changes along DEJ and can be expanded for other mineralized tissues.

Collagen cross linking: Current perspectives

Directory of Open Access Journals (Sweden)

Srinivas K Rao

2013-01-01

Full Text Available Keratoconus is a common ectatic disorder occurring in more than 1 in 1,000 individuals. The condition typically starts in adolescence and early adulthood. It is a disease with an uncertain cause and its progression is unpredictable, but in extreme cases, vision deteriorates and can require corneal transplant surgery. Corneal collagen cross-linking (CCL with riboflavin (C3R is a recent treatment option that can enhance the rigidity of the cornea and prevent disease progression. Since its inception, the procedure has evolved with newer instrumentation, surgical techniques, and is also now performed for expanded indications other than keratoconus. With increasing experience, newer guidelines regarding optimization of patient selection, the spectrum of complications and their management, and combination procedures are being described. This article in conjunction with the others in this issue, will try and explore the uses of collagen cross-linking (CXL in its current form.

Diffusion and Binding of Laponite Clay Nanoparticles into Collagen Fibers for the Formation of Leather Matrix.

Science.gov (United States)

Shi, Jiabo; Wang, Chunhua; Ngai, To; Lin, Wei

2018-06-13

Understanding accessibility and interactions of clay nanoparticles with collagen fibers is an important fundamental issue for the conversion of collagen to leather matrix. In this study, we have investigated the diffusion and binding of Laponite into the collagen fiber network. Our results indicate that the diffusion behaviors of Laponite into the collagen exhibit the Langmuir adsorption, verifying its affinity for collagen. The introduction of Laponite leads to a shift in the isoelectric point of collagen from ∼6.8 to ∼4.5, indicating the ionic bonding between the positively charged amino groups of the collagen and negatively charged Laponite under the tanning conditions. Fluorescence microscopy, atomic force microscopy, field-emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, and wide-angle X-ray diffraction analyses reveal that Laponite nanoparticles can penetrate into collagen microstructure and evenly distributed onto collagen fibrils, not altering native D-periodic banding patterns of collagen fibrils. Attenuated total reflectance-Fourier transform infrared and Raman spectroscopy detections further demonstrate the presence of noncovalent interactions, namely, ionic and hydrogen bonding, between Laponite and collagen. These findings provide a theoretical basis for the use of Laponite as an emerging tanning agent in leather manufacture.

Marine-derived collagen biomaterials from echinoderm connective tissues

KAUST Repository

Ferrario, Cinzia; Leggio, Livio; Leone, Roberta; Di Benedetto, Cristiano; Guidetti, Luca; Coccè , Valentina; Ascagni, Miriam; Bonasoro, Francesco; La Porta, Caterina A.M.; Candia Carnevali, M. Daniela; Sugni, Michela

2016-01-01

The use of marine collagens is a hot topic in the field of tissue engineering. Echinoderms possess unique connective tissues (Mutable Collagenous Tissues, MCTs) which can represent an innovative source of collagen to develop collagen barrier-membranes for Guided Tissue Regeneration (GTR). In the present work we used MCTs from different echinoderm models (sea urchin, starfish and sea cucumber) to produce echinoderm-derived collagen membranes (EDCMs). Commercial membranes for GTR or soluble/reassembled (fibrillar) bovine collagen substrates were used as controls. The three EDCMs were similar among each other in terms of structure and mechanical performances and were much thinner and mechanically more resistant than the commercial membranes. Number of fibroblasts seeded on sea-urchin membranes were comparable to the bovine collagen substrates. Cell morphology on all EDCMs was similar to that of structurally comparable (reassembled) bovine collagen substrates. Overall, echinoderms, and sea urchins particularly, are alternative collagen sources to produce efficient GTR membranes. Sea urchins display a further advantage in terms of eco-sustainability by recycling tissues from food wastes.

Marine-derived collagen biomaterials from echinoderm connective tissues

KAUST Repository

Ferrario, Cinzia

2016-03-31

The use of marine collagens is a hot topic in the field of tissue engineering. Echinoderms possess unique connective tissues (Mutable Collagenous Tissues, MCTs) which can represent an innovative source of collagen to develop collagen barrier-membranes for Guided Tissue Regeneration (GTR). In the present work we used MCTs from different echinoderm models (sea urchin, starfish and sea cucumber) to produce echinoderm-derived collagen membranes (EDCMs). Commercial membranes for GTR or soluble/reassembled (fibrillar) bovine collagen substrates were used as controls. The three EDCMs were similar among each other in terms of structure and mechanical performances and were much thinner and mechanically more resistant than the commercial membranes. Number of fibroblasts seeded on sea-urchin membranes were comparable to the bovine collagen substrates. Cell morphology on all EDCMs was similar to that of structurally comparable (reassembled) bovine collagen substrates. Overall, echinoderms, and sea urchins particularly, are alternative collagen sources to produce efficient GTR membranes. Sea urchins display a further advantage in terms of eco-sustainability by recycling tissues from food wastes.

LOX-mediated collagen crosslinking is responsible for fibrosis-enhanced metastasis

DEFF Research Database (Denmark)

Cox, Thomas R; Bird, Demelza; Baker, Ann-Marie

2013-01-01

of metastastic tumor cells. We show that LOX-dependent collagen crosslinking is involved in creating a growth-permissive fibrotic microenvironment capable of supporting metastatic growth by enhancing tumor cell persistence and survival. We show that therapeutic targeting of LOX abrogates not only the extent...... to which fibrosis manifests, but also prevents fibrosis-enhanced metastatic colonization. Finally, we show that the LOX-mediated collagen crosslinking directly increases tumor cell proliferation, enhancing metastatic colonization and growth manifesting in vivo as increased metastasis. This is the first...... time that crosslinking of collagen I has been shown to enhance metastatic growth. These findings provide an important link between ECM homeostasis, fibrosis, and cancer with important clinical implications for both the treatment of fibrotic disease and cancer....

Characterizing the collagen stabilizing effect of crosslinked chitosan nanoparticles against collagenase degradation.

Science.gov (United States)

Kishen, Anil; Shrestha, Suja; Shrestha, Annie; Cheng, Calvin; Goh, Cynthia

2016-08-01

Antibacterial and chelating properties of chitosan has been widely studied for various dental applications. To characterize the interaction between chitosan-nanoparticles (CSnp) and collagen, and understand their stabilizing effect against collagenase degradation for dentin matrix stabilization. Phase-1: a single Type I collagen-fibril model was used to study the interaction with CSnp along with carbodiimides crosslinking treatment. Degradation of the crosslinked fibrils was studied with bacterial collagenase enzyme and monitored using Fourier Transform Infrared (FTIR) spectroscopy, turbidity measurement (400nm), ninhydrin assay and Atomic Force Microscopy (AFM). Interaction of CSnp with collagenase and Type I collagen, were evaluated using SDS-PAGE, and proteolytic cleavage potential of a synthetic peptide. Phase-2: degradation of dentin collagen crosslinked with/without CSnp was evaluated using FTIR, ninhydrin assay and Scanning Electron Microscopy (SEM). Glutaraldehyde crosslinking was used as a positive control. Both native collagen-fibrils and dentin collagen after crosslinking showed higher resistance to collagenase degradation, as observed in turbidity measurements and FTIR spectra. AFM images showed the interaction of CSnp with single collagen-fibril and crosslinked collagen resisted collagenase degradation up to 54h. The collagen and collagenase both formed complexes with CSnp resulting in thickening of bands and reduction in collagen degradation. CSnp treated collagenase showed significantly reduced cleavage of the fluorescent peptides. Dentin collagen was coated with CSnp following crosslinking with significant increase in resistance to collagenase degradation. Crosslinked CSnp on collagen stabilized and enhanced the resistance of dentin matrix against bacterial collagenase degradation due to non-specific interaction with both collagen and collagenase. Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Differential control of collagen synthesis by the sympathetic and renin-angiotensin systems in the rat left ventricle.

Science.gov (United States)

Dab, Houcine; Hachani, Rafik; Hodroj, Wassim; Sakly, Mohsen; Bricca, Giampiero; Kacem, Kamel

2009-12-03

In the present study, we tested the hypothesis of the indirect (via the sympathetic nervous system (SNS)) and direct (via AT1 receptors) contributions of Angiotensin II (Ang II) on the synthesis of collagen types I and III in the left ventricle (LV) in vivo. Sympathectomy and blockade of the Ang II receptor AT1 were performed alone or in combination in normotensive rats. The mRNA and protein synthesis of collagen types I and III were examined by Q-RT-PCR and immunoblotting in the LV. Collagen types I and III mRNA were decreased respectively by 53% and 22% after sympathectomy and only collagen type I mRNA was increased by 52% after AT1 receptor blockade. mRNA was not changed for collagen type I but was decreased by 25% for collagen type III after double treatment. Only collagen protein type III was decreased after sympathectomy by 12%, but collagen proteins were increased respectively for types I and III by 145% and 52% after AT1 receptor blockade and by 45% and 60% after double treatment. Deducted interpretations from our experimental approach suggest that Ang II stimulates indirectly (via SNS) and inhibits directly (via AT1 receptors) the collagen type I at transcriptional and protein levels. For collagen type III, it stimulates indirectly the transcription and inhibited directly the protein level. Therefore, the Ang II regulates collagen synthesis differently through indirect and direct pathways.

Induction of PNAd and N-acetylglucosamine 6-O-sulfotransferases 1 and 2 in mouse collagen-induced arthritis

Directory of Open Access Journals (Sweden)

Rosen Steven D

2006-06-01

Full Text Available Abstract Background Leukocyte recruitment across blood vessels is fundamental to immune surveillance and inflammation. Lymphocyte homing to peripheral lymph nodes is mediated by the adhesion molecule, L-selectin, which binds to sulfated carbohydrate ligands on high endothelial venules (HEV. These glycoprotein ligands are collectively known as peripheral node addressin (PNAd, as defined by the function-blocking monoclonal antibody known as MECA-79. The sulfation of these ligands depends on the action of two HEV-expressed N-acetylglucosamine 6-O-sulfotransferases: GlcNAc6ST-2 and to a lesser degree GlcNAc6ST-1. Induction of PNAd has also been shown to occur in a number of human inflammatory diseases including rheumatoid arthritis (RA. Results In order to identify an animal model suitable for investigating the role of PNAd in chronic inflammation, we examined the expression of PNAd as well as GlcNAc6ST-1 and -2 in collagen-induced arthritis in mice. Here we show that PNAd is expressed in the vasculature of arthritic synovium in mice immunized with collagen but not in the normal synovium of control animals. This de novo expression of PNAd correlates strongly with induction of transcripts for both GlcNAc6ST-1 and GlcNAc6ST-2, as well as the expression of GlcNAc6ST-2 protein. Conclusion Our results demonstrate that PNAd and the sulfotransferases GlcNAc6ST-1 and 2 are induced in mouse collagen-induced arthritis and suggest that PNAd antagonists or inhibitors of the enzymes may have therapeutic benefit in this widely-used mouse model of RA.

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

International Nuclear Information System (INIS)

Yano, Hiroyuki; Hamanaka, Ryoji; Nakamura, Miki; Sumiyoshi, Hideaki; Matsuo, Noritaka; Yoshioka, Hidekatsu

2012-01-01

Highlights: ► We examine how radiation affects the expression level and signal pathway of collagen. ► TGF-β1 mRNA is elevated earlier than those of collagen genes after irradiation. ► Smad pathway mediates the expression of collagen in radiation induced fibrosis. ► MAPK pathways are not affected in the expression of collagen after irradiation. -- Abstract: Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Real time RT-RCR showed that both α1and α2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-β1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-β receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of α2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

Energy Technology Data Exchange (ETDEWEB)

Yano, Hiroyuki [Department of Matrix Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan); Division of Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan); Hamanaka, Ryoji; Nakamura, Miki [Cell Biology, Faculty of Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan); Sumiyoshi, Hideaki; Matsuo, Noritaka [Department of Matrix Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan); Yoshioka, Hidekatsu, E-mail: hidey@oita-u.ac.jp [Department of Matrix Medicine, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593 (Japan)

2012-02-17

Highlights: Black-Right-Pointing-Pointer We examine how radiation affects the expression level and signal pathway of collagen. Black-Right-Pointing-Pointer TGF-{beta}1 mRNA is elevated earlier than those of collagen genes after irradiation. Black-Right-Pointing-Pointer Smad pathway mediates the expression of collagen in radiation induced fibrosis. Black-Right-Pointing-Pointer MAPK pathways are not affected in the expression of collagen after irradiation. -- Abstract: Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Real time RT-RCR showed that both {alpha}1and {alpha}2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-{beta}1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-{beta} receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of {alpha}2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.

The collagen type I segment long spacing (SLS) and fibrillar forms: Formation by ATP and sulphonated diazo dyes.

Science.gov (United States)

Harris, J Robin; Lewis, Richard J

2016-07-01

The collagen type I segment long spacing (SLS) crystallite is a well-ordered rod-like molecular aggregate, ∼300nm in length, which is produced in vitro under mildly acidic conditions (pH 2.5-3.5) in the presence of 1mM ATP. The formation of the SLS crystallite amplifies the inherent linear structural features of individual collagen heterotrimers, due to the punctate linear distribution and summation of the bulkier amino acid side chains along the length of individual collagen heterotrimers. This can be correlated structurally with the 67nm D-banded collagen fibril that is found in vivo, and formed in vitro. Although first described many years ago, the range of conditions required for ATP-induced SLS crystallite formation from acid-soluble collagen have not been explored extensively. Consequently, we have addressed biochemical parameters such as the ATP concentration, pH, speed of formation and stability so as to provide a more complete structural understanding of the SLS crystallite. Treatment of collagen type I with 1mM ATP at neutral and higher pH (6.0-9.0) also induced the formation of D-banded fibrils. Contrary to previous studies, we have shown that the polysulphonated diazo dyes Direct red (Sirius red) and Evans blue, but not Congo red and Methyl blue, can also induce the formation of SLS-like aggregates of collagen, but under markedly different ionic conditions to those employed in the presence of ATP. Specifically, pre-formed D-banded collagen fibrils, prepared in a higher than the usual physiological NaCl concentration (e.g. 500mM NaCl, 20mM Tris-HCl pH7.4 or x3 PBS), readily form SLS aggregates when treated with 0.1mM Direct red and Evans blue, but this did not occur at lower NaCl concentrations. These new data are discussed in relation to the anion (Cl(-)) and polyanion (phosphate and sulphonate) binding by the collagen heterotrimer and their likely role in collagen fibrillogenesis and SLS formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protease-activatable collagen targeting based on protein cyclization

NARCIS (Netherlands)

Breurken, M.; Lempens, E.H.M.; Merkx, M.

2010-01-01

Threading collagen through a protein needle: The collagen-binding protein CNA35 operates by wrapping itself around the collagen triple helix. By connecting the N and C termini through an MMP recognition sequence, a dual-specific MMP-sensitive collagen-targeting ligand is obtained that can be used

Extraction Optimization and Characterization of Collagen from Yellow Pike Conger Swimbladder with Acid-Hydro-Exctraction Method

Directory of Open Access Journals (Sweden)

Fernandy Djailani

2016-08-01

Full Text Available Swim bladder is one of marine potential byproducts for alternative source of collagen. This study aimedto optimize hydro-extraction and characterized collagen. Extraction optimization of collagen was determinedusing Box-behnken design response surface method with three variables: CH3COOH concentration, soakingtime and extraction time to yield response. Hydro-extraction collagen was characterization based on theamino acid content, SDS-PAGE, FT-IR and DSC. Acording to the result, the concentration had significantlyinfluence yield. Optimum extraction conditions were variable combinations of acetic acid concentration of0.1 M, for 1 h and hydro-extraction time of 1 h to produce the highest yield 63.35%. collagen was classifiedas type I collagen by amino acid content, electrophoresis patterns and fourier transform infrared (FTIRspectra. The glass transition of collagen was 67.23oC, showed high thermal stability that can be applied tothe cosmetics industry and nutraceutical.

Artificially modified collagen fibril orientation affects leather tear strength.

Science.gov (United States)

Kelly, Susyn J; Wells, Hannah C; Sizeland, Katie H; Kirby, Nigel; Edmonds, Richard L; Ryan, Tim; Hawley, Adrian; Mudie, Stephen; Haverkamp, Richard G

2018-07-01

Ovine leather has around half the tear strength of bovine leather and is therefore not suitable for high-value applications such as shoes. Tear strength has been correlated with the natural collagen fibril alignment (orientation index, OI). It is hypothesized that it could be possible to artificially increase the OI of the collagen fibrils and that an artificial increase in OI could increase tear strength. Ovine skins, after pickling and bating, were strained biaxially during chrome tanning. The strain ranged from 2 to 15% of the initial sample length, either uniformly in both directions by 10% or with 3% in one direction and 15% in the other. Once tanned, the leather tear strengths were measured and the collagen fibril orientation was measured using synchrotron-based small-angle X-ray scattering. The OI increased as a result of strain during tanning from 0.48 to 0.79 (P = 0.001) measured edge-on and the thickness-normalized tear strength increased from 27 to 43 N mm -1 (P leather was strained 10% in two orthogonal directions. This is evidence to support a causal relationship between high OI (measured edge-on), highly influenced by thickness, and tear strength. It also provides a method to produce stronger leather. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Collagen Conduit Versus Microsurgical Neurorrhaphy

DEFF Research Database (Denmark)

Boeckstyns, Michel; Sørensen, Allan Ibsen; Viñeta, Joaquin Fores

2013-01-01

To compare repair of acute lacerations of mixed sensory-motor nerves in humans using a collagen tube versus conventional repair.......To compare repair of acute lacerations of mixed sensory-motor nerves in humans using a collagen tube versus conventional repair....

Structural properties of pepsin-solubilized collagen acylated by lauroyl chloride along with succinic anhydride

International Nuclear Information System (INIS)

Li, Conghu; Tian, Zhenhua; Liu, Wentao; Li, Guoying

2015-01-01

The structural properties of pepsin-solubilized calf skin collagen acylated by lauroyl chloride along with succinic anhydride were investigated in this paper. Compared with native collagen, acylated collagen retained the unique triple helix conformation, as determined by amino acid analysis, circular dichroism and X-ray diffraction. Meanwhile, the thermostability of acylated collagen using thermogravimetric measurements was enhanced as the residual weight increased by 5%. With the temperature increased from 25 to 115 °C, the secondary structure of native and acylated collagens using Fourier transform infrared spectroscopy measurements was destroyed since the intensity of the major amide bands decreased and the positions of the major amide bands shifted to lower wavenumber, respectively. Meanwhile, two-dimensional correlation spectroscopy revealed that the most sensitive bands for acylated and native collagens were amide I and II bands, respectively. Additionally, the corresponding order of the groups between native and acylated collagens was different and the correlation degree for acylated collagen was weaker than that of native collagen, suggesting that temperature played a small influence on the conformation of acylated collagen, which might be concluded that the hydrophobic interaction improved the thermostability of collagen. - Highlights: • Acylated collagen retained the unique triple helix conformation. • Acylated collagen had stronger thermostability than native collagen. • Amide I was the most sensitive band to the temperature for acylated collagen. • Amide II was the most sensitive band to the temperature for native collagen. • Auto-peak at 1680 cm −1 for acylated collagen disappeared at higher temperature

Disintegration of collagen fibrils by Glucono-δ-lactone: An implied lead for disintegration of fibrosis.

Science.gov (United States)

Jayamani, Jayaraman; Ravikanth Reddy, R; Madhan, Balaraman; Shanmugam, Ganesh

2018-02-01

Excess accumulation of collagen (fibrosis) undergoes self-aggregation, which leads to fibrillar collagen, on the extracellular matrix is the hallmark of a number of diseases such as keloids, hypertrophic scars, and systemic scleroderma. Direct inhibition or disintegration of collagen fibrils by small molecules offer a therapeutic approach to prevent or treat the diseases related to fibrosis. Herein, the anti-fibrotic property of Glucono-δ-lactone (GdL), known as acidifier, on the fibrillation and its disintegration of collagen was investigated. As collagen fibrillation is pH dependent, the pH modulation property of GdL is attractive to inhibit self-association of collagen. Optical density and microscopic data indicate that GdL elicits concentration-dependent fibril inhibition and also disintegrates pre-formed collagen fibrils. The simultaneous pH analysis showed that the modulation(lowering) of pH by GdL is the primary cause for its anti-fibrotic activity. The intact triple helical structure of collagen upon treatment of GdL suggests that collagen fibril disintegration can be achieved without affecting the native structure of collagen which is essential for any anti-fibrotic agents. Saturation transfer difference (STD) NMR result reveals that GdL is in proximity to collagen. The present results thus suggest that GdL provides a lead to design novel anti-fibrotic agents for the pathologies related to collagen deposition. Copyright © 2017 Elsevier B.V. All rights reserved.

Collagen targeting using multivalent protein-functionalized dendrimers

NARCIS (Netherlands)

Breurken, M.; Lempens, E.H.M.; Temming, R.P.; Helms, B.A.; Meijer, E.W.; Merkx, M.

2011-01-01

Collagen is an attractive marker for tissue remodeling in a variety of common disease processes. Here we report the preparation of protein dendrimers as multivalent collagen targeting ligands by native chemical ligation of the collagen binding protein CNA35 to cysteine-functionalized dendritic

Building blocks of Collagen based biomaterial devices

Indian Academy of Sciences (India)

First page Back Continue Last page Overview Graphics. Building blocks of Collagen based biomaterial devices. Collagen as a protein. Collagen in tissues and organs. Stabilizing and cross linking agents. Immunogenicity. Hosts (drugs). Controlled release mechanisms of hosts. Biodegradability, workability into devices ...

Polarized Raman anisotropic response of collagen in tendon: towards 3D orientation mapping of collagen in tissues.

Directory of Open Access Journals (Sweden)

Leonardo Galvis

Full Text Available In this study, polarized Raman spectroscopy (PRS was used to characterize the anisotropic response of the amide I band of collagen as a basis for evaluating three-dimensional collagen fibril orientation in tissues. Firstly, the response was investigated theoretically by applying classical Raman theory to collagen-like peptide crystal structures. The theoretical methodology was then tested experimentally, by measuring amide I intensity anisotropy in rat tail as a function of the orientation of the incident laser polarization. For the theoretical study, several collagen-like triple-helical peptide crystal structures obtained from the Protein Data Bank were rotated "in plane" and "out of plane" to evaluate the role of molecular orientation on the intensity of the amide I band. Collagen-like peptides exhibit a sinusoidal anisotropic response when rotated "in plane" with respect to the polarized incident laser. Maximal intensity was obtained when the polarization of the incident light is perpendicular to the molecule and minimal when parallel. In the case of "out of plane" rotation of the molecular structure a decreased anisotropic response was observed, becoming completely isotropic when the structure was perpendicular to the plane of observation. The theoretical Raman response of collagen was compared to that of alpha helical protein fragments. In contrast to collagen, alpha helices have a maximal signal when incident light is parallel to the molecule and minimal when perpendicular. For out-of-plane molecular orientations alpha-helix structures display a decreased average intensity. Results obtained from experiments on rat tail tendon are in excellent agreement with the theoretical predictions, thus demonstrating the high potential of PRS for experimental evaluation of the three-dimensional orientation of collagen fibers in biological tissues.

Changes in guinea-pig dermal collagen during development

International Nuclear Information System (INIS)

Shuttleworth, C.A.; Forrest, L.

1975-01-01

Guinea-pig dermis was digested with pepsin and the solubilized collagen molecules separated by differential salt precipitation at pH 7.5. Differences in subunit composition and amino acid analysis were noted between type I and type III collagen. Incorporation of radioactive proline into the developing foetus enabled isolation of labelled type I and type III collagens. Comparison of the specific activity of the isolated collagen molecules showed that type III collagen had a high specific activity in the early stages of foetal development, which decreased dramatically during foetal development. The specific activity of pepsin-solubilized type I collagen remained fairly constant during foetal development. (orig.) [de

Combined effects of moderately elevated blood glucose and locally produced TGF-beta1 on glomerular morphology and renal collagen production

DEFF Research Database (Denmark)

Krag, Søren; Nyengaard, Jens R; Wogensen, Lise

2007-01-01

BACKGROUND: There is a correlation between renal graft rejection and blood glucose (BG) levels. Furthermore, diabetic patients may develop non-diabetic renal diseases, which in some circumstances progress rapidly. Since transforming growth factor-beta1 (TGF-beta) levels are elevated in many renal...... diseases, the accelerated progression may be due to interactions between glucose and locally produced TGF-beta1. Therefore, we investigated the effect of mild hyperglycaemia on glomerular morphology and collagen production in TGF-beta1 transgenic mice. METHODS: To achieve BG concentrations of approximately...... 15 mmol/l in TGF-beta1 transgenic and non-transgenic mice, we used multiple streptozotocin (STZ) injections, and after 8 weeks, we measured the changes in glomerular morphology and total collagen content. We also analysed extracellular matrix (ECM) and protease mRNA levels using real-time polymerase...

RB1CC1 Protein Suppresses Type II Collagen Synthesis in Chondrocytes and Causes Dwarfism*

Science.gov (United States)

Nishimura, Ichiro; Chano, Tokuhiro; Kita, Hiroko; Matsusue, Yoshitaka; Okabe, Hidetoshi

2011-01-01

RB1-inducible coiled-coil 1 (RB1CC1) functions in various processes, such as cell growth, differentiation, senescence, apoptosis, and autophagy. The conditional transgenic mice with cartilage-specific RB1CC1 excess that were used in the present study were made for the first time by the Cre-loxP system. Cartilage-specific RB1CC1 excess caused dwarfism in mice without causing obvious abnormalities in endochondral ossification and subsequent skeletal development from embryo to adult. In vitro and in vivo analysis revealed that the dwarf phenotype in cartilaginous RB1CC1 excess was induced by reductions in the total amount of cartilage and the number of cartilaginous cells, following suppressions of type II collagen synthesis and Erk1/2 signals. In addition, we have demonstrated that two kinds of SNPs (T-547C and C-468T) in the human RB1CC1 promoter have significant influence on the self-transcriptional level. Accordingly, human genotypic variants of RB1CC1 that either stimulate or inhibit RB1CC1 transcription in vivo may cause body size variations. PMID:22049074

Lysyl oxidase activity is required for ordered collagen fibrillogenesis by tendon cells

DEFF Research Database (Denmark)

Herchenhan, Andreas; Uhlenbrock, Franziska Katharina; Eliasson, Pernilla

2015-01-01

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...

Laser welding and collagen crosslinks

Energy Technology Data Exchange (ETDEWEB)

Reiser, K.M.; Last, J.A. [California Univ., Davis, CA (United States). Dept. of Medicine; Small, W. IV; Maitland, D.J.; Heredia, N.J.; Da Silva, L.B.; Matthews, D.L. [Lawrence Livermore National Lab., CA (United States)

1997-02-20

Strength and stability of laser-welded tissue may be influenced, in part, by effects of laser exposure on collagen crosslinking. We therefore studied effects of diode laser exposure (805 nm, 1-8 watts, 30 seconds) + indocyanine green dye (ICG) on calf tail tendon collagen crosslinks. Effect of ICG dye alone on crosslink content prior to laser exposure was investigated; unexpectedly, we found that ICG-treated tissue had significantly increased DHLNL and OHP, but not HLNL. Laser exposure after ICG application reduced elevated DHLNL and OHP crosslink content down to their native levels. The monohydroxylated crosslink HLNL was inversely correlated with laser output (p<0.01 by linear regression analysis). DHLNL content was highly correlated with content of its maturational product, OHP, suggesting that precursor-product relations are maintained. We conclude that: (1)ICG alone induces DHLNL and OHP crosslink formation; (2)subsequent laser exposure reduces the ICG-induced crosslinks down to native levels; (3)excessive diode laser exposure destroys normally occurring HLNL crosslinks.

Dermatan Sulfate Epimerase 1-Deficient Mice Have Reduced Content and Changed Distribution of Iduronic Acids in Dermatan Sulfate and an Altered Collagen Structure in Skin

DEFF Research Database (Denmark)

Maccarana, M.; Kalamajski, S.; Kongsgaard, M.

2009-01-01

Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks......-derived chains. DS-epi1-deficient mice are smaller than their wild-type littermates but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans, and the consequences for skin collagen structure were initially analyzed. We found...... that the skin collagen architecture was altered, and electron microscopy showed that the DS-epi1-null fibrils have a larger diameter than the wild-type fibrils. The altered chondroitin/dermatan sulfate chains carried by decorin in skin are likely to affect collagen fibril formation and reduce the tensile...

uPARAP/endo180 directs lysosomal delivery and degradation of collagen IV

DEFF Research Database (Denmark)

Kjøller, Lars; Engelholm, Lars H; Høyer-Hansen, Maria

2004-01-01

Collagen turnover is crucial for tissue homeostasis and remodeling and pathological processes such as cancer invasion, but the underlying molecular mechanisms are poorly understood. A major pathway appears to be internalization and degradation by fibroblasts. We now show that the endocytic...... transmembrane glycoprotein urokinase plasminogen activator receptor-associated protein (uPARAP/endo180) directs collagen IV for lysosomal delivery and degradation. In wild-type fibroblasts, fluorescently labeled collagen IV was first internalized into vesicular structures with diffuse fluorescence eventually...... appearing uniformly within the wild-type cells after longer incubation times. In these cells, some collagen-containing vesicles were identified as lysosomes by staining for LAMP-1. In contrast, collagen IV remained extracellular and associated with fiber-like structures on uPARAP/endo180-deficient...

Immune responses to implanted human collagen graft in rats

International Nuclear Information System (INIS)

Quteish, D.; Dolby, A.E.

1991-01-01

Immunity to collagen implants may be mediated by cellular and humoral immune responses. To examine the possibility of such immunological reactivity and crossreactivity to collagen, 39 Sprague-Dawley rats (female, 10 weeks old, approximately 250 g wt) were implanted subcutaneously at thigh sites with crosslinked, freeze-dried human placental type I collagen grafts (4x4x2 mm) which had been irradiated (520 Gray) or left untreated. Blood was obtained by intracardiac sampling prior to implantation or from normal rats, and at various times afterwards when the animals were sacrificed. The sera from these animals were examined for circulating antibodies to human, bovine and rat tail (type I) collagens by enzyme-linked immunosorbent assay (ELISA). Also, the lymphoblastogenic responses of spleen lymphocytes from the irradiated collagen-implanted animals were assessed in culture by measuring thymidine uptake with autologous and normal rat sera in the presence of human bovine type I collagens. Implantation of the irradiated and non-irradiated collagen graft in rats led to a significant increase in the level of circulating antibodies to human collagen. Also antibody to bovine and rat tail collagens was detectable in the animals implanted with irradiated collagen grafts but at a lower level than the human collagen. There was a raised lymphoblastogenic response to both human and bovine collagens. The antibody level and lymphoblastogenesis to the tested collagens gradually decreased towards the end of the post-implantation period. (author)

Training-induced changes in peritendinous type I collagen turnover determined by microdialysis in humans

DEFF Research Database (Denmark)

Langberg, Henning; Rosendal, L; Kjaer, M

2001-01-01

1. Acute exercise is found to increase collagen type I formation locally in peritendinous connective tissue of the Achilles' tendon in humans, as determined from changes in interstitial concentrations of collagen propeptide (PICP) and a collagen degradation product (ICTP) by the use of microdialy...

Deficiency of CRTAP in non-lethal recessive osteogenesis imperfecta reduces collagen deposition into matrix.

Science.gov (United States)

Valli, M; Barnes, A M; Gallanti, A; Cabral, W A; Viglio, S; Weis, M A; Makareeva, E; Eyre, D; Leikin, S; Antoniazzi, F; Marini, J C; Mottes, M

2012-11-01

Deficiency of any component of the ER-resident collagen prolyl 3-hydroxylation complex causes recessive osteogenesis imperfecta (OI). The complex modifies the α1(I)Pro986 residue and contains cartilage-associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and cyclophilin B (CyPB). Fibroblasts normally secrete about 10% of CRTAP. Most CRTAP mutations cause a null allele and lethal type VII OI. We identified a 7-year-old Egyptian boy with non-lethal type VII OI and investigated the effects of his null CRTAP mutation on collagen biochemistry, the prolyl 3-hydroxylation complex, and collagen in extracellular matrix. The proband is homozygous for an insertion/deletion in CRTAP (c.118_133del16insTACCC). His dermal fibroblasts synthesize fully overmodified type I collagen, and 3-hydroxylate only 5% of α1(I)Pro986. CRTAP transcripts are 10% of control. CRTAP protein is absent from proband cells, with residual P3H1 and normal CyPB levels. Dermal collagen fibril diameters are significantly increased. By immunofluorescence of long-term cultures, we identified a severe deficiency (10-15% of control) of collagen deposited in extracellular matrix, with disorganization of the minimal fibrillar network. Quantitative pulse-chase experiments corroborate deficiency of matrix deposition, rather than increased matrix turnover. We conclude that defects of extracellular matrix, as well as intracellular defects in collagen modification, contribute to the pathology of type VII OI. © 2011 John Wiley & Sons A/S.

Fluorescence studies on the aggregation behaviors of collagen modified with NHS-activated poly(γ-glutamic acid).

Science.gov (United States)

Zhang, Min; Yang, Junhui; Yang, Qili; Huang, Liulian; Wu, Hui; Chen, Lihui; Ding, Cuicui

2018-06-01

The poly(γ-glutamic acid)-NHS (γ-PGA-NHS) esters were used to endow collagen with both of excellent water-solubility and thermal stability via cross-linking reaction between γ-PGA-NHS and collagen. In the present work, the effect of γ-PGA-NHS on the aggregation of collagen molecules was studied by fluorescence techniques. The fluorescence emission spectra of pyrene in collagen solutions and the intrinsic fluorescence emission spectra of collagen suggested different effects of γ-PGA-NHS on collagen molecules: inhibiting aggregation below critical aggregation concentration (CAC) and promoting aggregation above CAC. The two-dimensional (2D) fluorescence correlation spectra indicated that the intermolecular hydrogen bonding and cross-linking between γ-PGA-NHS and collagen would influence the aggregation of collagen molecules. By the ultra-sensitive differential scanning calorimeter (VP-DSC), it was found that the main denaturational transition temperature (T m2 ) of modified collagen increased, while its calorimetric enthalpy changes (ΔH 2 ) decreased compared to those of native collagen, further indicating that the modification of γ-PGA-NHS influenced the aggregation of collagen molecules. The study provide useful information for the utilizing and or the processing of water-soluble collagen in aqueous solution in the fields such as cosmetics, health care products, tissue engineering and biomedical materials, etc. Copyright © 2018 Elsevier B.V. All rights reserved.

Collagen Fingerprinting: A New Screening Technique for Radiocarbon Dating Ancient Bone.

Directory of Open Access Journals (Sweden)

Virginia L Harvey

Full Text Available Collagen is the dominant organic component of bone and is intimately locked within the hydroxyapatite structure of this ubiquitous biomaterial that dominates archaeological and palaeontological assemblages. Radiocarbon analysis of extracted collagen is one of the most common approaches to dating bone from late Pleistocene or Holocene deposits, but dating is relatively expensive compared to other biochemical techniques. Numerous analytical methods have previously been investigated for the purpose of screening out samples that are unlikely to yield reliable dates including histological analysis, UV-stimulated fluorescence and, most commonly, the measurement of percentage nitrogen (%N and ratio of carbon to nitrogen (C:N. Here we propose the use of collagen fingerprinting (also known as Zooarchaeology by Mass Spectrometry, or ZooMS, when applied to species identification as an alternative screening method for radiocarbon dating, due to its ability to provide information on collagen presence and quality, alongside species identification. The method was tested on a series of sub-fossil bone specimens from cave systems on Cayman Brac (Cayman Islands, chosen due to the observable range in diagenetic alteration, and in particular, the extent of mineralisation. Six (14C dates, of 18 initial attempts, were obtained from remains of extinct hutia, Capromys sp. (Rodentia; Capromyidae, recovered from five distinct caves on Cayman Brac, and ranging from 393 ± 25 to 1588 ± 26 radiocarbon years before present (yr BP. All of the bone samples that yielded radiocarbon dates generated excellent collagen fingerprints, and conversely those that gave poor fingerprints also failed dating. Additionally, two successfully fingerprinted bone samples were screened out from a set of 81. Both subsequently generated (14C dates, demonstrating successful utilisation of ZooMS as an alternative screening mechanism to identify bone samples that are suitable for 1(4C analysis.

Collagen Fingerprinting: A New Screening Technique for Radiocarbon Dating Ancient Bone.

Science.gov (United States)

Harvey, Virginia L; Egerton, Victoria M; Chamberlain, Andrew T; Manning, Phillip L; Buckley, Michael

2016-01-01

Collagen is the dominant organic component of bone and is intimately locked within the hydroxyapatite structure of this ubiquitous biomaterial that dominates archaeological and palaeontological assemblages. Radiocarbon analysis of extracted collagen is one of the most common approaches to dating bone from late Pleistocene or Holocene deposits, but dating is relatively expensive compared to other biochemical techniques. Numerous analytical methods have previously been investigated for the purpose of screening out samples that are unlikely to yield reliable dates including histological analysis, UV-stimulated fluorescence and, most commonly, the measurement of percentage nitrogen (%N) and ratio of carbon to nitrogen (C:N). Here we propose the use of collagen fingerprinting (also known as Zooarchaeology by Mass Spectrometry, or ZooMS, when applied to species identification) as an alternative screening method for radiocarbon dating, due to its ability to provide information on collagen presence and quality, alongside species identification. The method was tested on a series of sub-fossil bone specimens from cave systems on Cayman Brac (Cayman Islands), chosen due to the observable range in diagenetic alteration, and in particular, the extent of mineralisation. Six (14)C dates, of 18 initial attempts, were obtained from remains of extinct hutia, Capromys sp. (Rodentia; Capromyidae), recovered from five distinct caves on Cayman Brac, and ranging from 393 ± 25 to 1588 ± 26 radiocarbon years before present (yr BP). All of the bone samples that yielded radiocarbon dates generated excellent collagen fingerprints, and conversely those that gave poor fingerprints also failed dating. Additionally, two successfully fingerprinted bone samples were screened out from a set of 81. Both subsequently generated (14)C dates, demonstrating successful utilisation of ZooMS as an alternative screening mechanism to identify bone samples that are suitable for 1(4)C analysis.

[Three-dimensional parallel collagen scaffold promotes tendon extracellular matrix formation].

Science.gov (United States)

Zheng, Zefeng; Shen, Weiliang; Le, Huihui; Dai, Xuesong; Ouyang, Hongwei; Chen, Weishan

2016-03-01

To investigate the effects of three-dimensional parallel collagen scaffold on the cell shape, arrangement and extracellular matrix formation of tendon stem cells. Parallel collagen scaffold was fabricated by unidirectional freezing technique, while random collagen scaffold was fabricated by freeze-drying technique. The effects of two scaffolds on cell shape and extracellular matrix formation were investigated in vitro by seeding tendon stem/progenitor cells and in vivo by ectopic implantation. Parallel and random collagen scaffolds were produced successfully. Parallel collagen scaffold was more akin to tendon than random collagen scaffold. Tendon stem/progenitor cells were spindle-shaped and unified orientated in parallel collagen scaffold, while cells on random collagen scaffold had disorder orientation. Two weeks after ectopic implantation, cells had nearly the same orientation with the collagen substance. In parallel collagen scaffold, cells had parallel arrangement, and more spindly cells were observed. By contrast, cells in random collagen scaffold were disorder. Parallel collagen scaffold can induce cells to be in spindly and parallel arrangement, and promote parallel extracellular matrix formation; while random collagen scaffold can induce cells in random arrangement. The results indicate that parallel collagen scaffold is an ideal structure to promote tendon repairing.

[Research on ultrasonic permeability of low intensity pulsed ultrasound through PTFE membrane and Bio-Gide collagen membrane].

Science.gov (United States)

Chai, Zhaowu; Zhao, Chunliang; Song, Jinlin; Deng, Feng; Yang, Ji; Gao, Xiang; Liu, Minyi

2013-12-01

The aim of the present study was to detect the transmission rate of ultrasonic low intensity pulsed ultrasound (LIPUS) through polytetrafluoroethylene (PTFE) membrane (Thickness: 0.01 mm) and Bio-Gide collagen membrane, and to provide the basis for the barrier membrane selection on the study of LIPUS combined with guided tissue regeneration (GTR). The ultrasonic (LIPUS, frequency 1.5 MHz, pulse width 200 micros, repetition rate 1.0 kHz) transmission coefficient of the two kinds of barrier membrane were detected respectively through setting ten groups from 10 to 100mW/cm2 every other 10 mW/cm2. We found in the study that the ultrasonic transmission coefficient through 0.01 mm PTFE membrane was 78.1% to 92.%, and the ultrasonic transmission coefficient through Bio-Gide collagen membrane was 43.9% to 55.8%. The ultrasonic transmission coefficient through PTFE membrane was obviously higher than that through Bio-Gide collagen membrane. The transmission coefficient of the same barrier membrane of the ultrasonic ion was statistically different under different powers (P PTFE membrane and Bio-Gide collagen membrane were relatively high. We should select barrier membranes based on different experimental needs, and exercise ultrasonic transmission coefficient experiments to ensure effective power.

Fluorescent Labeling of Collagen Production by Cells for Noninvasive Imaging of Extracellular Matrix Deposition.

Science.gov (United States)

Bardsley, Katie; Yang, Ying; El Haj, Alicia J

2017-04-01

Extracellular matrix (ECM) is an essential component of tissues and provides both integrity and biological cues for cells. Collagen is one of the major proteins found within the ECM and therefore is an essential component of all engineered tissues. Therefore, in this article, we present a method for the online real-time monitoring of collagen deposition in three-dimensional engineered constructs. This method revolves around modification of collagen through the addition of azide-L-proline to cell culture media. The incorporation of azide-L-proline into the neocollagen produced by cells can then be detected by reaction with 10 mM of a Click-IT Alexa Fluor 488 DIBO Alkyne. The reaction was shown as being specific to the collagen as little background staining was observed in cultures, which did not contain the modified proline, and the staining was also depleted after treatment with collagenase and colocalization of collagen type I staining by immunochemistry assay. Real-time online staining of collagen deposition was observed under different culture conditions without affecting proliferation. Collagen deposition was observed to be increased under mechanical stimulation; however, the localization varied across stimulation regimes. This is a new technique for real-time monitoring of cell-produced collagen and will be a valuable addition to the tissue engineering field.

Vitamin C–enriched gelatin supplementation before intermittent activity augments collagen synthesis12

Science.gov (United States)

Shaw, Gregory; Lee-Barthel, Ann; Ross, Megan LR; Wang, Bing; Baar, Keith

2017-01-01

Background: Musculoskeletal injuries are the most common complaint in active populations. More than 50% of all injuries in sports can be classified as sprains, strains, ruptures, or breaks of musculoskeletal tissues. Nutritional and/or exercise interventions that increase collagen synthesis and strengthen these tissues could have an important effect on injury rates. Objective: This study was designed to determine whether gelatin supplementation could increase collagen synthesis. Design: Eight healthy male subjects completed a randomized, double-blinded, crossover-design study in which they consumed either 5 or 15 g of vitamin C–enriched gelatin or a placebo control. After the initial drink, blood was taken every 30 min to determine amino acid content in the blood. A larger blood sample was taken before and 1 h after consumption of gelatin for treatment of engineered ligaments. One hour after the initial supplement, the subjects completed 6 min of rope-skipping to stimulate collagen synthesis. This pattern of supplementation was repeated 3 times/d with ≥6 h between exercise bouts for 3 d. Blood was drawn before and 4, 24, 48, and 72 h after the first exercise bout for determination of amino-terminal propeptide of collagen I content. Results: Supplementation with increasing amounts of gelatin increased circulating glycine, proline, hydroxyproline, and hydroxylysine, peaking 1 h after the supplement was given. Engineered ligaments treated for 6 d with serum from samples collected before or 1 h after subjects consumed a placebo or 5 or 15 g gelatin showed increased collagen content and improved mechanics. Subjects who took 15 g gelatin 1 h before exercise showed double the amino-terminal propeptide of collagen I in their blood, indicating increased collagen synthesis. Conclusion: These data suggest that adding gelatin to an intermittent exercise program improves collagen synthesis and could play a beneficial role in injury prevention and tissue repair. This trial

Dwarfism in mice lacking collagen-binding integrins alpha 2 beta 1 and alpha 11 beta 1 is caused by severely diminished IGF-1 levels

OpenAIRE

Blumbach, Katrin; Niehoff, Anja; Belgardt, Bengt F.; Ehlen, Harald W.A.; Schmitz, Markus; Hallinger, Ralf; Schulz, Jan-Niklas; Brüning, Jens C.; Krieg, Thomas; Schubert, Markus; Gullberg, Donald; Eckes, Beate

2012-01-01

Mice with a combined deficiency in the α2β1 and α11β1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggest...

Understanding Collagen Organization in Breast Tumors to Predict and Prevent Metastasis

Science.gov (United States)

2015-11-01

mouse mammary tumor virus polyoma middle T (MMTV-PyMT) mice crossed with MMP13 KO mice, noted proportionately more “thin collagen fibers” (rela- tive to...mammary gland gene expression and increased tumor growth following social isolation. Cancer Prev. Res. 2, 850–861. Wohleb, E.S., Hanke, M.L., Corona , A.W...1:100 dilution of mouse anti-Collagen II (II-II6B3; Developmental Studies Hybridoma Bank, Iowa City, IA) or a mouse monoclonal anti-Collagen I ( Cat

Parametric imaging of collagen structural changes in human osteoarthritic cartilage using optical polarization tractography

Science.gov (United States)

Ravanfar, Mohammadreza; Pfeiffer, Ferris M.; Bozynski, Chantelle C.; Wang, Yuanbo; Yao, Gang

2017-12-01

Collagen degeneration is an important pathological feature of osteoarthritis. The purpose of this study is to investigate whether the polarization-sensitive optical coherence tomography (PSOCT)-based optical polarization tractography (OPT) can be useful in imaging collagen structural changes in human osteoarthritic cartilage samples. OPT eliminated the banding artifacts in conventional PSOCT by calculating the depth-resolved local birefringence and fiber orientation. A close comparison between OPT and PSOCT showed that OPT provided improved visualization and characterization of the zonal structure in human cartilage. Experimental results obtained in this study also underlined the importance of knowing the collagen fiber orientation in conventional polarized light microscopy assessment. In addition, parametric OPT imaging was achieved by quantifying the surface roughness, birefringence, and fiber dispersion in the superficial zone of the cartilage. These quantitative parametric images provided complementary information on the structural changes in cartilage, which can be useful for a comprehensive evaluation of collagen damage in osteoarthritic cartilage.

Chondroitin Sulfate Perlecan Enhances Collagen Fibril Formation

DEFF Research Database (Denmark)

Kvist, A. J.; Johnson, A. E.; Mörgelin, M.

2006-01-01

in collagen type II fibril assembly by perlecan-null chondrocytes. Cartilage perlecan is a heparin sulfate or a mixed heparan sulfate/chondroitin sulfate proteoglycan. The latter form binds collagen and accelerates fibril formation in vitro, with more defined fibril morphology and increased fibril diameters...... produced in the presence of perlecan. Interestingly, the enhancement of collagen fibril formation is independent on the core protein and is mimicked by chondroitin sulfate E but neither by chondroitin sulfate D nor dextran sulfate. Furthermore, perlecan chondroitin sulfate contains the 4,6-disulfated...... disaccharides typical for chondroitin sulfate E. Indeed, purified glycosaminoglycans from perlecan-enriched fractions of cartilage extracts contain elevated levels of 4,6-disulfated chondroitin sulfate disaccharides and enhance collagen fibril formation. The effect on collagen assembly is proportional...

Non-enzymatic glycosylation of a type I collagen matrix: effects on osteoblastic development and oxidative stress

Directory of Open Access Journals (Sweden)

Barrio Daniel A

2001-08-01

Full Text Available Abstract Background The tissue accumulation of protein-bound advanced glycation endproducts (AGE may be involved in the etiology of diabetic chronic complications, including osteopenia. The aim of this study was to investigate the effect of an AGE-modified type I collagen substratum on the adhesion, spreading, proliferation and differentiation of rat osteosarcoma UMR106 and mouse non-transformed MC3T3E1 osteoblastic cells. We also studied the role of reactive oxygen species (ROS and nitric oxide synthase (NOS expression on these AGE-collagen mediated effects. Results AGE-collagen decreased the adhesion of UMR106 cells, but had no effect on the attachment of MC3T3E1 cells. In the UMR106 cell line, AGE-collagen also inhibited cellular proliferation, spreading and alkaline phosphatase (ALP activity. In preosteoblastic MC3T3E1 cells (24-hour culture, proliferation and spreading were significantly increased by AGE-collagen. After one week of culture (differentiated MC3T3E1 osteoblasts AGE-collagen inhibited ALP activity, but had no effect on cell number. In mineralizing MC3T3E1 cells (3-week culture AGE-collagen induced a decrease in the number of surviving cells and of extracellular nodules of mineralization, without modifying their ALP activity. Intracellular ROS production, measured after a 48-hour culture, was decreased by AGE-collagen in MC3T3E1 cells, but was increased by AGE-collagen in UMR106 cells. After a 24-hour culture, AGE-collagen increased the expression of endothelial and inducible NOS, in both osteoblastic cell lines. Conclusions These results suggest that the accumulation of AGE on bone extracellular matrix could regulate the proliferation and differentiation of osteoblastic cells. These effects appear to depend on the stage of osteoblastic development, and possibly involve the modulation of NOS expression and intracellular ROS pathways.

Hydrolysates of Fish Skin Collagen: An Opportunity for Valorizing Fish Industry Byproducts.

Science.gov (United States)

Blanco, María; Vázquez, José Antonio; Pérez-Martín, Ricardo I; Sotelo, Carmen G

2017-05-05

During fish processing operations, such as skinning and filleting, the removal of collagen-containing materials can account for up to 30% of the total fish byproducts. Collagen is the main structural protein in skin, representing up to 70% of dry weight depending on the species, age and season. It has a wide range of applications including cosmetic, pharmaceutical, food industry, and medical. In the present work, collagen was obtained by pepsin extraction from the skin of two species of teleost and two species of chondrychtyes with yields varying between 14.16% and 61.17%. The storage conditions of the skins appear to influence these collagen extractions yields. Pepsin soluble collagen (PSC) was enzymatically hydrolyzed and the resultant hydrolysates were ultrafiltrated and characterized. Electrophoretic patterns showed the typical composition of type I collagen, with denaturation temperatures ranged between 23 °C and 33 °C. In terms of antioxidant capacity, results revealed significant intraspecific differences between hydrolysates, retentate, and permeate fractions when using β -Carotene and DPPH methods and also showed interspecies differences between those fractions when using DPPH and ABTS methods. Under controlled conditions, PSC hydrolysates from Prionace glauca , Scyliorhinus canicula , Xiphias gladius, and Thunnus albacares provide a valuable source of peptides with antioxidant capacities constituting a feasible way to efficiently upgrade fish skin biomass.

The anabolic effects of insulin on type II collagen synthesis of Swarm rat chondrosarcoma chondrocytes

International Nuclear Information System (INIS)

Bembenek, M.E.; Liberti, J.P.

1984-01-01

The anabolic effects of insulin on collagen production of freshly isolated Swarm rat chondrosarcoma chondrocytes were investigated. The specific radioactivity of newly synthesized collagen was not increased by insulin, indicating that the hormone has no effect on the specific radioactivity of the aminoacyl tRNA pool. Results of further studies obtained from collagen degradation experiments demonstrated that insulin did not alter the rate of [3H]collagen degradation. Together, these results clearly indicate that insulin stimulates collagen biosynthesis. Polyacrylamide gel analysis of the newly synthesized collagen of both control and insulin-stimulated cells revealed a large-molecular-weight component which migrated with authentic alpha 1(II) collagen and was collagenase-sensitive. Additional studies showed that, although insulin increased the processing and secretion of collagen, the hormone did not cause a shift in the distribution of the extracellular and intracellular collagen pools. Finally, results of studies conducted with the transcriptional inhibitor, actinomycin D, indicated that the anabolic effects of insulin on collagen and non-collagen proteins were mediated at a post-transcriptional site

A New Kind of Biomaterials-Bullfrog Skin Collagen

Institute of Scientific and Technical Information of China (English)

He LI; Bai Ling LIU; Hua Lin CHEN; Li Zhen GAO

2003-01-01

Pepsin-soluble collagen was prepared from bullfrog skin and partially characterized. This study revealed interesting differences, such as molecular weight, amino acid composition, denaturation temperature (Td), in the frog skin collagen when compared to the known vertebrate collagens. This study gives hints that bullfrog skin can be a potential, safe alternative source of collagen from cattle for use in various fields.

The decorin sequence SYIRIADTNIT binds collagen type I

DEFF Research Database (Denmark)

Kalamajski, Sebastian; Aspberg, Anders; Oldberg, Ake

2007-01-01

Decorin belongs to the small leucine-rich repeat proteoglycan family, interacts with fibrillar collagens, and regulates the assembly, structure, and biomechanical properties of connective tissues. The decorin-collagen type I-binding region is located in leucine-rich repeats 5-6. Site......-directed mutagenesis of this 54-residue-long collagen-binding sequence identifies Arg-207 and Asp-210 in leucine-rich repeat 6 as crucial for the binding to collagen. The synthetic peptide SYIRIADTNIT, which includes Arg-207 and Asp-210, inhibits the binding of full-length recombinant decorin to collagen in vitro....... These collagen-binding amino acids are exposed on the exterior of the beta-sheet-loop structure of the leucine-rich repeat. This resembles the location of interacting residues in other leucine-rich repeat proteins....

Tumor cell culture on collagen-chitosan scaffolds as three-dimensional tumor model: A suitable model for tumor studies.

Science.gov (United States)

Mahmoudzadeh, Aziz; Mohammadpour, Hemn

2016-07-01

Tumor cells naturally live in three-dimensional (3D) microenvironments, while common laboratory tests and evaluations are done in two-dimensional (2D) plates. This study examined the impact of cultured 4T1 cancer cells in a 3D collagen-chitosan scaffold compared with 2D plate cultures. Collagen-chitosan scaffolds were provided and passed confirmatory tests. 4T1 tumor cells were cultured on scaffolds and then tumor cells growth rate, resistance to X-ray radiation, and cyclophosphamide as a chemotherapy drug were analyzed. Furthermore, 4T1 cells were extracted from the scaffold model and were injected into the mice. Tumor growth rate, survival rate, and systemic immune responses were evaluated. Our results showed that 4T1 cells infiltrated the scaffolds pores and constructed a 3D microenvironment. Furthermore, 3D cultured tumor cells showed a slower proliferation rate, increased levels of survival to the X-ray irradiation, and enhanced resistance to chemotherapy drugs in comparison with 2D plate cultures. Transfer of extracted cells to the mice caused enhanced tumor volume and decreased life span. This study indicated that collagen-chitosan nanoscaffolds provide a suitable model of tumor that would be appropriate for tumor studies. Copyright © 2016. Published by Elsevier B.V.

Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling

Directory of Open Access Journals (Sweden)

Anthis Nicholas J

2006-12-01

Full Text Available Abstract Background Lysophosphatidic acid (LPA and sphingosine 1-phosphate (S1P are bioactive lipid signaling molecules implicated in tumor dissemination. Membrane-type matrix metalloproteinase 1 (MT1-MMP is a membrane-tethered collagenase thought to be involved in tumor invasion via extracellular matrix degradation. In this study, we investigated the molecular requirements for LPA- and S1P-regulated tumor cell migration in two dimensions (2D and invasion of three-dimensional (3D collagen matrices and, in particular, evaluated the role of MT1-MMP in this process. Results LPA stimulated while S1P inhibited migration of most tumor lines in Boyden chamber assays. Conversely, HT1080 fibrosarcoma cells migrated in response to both lipids. HT1080 cells also markedly invaded 3D collagen matrices (~700 μm over 48 hours in response to either lipid. siRNA targeting of LPA1 and Rac1, or S1P1, Rac1, and Cdc42 specifically inhibited LPA- or S1P-induced HT1080 invasion, respectively. Analysis of LPA-induced HT1080 motility on 2D substrates vs. 3D matrices revealed that synthetic MMP inhibitors markedly reduced the distance (~125 μm vs. ~45 μm and velocity of invasion (~0.09 μm/min vs. ~0.03 μm/min only when cells navigated 3D matrices signifying a role for MMPs exclusively in invasion. Additionally, tissue inhibitors of metalloproteinases (TIMPs-2, -3, and -4, but not TIMP-1, blocked lipid agonist-induced invasion indicating a role for membrane-type (MT-MMPs. Furthermore, MT1-MMP expression in several tumor lines directly correlated with LPA-induced invasion. HEK293s, which neither express MT1-MMP nor invade in the presence of LPA, were transfected with MT1-MMP cDNA, and subsequently invaded in response to LPA. When HT1080 cells were seeded on top of or within collagen matrices, siRNA targeting of MT1-MMP, but not other MMPs, inhibited lipid agonist-induced invasion establishing a requisite role for MT1-MMP in this process. Conclusion LPA is a

Sumoylation of the Tumor Suppressor Promyelocytic Leukemia Protein Regulates Arsenic Trioxide-Induced Collagen Synthesis in Osteoblasts.

Science.gov (United States)

Xu, Wen-Xiao; Liu, Sheng-Zhi; Wu, Di; Qiao, Guo-Fen; Yan, Jinglong

2015-01-01

Promyelocytic leukemia (PML) protein is a tumor suppressor that fuses with retinoic acid receptor-α (PML-RARα) to contribute to the initiation of acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) upregulates expression of TGF-β1, promoting collagen synthesis in osteoblasts, and ATO binds directly to PML to induce oligomerization, sumoylation, and ubiquitination. However, how ATO upregulates TGF-β1 expression is uncertain. Thus, we suggested that PML sumoylation is responsible for regulation of TGF-β1 protein expression. Kunming mice were treated with ATO, and osteoblasts were counted under scanning electron microscopy. Masson's staining was used to quantify collagen content. hFOB1.19 cells were transfected with siRNA against UBC9 or RNF4, and then treated with ATO or FBS. TGF-β1, PML expression, and sumoylation were quantified with Western blot, and collagen quantified via immunocytochemistry. ATO enhanced osteoblast accumulation, collagen synthesis, and PML-NB formation in vivo. Knocking down UBC9 in hFOB1.19 cells inhibited ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. Conversely, knocking down RNF4 enhanced ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. These data suggest that PML sumoylation is required for ATO-induced collagen synthesis in osteoblasts. © 2015 S. Karger AG, Basel.

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

Science.gov (United States)

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

2009-09-15

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.

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

Directory of Open Access Journals (Sweden)

Joseph P R O Orgel

2009-09-01

Full Text Available 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.

Evaluation of epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes and concerns on osteoblasts

Energy Technology Data Exchange (ETDEWEB)

Chu, Chenyu; Deng, Jia [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Xiang, Lin; Wu, Yingying [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Wei, Xiawei [State Key Laboratory of Biotherapy and Laboratory for Aging Research, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan 610041 (China); Qu, Yili [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Man, Yi, E-mail: manyi780203@126.com [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China); Department of Oral Implantology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041 (China)

2016-10-01

Collagen membranes have ideal biological and mechanical properties for supporting infiltration and proliferation of osteoblasts and play a vital role in guided bone regeneration (GBR). However, pure collagen can lead to inflammation, resulting in progressive bone resorption. Therefore, a method for regulating the level of inflammatory cytokines at surgical sites is paramount for the healing process. Epigallocatechin-3-gallate (EGCG) is a component extracted from green tea with numerous biological activities including an anti-inflammatory effect. Herein, we present a novel cross-linked collagen membrane containing different concentrations of EGCG (0.0064%, 0.064%, and 0.64%) to regulate the level of inflammatory factors secreted by pre-osteoblast cells; improve cell proliferation; and increase the tensile strength, wettability, and thermal stability of collagen membranes. Scanning electron microscope images show that the surfaces of collagen membranes became smoother and the collagen fiber diameters became larger with EGCG treatment. Measurement of the water contact angle demonstrated that introducing EGCG improved membrane wettability. Fourier transform infrared spectroscopy analyses indicated that the backbone of collagen was intact, and the thermal stability was significant improved in differential scanning calorimetry. The mechanical properties of 0.064% and 0.64% EGCG-treated collagen membranes were 1.5-fold greater than those of the control. The extent of cross-linking was significantly increased, as determined by a 2,4,6-trinitrobenzenesulfonic acid solution assay. The Cell Counting Kit-8 (CCK-8) and live/dead assays revealed that collagen membrane cross-linked by 0.0064% EGCG induced greater cell proliferation than pure collagen membranes. Additionally, real-time polymerase chain reaction and enzyme-linked immunosorbent assay results showed that EGCG significantly affected the production of inflammatory factors secreted by MC3T3-E1 cells. Taken together, our

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

International Nuclear Information System (INIS)

Walker, G.; Bourguignon, L.Y.

1990-01-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

Energy Technology Data Exchange (ETDEWEB)

Walker, G.; Bourguignon, L.Y. (Univ. of Miami Medical School, FL (USA))

1990-08-01

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

Structural aspects of fish skin collagen which forms ordered arrays via liquid crystalline states.

Science.gov (United States)

Giraud-Guille, M M; Besseau, L; Chopin, C; Durand, P; Herbage, D

2000-05-01

The ability of acid-soluble type I collagen extracts from Soleidae flat fish to form ordered arrays in condensed phases has been compared with data for calf skin collagen. Liquid crystalline assemblies in vitro are optimized by preliminary treatment of the molecular population with ultrasounds. This treatment requires the stability of the fish collagen triple helicity to be controlled by X-ray diffraction and differential scanning calorimetry and the effect of sonication to be evaluated by viscosity measurements and gel electrophoresis. The collagen solution in concentrations of at least 40 mg ml(-1) showed in polarized light microscopy birefringent patterns typical of precholesteric phases indicating long-range order within the fluid collagen phase. Ultrastructural data, obtained after stabilization of the liquid crystalline collagen into a gelated matrix, showed that neutralized acid-soluble fish collagen forms cross-striated fibrils, typical of type I collagen, following sine wave-like undulations in precholesteric domains. These ordered geometries, approximating in vivo situations, give interesting mechanical properties to the material.

Modification of fish skin collagen film and absorption property of tannic acid.

Science.gov (United States)

Liu, Haiying; Zhao, Lu; Guo, Shidong; Xia, Yu; Zhou, Peng

2014-06-01

Fish collagen is a biomacromolecule material and is usually used as a clarifying agent. However, fish collagen is not recyclable, and sedimentation usually occurs in the clarification process using fish collagen so that the filtration process is inevitable. This work aimed to provide a recyclable modified fish skin collagen film (MFCF) for adsorption of tannic acids. The collagen from channel catfish skin was extracted and used for preparation of the fish skin collagen film (FCF) and MFCF. The result indicated that the mechanical properties of MFCF were improved by addition of 2 ml/L glycerol, 6 ml/L polyvinyl alcohol (PVA) and 2 ml/L glutaraldehyde in 15 g/L collagen solution. As the most important property of adsorption material, the hydroscopicity of MFCF was only 54%, significantly lower than that of FCF (295%). Therefore, MFCF would not collapse in water. The infrared and thermal properties of MFCF were also investigated in this work. Results indicated that, in comparison to FCF, the physical and chemical properties of MFCF had been improved significantly. MFCF had higher shrink temperature (79.3 °C) and it did not collapse in distilled water at normal temperature. Furthermore, absorption and desorption properties of tannic acid were studied. MFCF showed good capability of absorption and desorption of tannic acid, which leaded to the suggestion that MFCF could have potential applications in adsorption material.

GH and IGF-I levels are positively associated with musculotendinous collagen expression: Experiments in acromegalic and GHD patients

DEFF Research Database (Denmark)

Doessing, Simon; Holm, Lars; Heinemeier, Katja

2010-01-01

OBJECTIVE: Disproportionate growth of musculoskeletal tissue is a major cause of morbidity in both acromegalic (ACRO) and GH-deficient (GHD) patients. GH/IGF1 is likely to play an important role in the regulation of tendon and muscle collagen. We hypothesized that the local production of collagen...... is associated with the level of GH/IGF1.DESIGN AND METHODS: As primary outcomes, collagen mRNA expression and collagen protein fractional synthesis rate (FSR) were determined locally in skeletal muscle and tendon in nine ACRO and nine GHD patients. Moreover, muscle myofibrillar protein synthesis and tendon...... collagen morphology were determined.RESULTS AND CONCLUSIONS: Muscle collagen I and III mRNA expression was higher in ACRO patients versus GHD patients (PIGF1Ea and IGF1Ec...

Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise

DEFF Research Database (Denmark)

Miller, Benjamin F; Olesen, Jens L; Hansen, Mette

2005-01-01

We hypothesized that an acute bout of strenuous, non-damaging exercise would increase rates of protein synthesis of collagen in tendon and skeletal muscle but these would be less than those of muscle myofibrillar and sarcoplasmic proteins. Two groups (n = 8 and 6) of healthy young men were studied...... collagen (0.077% h(-1)), muscle collagen (0.054% h(-1)), myofibrillar protein (0.121% h(-1)), and sarcoplasmic protein (0.134% h(-1))). The rates decreased toward basal values by 72 h although rates of tendon collagen and myofibrillar protein synthesis remained elevated. There was no tissue damage...... of muscle visible on histological evaluation. Neither tissue microdialysate nor serum concentrations of IGF-I and IGF binding proteins (IGFBP-3 and IGFBP-4) or procollagen type I N-terminal propeptide changed from resting values. Thus, there is a rapid increase in collagen synthesis after strenuous exercise...

MICROWAVE IRRADIATION AND CROSS-LINKING OF COLLAGEN

NARCIS (Netherlands)

VISSER, CE; VOUTE, ABE; OOSTING, J; BOON, ME; KOK, LP

1992-01-01

In a multifactorial experiment, dermal sheep collagen was treated in diluted glutaraldehyde solutions, 70% ethyl alcohol, Cialit 1:5000, and distilled water for 1, 3 and 5 min, respectively, in combination with microwave irradiation at different temperature settings. The shrinkage temperature

Measurement of skeletal muscle collagen breakdown by microdialysis

DEFF Research Database (Denmark)

Miller, B F; Ellis, D; Robinson, M M

2011-01-01

Exercise increases the synthesis of collagen in the extracellular matrix of skeletal muscle. Breakdown of skeletal muscle collagen has not yet been determined because of technical limitations. The purpose of the present study was to use local sampling to determine skeletal muscle collagen breakdown...... collagen breakdown 17–21 h post-exercise, and our measurement of OHP using GC–MS was in agreement with traditional assays....

Evaluation of a collagen-chitosan hydrogel for potential use as a pro-angiogenic site for islet transplantation.