Sample records for targets collagen type

  1. miRNA-29a targets COL3A1 to regulate the level of type III collagen in pig.

    PubMed

    Chuan-Hao, Li; Wei, Chen; Jia-Qing, Hu; Yan-Dong, Wang; Shou-Dong, Wang; Yong-Qing, Zeng; Hui, Wang

    2016-10-30

    COL3A1 encodes the protein, collagen type III alpha 1, which is an important component of collagen. Collagen can have a considerable effect on the processing quality of meat, and is nutritious. Bioinformatic analysis using Targetscan showed that COL3A1 could be a target gene of miRNA-29a. Moreover, we found that Laiwu pigs have higher levels of type III collagen and lower levels of miRNA-29a than Landrace pigs. Therefore, we hypothesized that miRNA-29a suppresses the expression of COL3A1 by targeting its 3'-UTR. miRNA-29a appears to play an inhibitory role in the regulation of COL3A1 in PK15 cells because of the following: (1) overexpression of miRNA-29a resulted in a significant down-regulation of COL3A1 protein levels (2) overexpression of miRNA-29a significantly decreased the level of COL3A1 mRNA. (3) The activity of a COL3A1 luciferase reporter was significant reduced by miRNA-29a. Furthermore, the levels of miRNA-29a and collagen type III in four tissues in Laiwu and Landrace pigs were consistent with the above observations. In this study, we identified COL3A1 as a direct target for miRNA-29a, which will inform further studies of meat quality. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Natural healing-inspired collagen-targeting surgical protein glue for accelerated scarless skin regeneration.

    PubMed

    Jeon, Eun Young; Choi, Bong-Hyuk; Jung, Dooyup; Hwang, Byeong Hee; Cha, Hyung Joon

    2017-07-01

    Skin scarring after deep dermal injuries is a major clinical problem due to the current therapies limited to established scars with poor understanding of healing mechanisms. From investigation of aberrations within the extracellular matrix involved in pathophysiologic scarring, it was revealed that one of the main factors responsible for impaired healing is abnormal collagen reorganization. Here, inspired by the fundamental roles of decorin, a collagen-targeting proteoglycan, in collagen remodeling, we created a scar-preventive collagen-targeting glue consisting of a newly designed collagen-binding mussel adhesive protein and a specific glycosaminoglycan. The collagen-targeting glue specifically bound to type I collagen in a dose-dependent manner and regulated the rate and the degree of fibrillogenesis. In a rat skin excisional model, the collagen-targeting glue successfully accelerated initial wound regeneration as defined by effective reepithelialization, neovascularization, and rapid collagen synthesis. Moreover, the improved dermal collagen architecture was demonstrated by uniform size of collagen fibrils, their regular packing, and a restoration of healthy tissue component. Collectively, our natural healing-inspired collagen-targeting glue may be a promising therapeutic option for improving the healing rate with high-quality and effective scar inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Type I collagen-targeted PET probe for pulmonary fibrosis detection and staging in preclinical models

    PubMed Central

    Désogère, Pauline; Tapias, Luis F.; Hariri, Lida P.; Rotile, Nicholas J.; Rietz, Tyson A.; Probst, Clemens K.; Blasi, Francesco; Day, Helen; Mino-Kenudson, Mari; Weinreb, Paul; Violette, Shelia M.; Fuchs, Bryan C.; Tager, Andrew M.; Lanuti, Michael; Caravan, Peter

    2017-01-01

    Pulmonary fibrosis is a scarring of the lungs that can arise from radiation injury, drug toxicity, environmental or genetic causes, and for unknown reasons [idiopathic pulmonary fibrosis (IPF)]. Overexpression of collagen is a hallmark of organ fibrosis. Here, we describe a peptide-based PET probe (68Ga-CBP8) that targets collagen type I. We evaluated 68Ga-CBP8 in vivo in the bleomycin-induced mouse model of pulmonary fibrosis. 68Ga-CBP8 showed high specificity for pulmonary fibrosis and high target:background ratios in diseased animals. The lung PET signal and lung 68Ga-CBP8 uptake (quantified ex vivo) correlated linearly (r2=0.80) with the amount of lung collagen in mice with fibrosis. We further demonstrated that the 68Ga-CBP8 probe could be used to monitor response to treatment in a second mouse model of pulmonary fibrosis associated with vascular leak. Ex vivo analysis of lung tissue from patients with IPF supported the animal findings. These studies indicate that 68Ga-CBP8 is a promising candidate for non-invasive imaging of human pulmonary fibrosis. PMID:28381537

  4. Collagen mimetic peptide engineered M13 bacteriophage for collagen targeting and imaging in cancer.

    PubMed

    Jin, Hyo-Eon; Farr, Rebecca; Lee, Seung-Wuk

    2014-11-01

    Collagens are over-expressed in various human cancers and subsequently degraded and denatured by proteolytic enzymes, thus making them a target for diagnostics and therapeutics. Genetically engineered bacteriophage (phage) is a promising candidate for the development of imaging or therapeutic materials for cancer collagen targeting due to its promising structural features. We genetically engineered M13 phages with two functional peptides, collagen mimetic peptide and streptavidin binding peptide, on their minor and major coat proteins, respectively. The resulting engineered phage functions as a therapeutic or imaging material to target degraded and denatured collagens in cancerous tissues. We demonstrated that the engineered phages are able to target and label abnormal collagens expressed on A549 human lung adenocarcinoma cells after the conjugation with streptavidin-linked fluorescent agents. Our engineered collagen binding phage could be a useful platform for abnormal collagen imaging and drug delivery in various collagen-related diseases. Published by Elsevier Ltd.

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

    PubMed

    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.

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

    PubMed Central

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

    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 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. PMID:26883295

  7. In vitro formation and thermal transition of novel hybrid fibrils from type I fish scale collagen and type I porcine collagen

    NASA Astrophysics Data System (ADS)

    Chen, Song; Ikoma, Toshiyuki; Ogawa, Nobuhiro; Migita, Satoshi; Kobayashi, Hisatoshi; Hanagata, Nobutaka

    2010-06-01

    Novel type I collagen hybrid fibrils were fabricated by neutralizing a mixture of type I fish scale collagen solution and type I porcine collagen solution with a phosphate buffer saline at 28 °C. Their structure was discussed in terms of the volume ratio of fish/porcine collagen solution. Scanning electron and atomic force micrographs showed that the diameter of collagen fibrils derived from the collagen mixture was larger than those derived from each collagen, and all resultant fibrils exhibited a typical D-periodic unit of ~67 nm, irrespective of volume ratio of both collagens. Differential scanning calorimetry revealed only one endothermic peak for the fibrils derived from collagen mixture or from each collagen solution, indicating that the resultant collagen fibrils were hybrids of type I fish scale collagen and type I porcine collagen.

  8. Dysregulated miR-127-5p contributes to type II collagen degradation by targeting matrix metalloproteinase-13 in human intervertebral disc degeneration.

    PubMed

    Hua, Wen-Bin; Wu, Xing-Huo; Zhang, Yu-Kun; Song, Yu; Tu, Ji; Kang, Liang; Zhao, Kang-Cheng; Li, Shuai; Wang, Kun; Liu, Wei; Shao, Zeng-Wu; Yang, Shu-Hua; Yang, Cao

    2017-08-01

    Intervertebral disc degeneration (IDD) is a chronic disease associated with the degradation of extracellular matrix (ECM). Matrix metalloproteinase (MMP)-13 is a major enzyme that mediates the degradation of ECM components. MMP-13 has been predicted to be a potential target of miR-127-5p. However, the exact function of miR-127-5p in IDD is still unclear. We designed this study to evaluate the correlation between miR-127-5p level and the degeneration of human intervertebral discs and explore the potential mechanisms. miR-127-5p levels and MMP-13 mRNA levels were detected by quantitative real-time polymerase chain reaction (qPCR). To determine whether MMP-13 is a target of miR-127-5p, dual luciferase reporter assays were performed. miR-127-5p mimic and miR-127-5p inhibitor were used to overexpress or downregulate miR-127-5p expression in human NP cells, respectively. Small interfering RNA (siRNA) was used to knock down MMP-13 expression in human NP cells. Type II collagen expression in human NP cells was detected by qPCR, western blotting, and immunofluorescence staining. We confirmed that miR-127-5p was significantly downregulated in nucleus pulposus (NP) tissue of degenerative discs and its expression was inversely correlated with MMP-13 mRNA levels. We reveal that MMP-13 may act as a target of miR-127-5p. Expression of miR-127-5p was inversely correlated with type II collagen expression in human NP cells. Moreover, suppression of MMP-13 expression by siRNA blocked downstream signaling and increased type II collagen expression. Dysregulated miR-127-5p contributed to the degradation of type II collagen by targeting MMP-13 in human IDD. Our findings highlight that miR-127-5p may serve as a new therapeutic target in IDD. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

  10. Identification of a collagen type I adhesin of Bacteroides fragilis.

    PubMed

    Galvão, Bruna P G V; Weber, Brandon W; Rafudeen, Mohamed S; Ferreira, Eliane O; Patrick, Sheila; Abratt, Valerie R

    2014-01-01

    Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼ 31 and ∼ 34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼ 31 kDa and the ∼ 34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼ 31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein.

  11. Identification of a Collagen Type I Adhesin of Bacteroides fragilis

    PubMed Central

    Galvão, Bruna P. G. V.; Weber, Brandon W.; Rafudeen, Mohamed S.; Ferreira, Eliane O.; Patrick, Sheila; Abratt, Valerie R.

    2014-01-01

    Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼31 and ∼34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼31 kDa and the ∼34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein. PMID:24618940

  12. Modulation of collagen-induced arthritis by adenovirus-mediated intra-articular expression of modified collagen type II.

    PubMed

    Tang, Bo; Cullins, David L; Zhou, Jing; Zawaski, Janice A; Park, Hyelee; Brand, David D; Hasty, Karen A; Gaber, M Waleed; Stuart, John M; Kang, Andrew H; Myers, Linda K

    2010-01-01

    Rheumatoid arthritis (RA) is a systemic disease manifested by chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. We have developed a unique modification to a clinically accepted method for delivering therapies directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to type II collagen (CII) and arthritis in the collagen-induced arthritis model (CIA). We engineered an adenoviral vector to contain the CB11 portion of recombinant type II collagen and used PCR to introduce point mutations at three sites within (CII124-402, 260A, 261B, 263D), (rCB11-A9) so that the resulting molecule contained the A9 sequence at the exact site of the wild-type sequence. We used this construct to target intra-articular tissues of mice and utilized the collagen-induced arthritis model to show that this treatment strategy provided a sustained, local therapy for individual arthritic joints, effective whether given to prevent arthritis or as a treatment. We also developed a novel system for in vivo bioimaging, using the firefly luciferase reporter gene to allow serial bioluminescence imaging to show that luciferase can be detected as late as 18 days post injection into the joint. Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells which secrete interleukin (IL)-4. We believe this approach has potential to effectively suppress RA with minimal side effects.

  13. Modulation of collagen-induced arthritis by adenovirus-mediated intra-articular expression of modified collagen type II

    PubMed Central

    2010-01-01

    Introduction Rheumatoid arthritis (RA) is a systemic disease manifested by chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. We have developed a unique modification to a clinically accepted method for delivering therapies directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to type II collagen (CII) and arthritis in the collagen-induced arthritis model (CIA). Methods We engineered an adenoviral vector to contain the CB11 portion of recombinant type II collagen and used PCR to introduce point mutations at three sites within (CII124-402, 260A, 261B, 263D), (rCB11-A9) so that the resulting molecule contained the A9 sequence at the exact site of the wild-type sequence. Results We used this construct to target intra-articular tissues of mice and utilized the collagen-induced arthritis model to show that this treatment strategy provided a sustained, local therapy for individual arthritic joints, effective whether given to prevent arthritis or as a treatment. We also developed a novel system for in vivo bioimaging, using the firefly luciferase reporter gene to allow serial bioluminescence imaging to show that luciferase can be detected as late as 18 days post injection into the joint. Conclusions Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells which secrete interleukin (IL)-4. We believe this approach has potential to effectively suppress RA with minimal side effects. PMID:20615221

  14. Normal Collagen and Bone Production by Gene-targeted Human Osteogenesis Imperfecta iPSCs

    PubMed Central

    Deyle, David R; Khan, Iram F; Ren, Gaoying; Wang, Pei-Rong; Kho, Jordan; Schwarze, Ulrike; Russell, David W

    2012-01-01

    Osteogenesis imperfecta (OI) is caused by dominant mutations in the type I collagen genes. In principle, the skeletal abnormalities of OI could be treated by transplantation of patient-specific, bone-forming cells that no longer express the mutant gene. Here, we develop this approach by isolating mesenchymal cells from OI patients, inactivating their mutant collagen genes by adeno-associated virus (AAV)-mediated gene targeting, and deriving induced pluripotent stem cells (iPSCs) that were expanded and differentiated into mesenchymal stem cells (iMSCs). Gene-targeted iMSCs produced normal collagen and formed bone in vivo, but were less senescent and proliferated more than bone-derived MSCs. To generate iPSCs that would be more appropriate for clinical use, the reprogramming and selectable marker transgenes were removed by Cre recombinase. These results demonstrate that the combination of gene targeting and iPSC derivation can be used to produce potentially therapeutic cells from patients with genetic disease. PMID:22031238

  15. Collagen like peptide bioconjugates for targeted drug delivery applications

    NASA Astrophysics Data System (ADS)

    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

  16. Techniques for Type I Collagen Organization

    NASA Astrophysics Data System (ADS)

    Anderson-Jackson, LaTecia Diamond

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

  17. Increased cartilage type II collagen degradation in patients with osteogenesis imperfecta used as a human model of bone type I collagen alterations.

    PubMed

    Rousseau, Jean-Charles; Chevrel, Guillaume; Schott, Anne-Marie; Garnero, Patrick

    2010-04-01

    We investigated whether cartilage degradation is altered in adult patients with mild osteogenesis imperfecta (OI) used as a human model of bone type I collagen-related osteoarthritis (OA). Sixty-four adult patients with OI (39% women, mean age+/-SD: 37+/-12 years) and 64 healthy age-matched controls (54% women, 39+/-7 years) were included. We also compared data in 87 patients with knee OA (73% women, 63+/-8 years, mean disease duration: 6 years) and 291 age-matched controls (80% women, 62+/-10 years). Urinary C-terminal cross-linked telopeptide of type II collagen (CTX-II), a marker of cartilage degradation, urinary helical peptide of type I collagen (Helix-I), a marker of bone resorption, and the urinary ratio between non-isomerised/isomerised (alpha/beta CTX-I) type I collagen C-telopeptide, a marker of type I collagen maturation, were measured. Patients with OI had CTX-II levels similar to those of subjects with knee OA (p=0.89; mean+/-SEM; 460+/-57 ng/mmol Cr for OI group and 547+/-32 ng/mmol Cr for OA group) and significantly higher than both young (144+/-7.8 ng/mmol Cr, p<0.0001) and old controls (247+/-7 ng/mmol Cr, p<0.0001). In patients with OI, increased Helix-I (p<0.0001) and alpha/beta CTX-I (p=0.0067) were independently associated with increased CTX-II and together explained 26% of its variance (p< 0.0001). In patients with knee OA, increased levels of alpha/beta CTX-I ratio were also associated with higher CTX-II levels. Adult patients with OI or knee OA are characterized by increased cartilage type II collagen degradation, which is associated with increased type I collagen degradation for OI and lower type I collagen maturation for both OI and OA. These data suggest that both quantitative and qualitative alterations of bone type I collagen metabolism are involved in increased cartilage degradation in patients with OI or knee OA. Copyright 2009 Elsevier Inc. All rights reserved.

  18. Type V Collagen is Persistently Altered after Inguinal Hernia Repair.

    PubMed

    Lorentzen, L; Henriksen, N A; Juhl, P; Mortensen, J H; Ågren, M S; Karsdal, M A; Jorgensen, L N

    2018-04-01

    Hernia formation is associated with alterations of collagen metabolism. Collagen synthesis and degradation cause a systemic release of products, which are measurable in serum. Recently, we reported changes in type V and IV collagen metabolisms in patients with inguinal and incisional hernia. The aim of this study was to determine if the altered collagen metabolism was persistent after hernia repair. Patients who had undergone repairs for inguinal hernia (n = 11) or for incisional hernia (n = 17) were included in this study. Patients who had undergone elective cholecystectomy served as controls (n = 10). Whole venous blood was collected 35-55 months after operation. Biomarkers for type V collagen synthesis (Pro-C5) and degradation (C5M) and those for type IV collagen synthesis (P4NP) and degradation (C4M2) were measured by a solid-phase competitive assay. The turnover of type V collagen (Pro-C5/C5M) was slightly higher postoperatively when compared to preoperatively in the inguinal hernia group (P = 0.034). In addition, the results revealed a postoperatively lower type V collagen turnover level in the inguinal hernia group compared to controls (P = 0.012). In the incisional hernia group, the type V collagen turnover was higher after hernia repair (P = 0.004) and the postoperative turnover level was not different from the control group (P = 0.973). Patients with an inguinal hernia demonstrated a systemic and persistent type V collagen turnover alteration. This imbalance of the collagen metabolism may be involved in the development of inguinal hernias.

  19. In Situ D-periodic Molecular Structure of Type II Collagen

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    Collagens are essential components of extracellular matrices in multicellular animals. Fibrillar type II collagen is the most prominent component of articular cartilage and other cartilage-like tissues such as notochord. Its in situ macromolecular and packing structures have not been fully characterized, but an understanding of these attributes may help reveal mechanisms of tissue assembly and degradation (as in osteo- and rheumatoid arthritis). In some tissues such as lamprey notochord, the collagen fibrillar organization is naturally crystalline and may be studied by x-ray diffraction. We used diffraction data from native and derivative notochord tissue samples to solve the axial, D-periodic structuremore » of type II collagen via multiple isomorphous replacement. The electron density maps and heavy atom data revealed the conformation of the nonhelical telopeptides and the overall D-periodic structure of collagen type II in native tissues, data that were further supported by structure prediction and transmission electron microscopy. These results help to explain the observed differences in collagen type I and type II fibrillar architecture and indicate the collagen type II cross-link organization, which is crucial for fibrillogenesis. Transmission electron microscopy data show the close relationship between lamprey and mammalian collagen fibrils, even though the respective larger scale tissue architecture differs.« less

  20. Edaravone suppresses degradation of type II collagen.

    PubMed

    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 typecollagen. And our results indicated that TNF-α treatment resulted in degradation of typecollagen, 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 typecollagen 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.

  1. Blister-inducing antibodies target multiple epitopes on collagen VII in mice

    PubMed Central

    Csorba, Kinga; Chiriac, Mircea Teodor; Florea, Florina; Ghinia, Miruna Georgiana; Licarete, Emilia; Rados, Andreea; Sas, Alexandra; Vuta, Vlad; Sitaru, Cassian

    2014-01-01

    Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non-collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen-specific B- and T cell-targeted therapies in EBA. PMID:25091020

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

    PubMed

    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.

  3. Second harmonic generation microscopy differentiates collagen type I and type III in COPD

    NASA Astrophysics Data System (ADS)

    Suzuki, Masaru; Kayra, Damian; Elliott, W. Mark; Hogg, James C.; Abraham, Thomas

    2012-03-01

    The structural remodeling of extracellular matrix proteins in peripheral lung region is an important feature in chronic obstructive pulmonary disease (COPD). Multiphoton microscopy is capable of inducing specific second harmonic generation (SHG) signal from non-centrosymmetric structural proteins such as fibrillar collagens. In this study, SHG microscopy was used to examine structural remodeling of the fibrillar collagens in human lungs undergoing emphysematous destruction (n=2). The SHG signals originating from these diseased lung thin sections from base to apex (n=16) were captured simultaneously in both forward and backward directions. We found that the SHG images detected in the forward direction showed well-developed and well-structured thick collagen fibers while the SHG images detected in the backward direction showed striking different morphological features which included the diffused pattern of forward detected structures plus other forms of collagen structures. Comparison of these images with the wellestablished immunohistochemical staining indicated that the structures detected in the forward direction are primarily the thick collagen type I fibers and the structures identified in the backward direction are diffusive structures of forward detected collagen type I plus collagen type III. In conclusion, we here demonstrate the feasibility of SHG microscopy in differentiating fibrillar collagen subtypes and understanding their remodeling in diseased lung tissues.

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

    PubMed

    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 < 0.05 or p < 0.01). Inhibition of miR-214-5p increased the viability of MC3T3-E1 cells and the expressions of COL1A1 and COL-X, but decreased the apoptotic rate and COL-II expression (p < 0.05 or p < 0.01). COL4A1 was a target of miR-214-5p, and was negatively regulated by miR-214-5p (p < 0.05 or p < 0.01). Overexpression of COL4A1 showed a similar impact on cell viability, apoptotic rate, and COL1A1, COL-II, and COL-X expressions inhibiting miR-214-5p (p < 0.01). Inhibition of miR-214-5p promotes cell survival and 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.

  5. Withaferin-A Reduces Type I Collagen Expression In Vitro and Inhibits Development of Myocardial Fibrosis In Vivo

    PubMed Central

    Challa, Azariyas A.; Vukmirovic, Milica; Blackmon, John; Stefanovic, Branko

    2012-01-01

    Type I collagen is the most abundant protein in the human body. Its excessive synthesis results in fibrosis of various organs. Fibrosis is a major medical problem without an existing cure. Excessive synthesis of type I collagen in fibrosis is primarily due to stabilization of collagen mRNAs. We recently reported that intermediate filaments composed of vimentin regulate collagen synthesis by stabilizing collagen mRNAs. Vimentin is a primary target of Withaferin-A (WF-A). Therefore, we hypothesized that WF-A may reduce type I collagen production by disrupting vimentin filaments and decreasing the stability of collagen mRNAs. This study is to determine if WF-A exhibits anti-fibrotic properties in vitro and in vivo and to elucidate the molecular mechanisms of its action. In lung, skin and heart fibroblasts WF-A disrupted vimentin filaments at concentrations of 0.5–1.5 µM and reduced 3 fold the half-lives of collagen α1(I) and α2(I) mRNAs and protein expression. In addition, WF-A inhibited TGF-β1 induced phosphorylation of TGF-β1 receptor I, Smad3 phosphorylation and transcription of collagen genes. WF-A also inhibited in vitro activation of primary hepatic stellate cells and decreased their type I collagen expression. In mice, administration of 4 mg/kg WF-A daily for 2 weeks reduced isoproterenol-induced myocardial fibrosis by 50%. Our findings provide strong evidence that Withaferin-A could act as an anti-fibrotic compound against fibroproliferative diseases, including, but not limited to, cardiac interstitial fibrosis. PMID:22900077

  6. Effect of Fish Collagen Hydrolysates on Type I Collagen mRNA Levels of Human Dermal Fibroblast Culture

    PubMed Central

    Sanchez, Ana; Blanco, Maria; Correa, Begoña

    2018-01-01

    Fish discards and subproducts may represent an important source of raw material, not only for the food industry, but for other different kind of industries, such as the nutraceutical and cosmetic industries. Collagen, which is mainly obtained from animal skins, is an important structural protein in the animal kingdom having many different applications. It is well known that fish skins constitute a significant subproduct in the fishery industry, especially in the case of some species, where fish skins may represent up to 20% of the total body weight of fish. Peptides from collagen hydrolysates have been described to be useful for preventing skin aging and osteoarthritis, however, the mechanism for these biological activities is not well known. Fibroblasts are the main cell types involved in the collagen synthesis, and in the present work, human dermal fibroblasts have been exposed to the treatment of collagen peptides of two different molecular weight ranges. Results show that higher molecular weight collagen peptides produce higher synthesis of collagen type I mRNA and, therefore, it may suggest that prior molecular weight selection may be an important step to maximize the effect of collagen hydrolysates on collagen type I synthesis by dermal fibroblasts. PMID:29701725

  7. Crosslinked type II collagen matrices: preparation, characterization, and potential for cartilage engineering.

    PubMed

    Pieper, J S; van der Kraan, P M; Hafmans, T; Kamp, J; Buma, P; van Susante, J L C; van den Berg, W B; Veerkamp, J H; van Kuppevelt, T H

    2002-08-01

    The limited intrinsic repair capacity of articular cartilage has stimulated continuing efforts to develop tissue engineered analogues. Matrices composed of type II collagen and chondroitin sulfate (CS), the major constituents of hyaline cartilage, may create an appropriate environment for the generation of cartilage-like tissue. In this study, we prepared, characterized, and evaluated type 11 collagen matrices with and without CS. Type II collagen matrices were prepared using purified, pepsin-treated, type II collagen. Techniques applied to prepare type I collagen matrices were found unsuitable for type II collagen. Crosslinking of collagen and covalent attachment of CS was performed using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide. Porous matrices were prepared by freezing and lyophilization, and their physico-chemical characteristics (degree of crosslinking, denaturing temperature, collagenase-resistance, amount of CS incorporated) established. Matrices were evaluated for their capacity to sustain chondrocyte proliferation and differentiation in vitro. After 7 d of culture, chondrocytes were mainly located at the periphery of the matrices. In contrast to type I collagen, type II collagen supported the distribution of cells throughout the matrix. After 14 d of culture, matrices were surfaced with a cartilagenous-like layer, and occasionally clusters of chondrocytes were present inside the matrix. Chondrocytes proliferated and differentiated as indicated by biochemical analyses, ultrastructural observations, and reverse transcriptase PCR for collagen types I, II and X. No major differences were observed with respect to the presence or absence of CS in the matrices.

  8. Distribution of type IV collagen in pancreatic adenocarcinoma and chronic pancreatitis.

    PubMed Central

    Lee, C. S.; Montebello, J.; Georgiou, T.; Rode, J.

    1994-01-01

    Changes in the basement membrane are present in various neoplastic conditions such as neurofibrosarcoma, cervical carcinoma, colorectal cancers and hepatoblastoma. This study examines the expression of type IV collagen in the basement membrane, using an immunohistochemical method, in the normal pancreas (n = 10), chronic pancreatitis (n = 15) and pancreatic adenocarcinoma (n = 30). The formalin fixed, paraffin embedded tissue was sectioned and pretreated with protease prior to immunostaining for type IV collagen. There was a statistically significant difference in type IV collagen expression between pancreatic carcinoma and chronic pancreatitis (P = 0.0001; chi 2 test with continuity correction). In pancreatic adenocarcinoma, type IV collagen distribution in the basement membrane was discontinuous and irregular or absent around individual or groups of neoplastic cells (n = 30). Most cases of chronic pancreatitis showed continuous pattern of basement membrane type IV collagen around residual ducts (n = 10). In the normal pancreas, only one of the ten cases showed discontinuous basement membrane around pancreatic ducts, while in the rest of the cases, the pattern was continuous. This study suggests that there is abnormal distribution of type IV collagen in the basement membrane in pancreatic carcinoma, which may be related to either abnormal deposition or degradation of the collagen. Immunostaining for type IV collagen may be of some diagnostic use for distinguishing pancreatic adenocarcinoma from problematic cases of chronic pancreatitis. Images Figure 1 Figure 2 Figure 3 PMID:8199008

  9. Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs.

    PubMed

    Lu, Yongbo; Kamel-El Sayed, Suzan A; Wang, Kun; Tiede-Lewis, LeAnn M; Grillo, Michael A; Veno, Patricia A; Dusevich, Vladimir; Phillips, Charlotte L; Bonewald, Lynda F; Dallas, Sarah L

    2018-06-01

    Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel

  10. Collagen type IX from human cartilage: a structural profile of intermolecular cross-linking sites.

    PubMed Central

    Diab, M; Wu, J J; Eyre, D R

    1996-01-01

    Type IX collagen, a quantitatively minor collagenous component of cartilage, is known to be associated with and covalently cross-linked to type II collagen fibrils in chick and bovine cartilage. Type IX collagen molecules have also been shown to form covalent cross-links with each other in bovine cartilage. In the present study we demonstrate by structural analysis and location of cross-linking sites that, in human cartilage, type IX collagen is covalently cross-linked to type II collagen and to other molecules of type IX collagen. We also present evidence that, if the proteoglycan form of type IX collagen is present in human cartilage, it can only be a minor component of the matrix, similar to findings with bovine cartilage. PMID:8660302

  11. Protease nexin 1 is a potent urinary plasminogen activator inhibitor in the presence of collagen type IV.

    PubMed

    Crisp, Robert J; Knauer, Mary F; Knauer, Daniel J

    2002-12-06

    Protease nexin 1 (PN1) in solution forms inhibitory complexes with thrombin or urokinase, which have opposing effects on the blood coagulation cascade. An initial report provided data supporting the idea that PN1 target protease specificity is under the influence of collagen type IV (1). Although collagen type IV demonstrated no effect on the association rate between PN1 and thrombin, the study reported that the association rate between PN1 and urokinase was allosterically reduced 10-fold. This has led to the generally accepted idea that the primary role of PN1 in the brain is to act as a rapid thrombin inhibition and clearance mechanism during trauma and loss of vascular integrity. In studies to identify the structural determinants of PN1 that mediate the allosteric interaction with collagen type IV, we found that protease specificity was only affected after transient exposure of PN1 to acidic conditions that mimic the elution protocol from a monoclonal antibody column. Because PN1 used in previous studies was purified over a monoclonal antibody column, we propose that the allosteric regulation of PN1 target protease specificity by collagen type IV is a result of the purification protocol. We provide both biochemical and kinetic data to support this conclusion. This finding is significant because it implies that PN1 may play a much larger role in the modeling and remodeling of brain tissues during development and is not simply an extravasated thrombin clearance mechanism as previously suggested.

  12. Activation of platelet-rich plasma using soluble type I collagen.

    PubMed

    Fufa, Duretti; Shealy, Blake; Jacobson, May; Kevy, Sherwin; Murray, Martha M

    2008-04-01

    Platelet-rich plasma (PRP) has recently been found to be a useful delivery system for growth factors important to oral tissue healing. But application of PRP in a liquid form to a wound site within the oral cavity can be complicated by significant loss of the PRP into the surrounding oral space unless gelation through the clotting mechanism is accomplished. Gelation is currently accomplished using bovine thrombin; however, rare but serious complications of this method have led to the search for alternative clotting mechanisms, including the use of soluble collagen as a clotting activator. In this work, our hypothesis was that soluble type I collagen would be as effective as bovine thrombin in causing clotting of the PRP and stimulating growth factor release from the platelets and granulocytes. PRP from human donors was clotted using type I collagen or bovine thrombin. Clot retraction was determined by measuring clot diameters over time. The release of platelet-derived growth factor (PDGF)-AB, transforming growth factor (TGF)-beta1, and vascular endothelial growth factor (VEGF) from both types of clots was measured over 10 days using enzyme-linked immunosorbent assasy. Clots formed using type I collagen exhibited far less retraction than those formed with bovine thrombin. Bovine thrombin and type I collagen stimulated similar release of PDGF-AB and VEGF between 1 and 10 days; however, thrombin activation resulted in a greater release of TGF-beta1 during the first 5 days after activation. The use of type I collagen to activate clotting of PRP may be a safe and effective alternative to bovine thrombin. The use of collagen results in less clot retraction and equal release of PDGF-AB and VEGF compared with currently available methods of clot activation.

  13. ACTIVATION OF PLATELET-RICH PLASMA USING SOLUBLE TYPE I COLLAGEN

    PubMed Central

    Fufa, Duretti; Shealy, Blake; Jacobson, May; Kevy, Sherwin; Murray, Martha M.

    2008-01-01

    PURPOSE Platelet-rich plasma (PRP) has recently been found to be a useful delivery system for growth factors important in oral tissue healing. However, application of PRP in a liquid form to a wound site within the oral cavity can be complicated by significant loss of the PRP into the surrounding oral space unless gelation via the clotting mechanism is accomplished. Gelation is currently accomplished using bovine thrombin; however, rare but serious complications of this method have led to the search for alternative clotting mechanisms, including the use of soluble collagen as a clotting activator. In this paper, our hypothesis was that soluble Type I collagen would be as effective as bovine thrombin in causing clotting of the PRP and of stimulating growth factor release from the platelets and granulocytes. MATERIALS AND METHODS PRP from human donors was clotted using Type I collagen or bovine thrombin. Clot retraction was determined by measuring clot diameters over time. The release of PDGF-AB, TGF-β1 and VEGF from both types of clots was measured over 10 days using ELISA. RESULTS Clots formed using Type I collagen had far less retraction than those formed with bovine thrombin. Bovine thrombin and Type I collagen stimulated similar release of PDGF-AB and VEGF between 1 and 10 days; however, thrombin activation resulted in a greater release of TGF-β1 during the first five days after activation. CONCLUSIONS The use of Type I collagen to activate clotting of PRP may be a safe and effective alternative to bovine thrombin. The use of collagen results in less clot retraction and equal release of PDGF-AB and VEGF when compared to currently available methods of clot activation. PMID:18355591

  14. Changes in type I collagen following laser welding.

    PubMed

    Bass, L S; Moazami, N; Pocsidio, J; Oz, M C; LoGerfo, P; Treat, M R

    1992-01-01

    Selection of ideal laser parameters for tissue welding is inhibited by poor understanding of the mechanism. We investigated structural changes in collagen molecules extracted from rat tail tendon (> 90% type I collagen) after tissue welding using an 808 nm diode laser and indocyanine green dye applied to the weld site. Mobility patterns on SDS-PAGE were identical in the lasered and untreated tendon extracts with urea or acetic acid. Pepsin incubation after acetic acid extraction revealed a reduction of collagen alpha and beta bands in lasered compared with untreated specimens. Circular dichroism studies of rat tail tendon showed absence of helical structure in collagen from lasered tendon. No evidence for covalent bonding was present in laser-treated tissues. Collagen molecules are denatured by the laser wavelength and parameters used in this study. No significant amount of helical structure is regenerated on cooling. We conclude that non-covalent interactions between denatured collagen molecules may be responsible for the creation of tissue welding.

  15. Gentamicin induces functional type VII collagen in recessive dystrophic epidermolysis bullosa patients

    PubMed Central

    Woodley, David T.; Cogan, Jon; Hou, Yingping; Lyu, Chao; Marinkovich, M. Peter; Keene, Douglas

    2017-01-01

    BACKGROUND. Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable disease caused by mutations in the gene encoding type VII collagen, the major component of anchoring fibrils (AF). We previously demonstrated that gentamicin produced functional type VII collagen in RDEB cells harboring nonsense mutations. Herein, we determined whether topical or intradermal gentamicin administration induces type VII collagen and AFs in RDEB patients. METHODS. A double-blind, placebo-controlled pilot trial assessed safety and efficacy of topical and intradermal gentamicin in 5 RDEB patients with nonsense mutations. The topical arm tested 0.1% gentamicin ointment or placebo application 3 times daily at 2 open erosion sites for 2 weeks. The intradermal arm tested daily intradermal injection of gentamicin solution (8 mg) or placebo into 2 intact skin sites for 2 days in 4 of 5 patients. Primary outcomes were induction of type VII collagen and AFs at the test sites and safety assessment. A secondary outcome assessed wound closure of topically treated erosions. RESULTS. Both topical and intradermal gentamicin administration induced type VII collagen and AFs at the dermal-epidermal junction of treatment sites. Newly created type VII collagen varied from 20% to 165% of that expressed in normal human skin and persisted for 3 months. Topical gentamicin corrected dermal-epidermal separation, improved wound closure, and reduced blister formation. There were no untoward side effects from gentamicin treatments. Type VII collagen induction did not generate anti–type VII collagen autoantibodies in patients’ blood or skin. CONCLUSION. Topical and intradermal gentamicin suppresses nonsense mutations and induces type VII collagen and AFs in RDEB patients. Gentamicin therapy may provide a readily available treatment for RDEB patients with nonsense mutations. TRIAL REGISTRATION. ClinicalTrials.gov NCT02698735. FUNDING. Epidermolysis Bullosa Research Partnership, Epidermolysis Bullosa

  16. Alveolar type II cell-fibroblast interactions, synthesis and secretion of surfactant and type I collagen.

    PubMed

    Griffin, M; Bhandari, R; Hamilton, G; Chan, Y C; Powell, J T

    1993-06-01

    During alveolar development and alveolar repair close contacts are established between fibroblasts and lung epithelial cells through gaps in the basement membrane. Using co-culture systems we have investigated whether these close contacts influence synthesis and secretion of the principal surfactant apoprotein (SP-A) by cultured rat lung alveolar type II cells and the synthesis and secretion of type I collagen by fibroblasts. The alveolar type II cells remained cuboidal and grew in colonies on fibroblast feeder layers and on Matrigel-coated cell culture inserts but were progressively more flattened on fixed fibroblast monolayers and plastic. Alveolar type II cells cultured on plastic released almost all their SP-A into the medium by 4 days. Alveolar type II cells cultured on viable fibroblasts or Matrigel-coated inserts above fibroblasts accumulated SP-A in the medium at a constant rate for the first 4 days, and probably recycle SP-A by endocytosis. The amount of mRNA for SP-A was very low after 4 days of culture of alveolar type II cells on plastic, Matrigel-coated inserts or fixed fibroblast monolayers: relatively, the amount of mRNA for SP-A was increased 4-fold after culture of alveolar type II cells on viable fibroblasts. Co-culture of alveolar type II cells with confluent human dermal fibroblasts stimulated by 2- to 3-fold the secretion of collagen type I into the culture medium, even after the fibroblasts' growth had been arrested with mitomycin C. Collagen secretion, by fibroblasts, also was stimulated 2-fold by conditioned medium from alveolar type II cells cultured on Matrigel. The amount of mRNA for type I collagen increased only modestly when fibroblasts were cultured in this conditioned medium. This stimulation of type I collagen secretion diminished as the conditioned medium was diluted out, but at high dilutions further stimulation occurred, indicating that a factor that inhibited collagen secretion also was being diluted out. The conditioned medium

  17. Fluorescence self-quenching assay for the detection of target collagen sequences using a short probe peptide.

    PubMed

    Nian, Linge; Hu, Yue; Fu, Caihong; Song, Chen; Wang, Jie; Xiao, Jianxi

    2018-01-01

    The development of novel assays to detect collagen fragments is of utmost importance for diagnostic, prognostic and therapeutic decisions in various collagen-related diseases, and one essential question is to discover probe peptides that can specifically recognize target collagen sequences. Herein we have developed the fluorescence self-quenching assay as a convenient tool to screen the capability of a series of fluorescent probe peptides of variable lengths to bind with target collagen peptides. We have revealed that the targeting ability of probe peptides is length-dependent, and have discovered a relatively short probe peptide FAM-G(POG) 8 capable to identify the target peptide. We have further demonstrated that fluorescence self-quenching assay together with this short probe peptide can be applied to specifically detect the desired collagen fragment in complex biological media. Fluorescence self-quenching assay provides a powerful new tool to discover effective peptides for the recognition of collagen biomarkers, and it may have great potential to identify probe peptides for various protein biomarkers involved in pathological conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Ultrastructural sinusoidal changes in extrahepatic cholestasis. Light and electron microscopic immunohistochemical localization of collagen type III and type IV.

    PubMed

    Gulubova, M V

    1996-07-01

    Extrahepatic cholestasis causes excessive extracellular matrix formation perisinusoidally. Ito cells, transitional and endothelial cells are considered to be a source of extracellular matrix proteins in experimental cholestasis. The localization of collagens type III and type IV in human liver in extrahepatic cholestasis was investigated immunohistochemically in the present study. Immersion fixation was used after modification to be applied to surgical biopsies with commercially available kits. Sinusoidal changes were observed that indicated excessive collagen and matrix formation. Light microscopically, increased immunostaining with the two collagen antibodies was found perisinusoidally and portally. Ultrastructurally, collagen type III positive fibres were found beneath basement membranes of vessels, in collagen bundles and as a fibrillar network in the space of Disse. Collagen type IV immunostaining was located in portal tracts and near hepatocyte microvilli. Intracellular staining with collagen type IV was detected in the rough endoplasmic reticulum of some transitional cells. Immunostaining was located around transitional cells, Ito cells or endothelial cells mainly. Our study indicates that Ito cells, transitional and endothelial cells are the main source of collagens type III and IV in the space of Disse in extrahepatic cholestasis in humans.

  19. Type IV collagen is a novel DEJ biomarker that is reduced by radiotherapy.

    PubMed

    McGuire, J D; Gorski, J P; Dusevich, V; Wang, Y; Walker, M P

    2014-10-01

    The dental basement membrane (BM) is composed of collagen types IV, VI, VII, and XVII, fibronectin, and laminin and plays an inductive role in epithelial-mesenchymal interactions during tooth development. The BM is degraded and removed during later-stage tooth morphogenesis; however, its original position defines the location of the dentin-enamel junction (DEJ) in mature teeth. We recently demonstrated that type VII collagen is a novel component of the inner enamel organic matrix layer contiguous with the DEJ. Since it is frequently co-expressed with and forms functional complexes with type VII collagen, we hypothesized that type IV collagen should also be localized to the DEJ in mature human teeth. To identify collagen IV, we first evaluated defect-free erupted teeth from various donors. To investigate a possible stabilizing role, we also evaluated extracted teeth exposed to high-dose radiotherapy--teeth that manifest post-radiotherapy DEJ instability. We now show that type IV collagen is a component within the morphological DEJ of posterior and anterior teeth from individuals aged 18 to 80 yr. Confocal microscopy revealed that immunostained type IV collagen was restricted to the 5- to 10-µm-wide optical DEJ, while collagenase treatment or previous in vivo tooth-level exposure to > 60 Gray irradiation severely reduced immunoreactivity. This assignment was confirmed by Western blotting with whole-tooth crown and enamel extracts. Without reduction, type IV collagen contained macromolecular α-chains of 225 and 250 kDa. Compositionally, our results identify type IV collagen as the first macromolecular biomarker of the morphological DEJ of mature teeth. Given its network structure and propensity to stabilize the dermal-epidermal junction, we propose that a collagen-IV-enriched DEJ may, in part, explain its well-known fracture toughness, crack propagation resistance, and stability. In contrast, loss of type IV collagen may represent a biochemical rationale for the DEJ

  20. Type IV Collagen is a Novel DEJ Biomarker that is Reduced by Radiotherapy

    PubMed Central

    McGuire, J.D.; Gorski, J.P.; Dusevich, V.; Wang, Y.; Walker, M.P.

    2014-01-01

    The dental basement membrane (BM) is composed of collagen types IV, VI, VII, and XVII, fibronectin, and laminin and plays an inductive role in epithelial-mesenchymal interactions during tooth development. The BM is degraded and removed during later-stage tooth morphogenesis; however, its original position defines the location of the dentin-enamel junction (DEJ) in mature teeth. We recently demonstrated that type VII collagen is a novel component of the inner enamel organic matrix layer contiguous with the DEJ. Since it is frequently co-expressed with and forms functional complexes with type VII collagen, we hypothesized that type IV collagen should also be localized to the DEJ in mature human teeth. To identify collagen IV, we first evaluated defect-free erupted teeth from various donors. To investigate a possible stabilizing role, we also evaluated extracted teeth exposed to high-dose radiotherapy – teeth that manifest post-radiotherapy DEJ instability. We now show that type IV collagen is a component within the morphological DEJ of posterior and anterior teeth from individuals aged 18 to 80 yr. Confocal microscopy revealed that immunostained type IV collagen was restricted to the 5- to 10-µm-wide optical DEJ, while collagenase treatment or previous in vivo tooth-level exposure to > 60 Gray irradiation severely reduced immunoreactivity. This assignment was confirmed by Western blotting with whole-tooth crown and enamel extracts. Without reduction, type IV collagen contained macromolecular α-chains of 225 and 250 kDa. Compositionally, our results identify type IV collagen as the first macromolecular biomarker of the morphological DEJ of mature teeth. Given its network structure and propensity to stabilize the dermal-epidermal junction, we propose that a collagen-IV-enriched DEJ may, in part, explain its well-known fracture toughness, crack propagation resistance, and stability. In contrast, loss of type IV collagen may represent a biochemical rationale for the

  1. An {alpha}1(II) Gly{sup 913} to cys substitution prevents the matrix incorporation of type II collagen which is replaced with type I and III collagens in cartilage from a patient with hypochondrogenesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mundlos, S.; Chan, D.; Bateman, J.F.

    1996-05-03

    A heterozygous mutation in the COL2A1 gene was identified in a patient with hypochondrogenesis. The mutation was a single nucleotide transition of G3285T that resulted in an amino acid substitution of Cys for Gly{sup 913} in the {alpha}1(II) chain of type II collagen. This amino acid change disrupted the obligatory Gly-X-Y triplet motif required for the normal formation of a stable collagen triple helix and prevented the deposition of type II collagen into the proposita`s cartilage, which contained predominantly type I and III collagens and minor amounts of type XI collagen. Biosynthetic analysis of collagens produced and secreted by themore » patient`s chondrocytes cultured in alginate beads was consistent with the in vivo matrix composition, demonstrating that the main products were type I and III collagens, along with type XI collagen. The synthesis of the cartilage-specific type XI collagen at similar levels to controls indicated that the isolated cartilage cells had re-differentiated to the chondrocyte phenotype. The chondrocytes also produced small amounts of type II collagen, but this was post-translationally overmodified and not secreted. These data further delineate the biochemical and phenotypic consequences of mutations in the COL2A1 gene and suggest that cartilage formation and bone development can take place in the absence of type II collagen. 23 refs., 5 figs.« less

  2. Dental pulp stem cell-derived chondrogenic cells demonstrate differential cell motility in type I and type II collagen hydrogels.

    PubMed

    Yao, Li; Flynn, Nikol

    2018-06-01

    Advances in the development of biomaterials and stem cell therapy provide a promising approach to regenerating degenerated discs. The normal nucleus pulposus (NP) cells exhibit similar phenotype to chondrocytes. Because dental pulp stem cells (DPSCs) can be differentiated into chondrogenic cells, the DPSCs and DPSCs-derived chondrogenic cells encapsulated in type I and type II collagen hydrogels can potentially be transplanted into degenerated NP to repair damaged tissue. The motility of transplanted cells is critical because the cells need to migrate away from the hydrogels containing the cells of high density and disperse through the NP tissue after implantation. The purpose of this study was to determine the motility of DPSC and DPSC-derived chondrogenic cells in type I and type II collagen hydrogels. The time lapse imaging that recorded cell migration was analyzed to quantify the cell migration velocity and distance. The cell viability of DPSCs in native or poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-StarPEG)-crosslinked type I and type II collagen hydrogels was determined using LIVE/DEAD cell viability assay and AlamarBlue assay. DPSCs were differentiated into chondrogenic cells. The migration of DPSCs and DPSC-derived chondrogenic cells in these hydrogels was recorded using a time lapse imaging system. This study was funded by the Regional Institute on Aging and Wichita Medical Research and Education Foundation, and the authors declare no competing interest. DPSCs showed high cell viability in non-crosslinked and crosslinked collagen hydrogels. DPSCs migrated in collagen hydrogels, and the cell migration speed was not significantly different in either type I collagen or type II collagen hydrogels. The migration speed of DPSC-derived chondrogenic cells was higher in type I collagen hydrogel than in type II collagen hydrogel. Crosslinking of type I collagen with 4S-StarPEG significantly reduced the cell migration speed of DPSC

  3. Collagen type I as a ligand for receptor-mediated signaling

    NASA Astrophysics Data System (ADS)

    Boraschi-Diaz, Iris; Wang, Jennifer; Mort, John S.; Komarova, Svetlana V.

    2017-05-01

    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 and mannose family receptor uPARAP/Endo 180. 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.

  4. Type VII Collagen Expression in the Human Vitreoretinal Interface, Corpora Amylacea and Inner Retinal Layers

    PubMed Central

    Wullink, Bart; Pas, Hendri H.; Van der Worp, Roelofje J.; Kuijer, Roel; Los, Leonoor I.

    2015-01-01

    Type VII collagen, as a major component of anchoring fibrils found at basement membrane zones, is crucial in anchoring epithelial tissue layers to their underlying stroma. Recently, type VII collagen was discovered in the inner human retina by means of immunohistochemistry, while proteomic investigations demonstrated type VII collagen at the vitreoretinal interface of chicken. Because of its potential anchoring function at the vitreoretinal interface, we further assessed the presence of type VII collagen at this site. We evaluated the vitreoretinal interface of human donor eyes by means of immunohistochemistry, confocal microscopy, immunoelectron microscopy, and Western blotting. Firstly, type VII collagen was detected alongside vitreous fibers6 at the vitreoretinal interface. Because of its known anchoring function, it is likely that type VII collagen is involved in vitreoretinal attachment. Secondly, type VII collagen was found within cytoplasmic vesicles of inner retinal cells. These cells resided most frequently in the ganglion cell layer and inner plexiform layer. Thirdly, type VII collagen was found in astrocytic cytoplasmic inclusions, known as corpora amylacea. The intraretinal presence of type VII collagen was confirmed by Western blotting of homogenized retinal preparations. These data add to the understanding of vitreoretinal attachment, which is important for a better comprehension of common vitreoretinal attachment pathologies. PMID:26709927

  5. Gap junction communications influence upon fibroblast synthesis of Type I collagen and fibronectin.

    PubMed

    Ehrlich, H Paul; Sun, Bonnie; Saggers, Gregory C; Kromath, Fatuma

    2006-07-01

    In rats polyvinyl alcohol sponge subcutaneous implants treated with gap junctional intercellular communications (GJIC) uncouplers showed reduced deposition of connective tissue. Do uncouplers inhibit the synthesis and deposition of a new connective tissue by fibroblasts? Confluent human dermal fibroblasts in serum-free medium received either endosulfan or oleamide, GJIC uncouplers. Collected media were subjected to Dot Blot analysis for native Type I collagen and fibronectin. Uncoupler-treated fibroblasts released less Type I collagen, while there was no change in fibronectin release. Collagen synthesis was restored to normal, when the uncouplers were removed, showing that these uncouplers were reversible and not toxic to cells. Northern blot analysis revealed procollagen alpha1 (I) mRNA was minimally affected by endosulfan. Oleamide-treated 17-day chick embryo calvaria explants were incubated with Type I collagen antibody, frozen, cryosectioned, and then subjected to rhodamine (Rh) tagged anti-mouse-IgG antibody, to detect newly deposited Type I collagen. Fluorescent antibody-collagen complexes were localized on the periphery of cells in control calvaria, but absent around cells in oleamide-treated calvaria. GJIC optimize collagen synthesis but not fibronectin synthesis. The lack of connective tissue deposited in granulation tissues treated with uncouplers appears related to the inhibition of collagen synthesis. These findings suggest that altering GJIC might control collagen deposition in scarring. 2006 Wiley-Liss, Inc.

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

    PubMed

    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.

  7. Absence of FKBP10 in Recessive Type XI Osteogenesis Imperfecta Leads to Diminished Collagen Cross-Linking and Reduced Collagen Deposition in Extracellular Matrix

    PubMed Central

    Barnes, Aileen M.; Cabral, Wayne A.; Weis, MaryAnn; Makareeva, Elena; Mertz, Edward L.; Leikin, Sergey; Eyre, David; Trujillo, Carlos; Marini, Joan C.

    2012-01-01

    Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% overmodification. Normal chain incorporation, helix folding, and collagen Tm support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix. PMID:22718341

  8. Structural requirements for fibromodulin binding to collagen and the control of type I collagen fibrillogenesis--critical roles for disulphide bonding and the C-terminal region.

    PubMed

    Font, B; Eichenberger, D; Goldschmidt, D; Boutillon, M M; Hulmes, D J

    1998-06-15

    Fibromodulin belongs to the family of small, leucine-rich proteoglycans which have been reported to interact with collagens and to inhibit type I collagen fibrillogenesis. Decorin and fibromodulin exhibit a noticeable degree of sequence similarity. However, as previously reported [Font, B., Eichenberger, D., Rosenberg, L. M. & van der Rest, M. (1996) Matrix Biol. 15, 341-348] the domains of these molecules implicated in the interactions with type XII and type XIV collagens are different, these being the dermatan sulphate/chondroitin sulphate chain for decorin and the core protein for fibromodulin. At the present time the fibromodulin domains implicated in the interactions with fibrillar collagens remain unknown. In experiments reported here, we have sought to identify the structural requirements for fibromodulin interaction with collagen and for the control of type I collagen fibrillogenesis. Circular dichroism spectra and fibrillogenesis inhibition studies show that fibromodulin structure and its collagen fibrillogenesis control function are strictly dependent on the presence of intact disulphide bridge(s). In addition, we show that the binding of fibromodulin (or fibromodulin-derived fragments) to type I collagen is not necessarily correlated with fibrillogenesis inhibition. To isolate fibromodulin domains, the native proteoglycan was submitted to mild proteolysis. We have isolated an alpha-chymotrypsin-resistant fragment which contains the bulk of the N-terminal and central region of the molecule including the leucine-rich repeats 4 and 6 reported for decorin to be involved in type I collagen binding. This fragment does not bind to type I collagen. Using enzymes with different specificities, a number of large fragments of fibromodulin were obtained, suggesting a compact structure for this molecule which is relatively resistant to proteolysis. None of these N-glycosylated fragments were able to bind to type I collagen in co-sedimentation experiments. Taken

  9. Collagen type IV at the fetal-maternal interface.

    PubMed

    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.

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

    PubMed Central

    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

  11. Label-free nonenzymatic glycation monitoring of collagen scaffolds in type 2 diabetic mice by confocal Raman microspectroscopy

    NASA Astrophysics Data System (ADS)

    Shi, Panpan; Liu, Hanping; Deng, Xiaoyuan; Jin, Ying; Wang, Qiannan; Liu, Hao; Chen, Maosheng; Han, Xue

    2015-02-01

    Collagen is the key target of nonenzymatic glycation during physiopathological processes such as diabetes. The induced changes in the biochemical property of collagen by nonenzymatic glycation remain a major challenge to probe. This study investigated the use of confocal Raman microspectroscopy to label-free monitor the nonenzymatic glycation of collagen scaffolds from type 2 diabetic (T2D) mice at different timepoints (0, 4, 8, and 12 weeks). The glycated collagen scaffolds were obtained through the decellularized dermal matrix method to remove the epidermis layer, subcutaneous tissue, and cells in the dermis and to retain the collagen fibrils. Raman spectra showed no changes in Raman peak positions, which indicated that nonenzymatic glycation could produce no significant changes in the triple-helix structure of collagen in T2D mice. However, the relative intensity of the Raman bands at 921, 1033, 1244, 1274, 1346, 1635, and 1672 cm-1 increased as diabetic time progressed. Correlation analysis suggested that the spectra of these bands had a high positive correlation with the expression of anti-advanced glycation end products obtained by immunofluorescence imaging of the same collagen scaffolds. Confocal Raman microspectroscopy proves a potential tool to label-free monitor the collagen changes caused by nonenzymatic glycation in T2D mice.

  12. Absence of FKBP10 in recessive type XI osteogenesis imperfecta leads to diminished collagen cross-linking and reduced collagen deposition in extracellular matrix.

    PubMed

    Barnes, Aileen M; Cabral, Wayne A; Weis, MaryAnn; Makareeva, Elena; Mertz, Edward L; Leikin, Sergey; Eyre, David; Trujillo, Carlos; Marini, Joan C

    2012-11-01

    Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% over-modification. Normal chain incorporation, helix folding, and collagen T(m) support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix. Published 2012 Wiley Periodicals, Inc.*This article is a US Government work and, as such, is in the public domain of the United States of America.

  13. Propolis Modifies Collagen Types I and III Accumulation in the Matrix of Burnt Tissue.

    PubMed

    Olczyk, Pawel; Wisowski, Grzegorz; Komosinska-Vassev, Katarzyna; Stojko, Jerzy; Klimek, Katarzyna; Olczyk, Monika; Kozma, Ewa M

    2013-01-01

    Wound healing represents an interactive process which requires highly organized activity of various cells, synthesizing cytokines, growth factors, and collagen. Collagen types I and III, serving as structural and regulatory molecules, play pivotal roles during wound healing. The aim of this study was to compare the propolis and silver sulfadiazine therapeutic efficacy throughout the quantitative and qualitative assessment of collagen types I and III accumulation in the matrix of burnt tissues. Burn wounds were inflicted on pigs, chosen for the evaluation of wound repair because of many similarities between pig and human skin. Isolated collagen types I and III were estimated by the surface plasmon resonance method with a subsequent collagenous quantification using electrophoretic and densitometric analyses. Propolis burn treatment led to enhanced collagens and its components expression, especially during the initial stage of the study. Less expressed changes were observed after silver sulfadiazine (AgSD) application. AgSD and, with a smaller intensity, propolis stimulated accumulation of collagenous degradation products. The assessed propolis therapeutic efficacy, throughout quantitatively and qualitatively analyses of collagen types I and III expression and degradation in wounds matrix, may indicate that apitherapeutic agent can generate favorable biochemical environment supporting reepithelization.

  14. In vitro, interaction of homotrimers with heterotrimers of type I collagen

    NASA Astrophysics Data System (ADS)

    Han, Sejin; Losert, Wolfgang; Leikin, Sergey

    2007-03-01

    The dominant mutations in type I collagen cause a group of diseases, often termed collagen, or connective tissue, diseases: for example, Osteogenesis Imperfecta (OI) characterized by bone fragility and skeletal deformity. The mechanism in which collagen mutations affect on the diseases is still unknown. To understand the fibril assembly and their interactions might provide a key to approaching the cause of the collagen diseases. This study demonstrates that the self-assembly, termed fibrillogenesis, of type I collagen homozygous mutations revealed substantial differences in the kinetics with the absence of lag time and in the morphology of 3D fibril network structure. The heterotrimers (normal) and homotrimers (mutant) in mixtures were segregated within the same fibrils during fibrillogenesis, in correspondence between confocal microscopy and thermodynamic measurements. The efficiency for self-assembly of the homotrimers into fibrils was markedly reduced, while that of the heterotrimers was not affected by the presence of homotrimers with no change in solubility.

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

    PubMed

    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.

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

    PubMed

    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.

  17. Vascular Ehlers-Danlos syndrome mutations in type III collagen differently stall the triple helical folding.

    PubMed

    Mizuno, Kazunori; Boudko, Sergei; Engel, Jürgen; Bächinger, Hans Peter

    2013-06-28

    Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)7 with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)7-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.

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

    PubMed

    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.

  19. Relative orientation of collagen molecules within a fibril: a homology model for homo sapiens type I collagen.

    PubMed

    Collier, Thomas A; Nash, Anthony; Birch, Helen L; de Leeuw, Nora H

    2018-02-15

    Type I collagen is an essential extracellular protein that plays an important structural role in tissues that require high tensile strength. However, owing to the molecule's size, to date no experimental structural data are available for the Homo sapiens species. Therefore, there is a real need to develop a reliable homology model and a method to study the packing of the collagen molecules within the fibril. Through the use of the homology model and implementation of a novel simulation technique, we have ascertained the orientations of the collagen molecules within a fibril, which is currently below the resolution limit of experimental techniques. The longitudinal orientation of collagen molecules within a fibril has a significant effect on the mechanical and biological properties of the fibril, owing to the different amino acid side chains available at the interface between the molecules.

  20. Ascorbic acid induces alkaline phosphatase, type X collagen, and calcium deposition in cultured chick chondrocytes.

    PubMed

    Leboy, P S; Vaias, L; Uschmann, B; Golub, E; Adams, S L; Pacifici, M

    1989-10-15

    During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic chondrocytes, which then deposit a mineralized matrix to form calcified cartilage. Chondrocyte hypertrophy and matrix mineralization are associated with expression of type X collagen and the induction of high levels of the bone/liver/kidney isozyme of alkaline phosphatase. To determine what role vitamin C plays in these processes, chondrocytes derived from the cephalic portion of 14-day chick embryo sternae were grown in the absence or presence of exogenous ascorbic acid. Control untreated cells displayed low levels of type X collagen and alkaline phosphatase activity throughout the culture period. However, cells grown in the presence of ascorbic acid produced increasing levels of alkaline phosphatase activity and type X collagen mRNA and protein. Both alkaline phosphatase activity and type X collagen mRNA levels began to increase within 24 h of ascorbate treatment; by 9 days, the levels of both alkaline phosphatase activity and type X collagen mRNA were 15-20-fold higher than in non-ascorbate-treated cells. Ascorbate treatment also increased calcium deposition in the cell layer and decreased the levels of types II and IX collagen mRNAs; these effects lagged significantly behind the elevation of alkaline phosphatase and type X collagen. Addition of beta-glycerophosphate to the medium increased calcium deposition in the presence of ascorbate but had no effect on levels of collagen mRNAs or alkaline phosphatase. The results suggest that vitamin C may play an important role in endochondral bone formation by modulating gene expression in hypertrophic chondrocytes.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yano, Hiroyuki; Division of Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593; Hamanaka, Ryoji

    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. Realmore » 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.« less

  2. Characterization of the interactions of type XII collagen with two small proteoglycans from fetal bovine tendon, decorin and fibromodulin.

    PubMed

    Font, B; Eichenberger, D; Rosenberg, L M; van der Rest, M

    1996-11-01

    In addition to the major collagens, such as type I or type II, connective tissues contain a number of less abundant collagens and proteoglycans, whose association contributes to the different properties of the tissues. Type XII and type XIV collagens have been described in soft connective tissues, and type XIV collagen has been shown to interact specifically with decorin through its glycosaminoglycan chain (Font et al., J. Biol. Chem. 268, 25015-25018, 1993). Interactions between these collagens and the small proteoglycans have been characterized further by studying the binding of type XII collagen to decorin by solid phase assays. Our results show a saturable binding of the proteoglycan through its glycosaminoglycan chain to type XII collagen, which does not seem to involve the large non-collagenous NC3 domain of the molecule. This interaction is strongly inhibited by heparin. Furthermore, we report that another small proteoglycan, fibromodulin, isolated from tendon under non-denaturing conditions, is able to bind to type XII collagen. This interaction has been characterized and, unlike that observed with decorin, type XII collagen-fibromodulin interaction seems to take place with the core protein of the proteoglycan. In addition, we report that type XII-type I collagen interactions are not necessarily mediated by decorin as previously suggested.

  3. Distribution of Basement Membrane Molecules, Laminin and Collagen Type IV, in Normal and Degenerated Cartilage Tissues

    PubMed Central

    Toh, Wei Seong; Gomoll, Andreas H.; Olsen, Bjørn Reino; Spector, Myron

    2014-01-01

    Objective: The objective of the present study was to investigate the presence and distribution of 2 basement membrane (BM) molecules, laminin and collagen type IV, in healthy and degenerative cartilage tissues. Design: Normal and degenerated tissues were obtained from goats and humans, including articular knee cartilage, the intervertebral disc, and meniscus. Normal tissue was also obtained from patella-tibial enthesis in goats. Immunohistochemical analysis was performed using anti-laminin and anti–collagen type IV antibodies. Human and goat skin were used as positive controls. The percentage of cells displaying the pericellular presence of the protein was graded semiquantitatively. Results: When present, laminin and collagen type IV were exclusively found in the pericellular matrix, and in a discrete layer on the articulating surface of normal articular cartilage. In normal articular (hyaline) cartilage in the human and goat, the proteins were found co-localized pericellularly. In contrast, in human osteoarthritic articular cartilage, collagen type IV but not laminin was found in the pericellular region. Nonpathological fibrocartilaginous tissues from the goat, including the menisci and the enthesis, were also positive for both laminin and collagen type IV pericellularly. In degenerated fibrocartilage, including intervertebral disc, as in degenerated hyaline cartilage only collagen type IV was found pericellularly around chondrocytes but with less intense staining than in non-degenerated tissue. In calcified cartilage, some cells were positive for laminin but not type IV collagen. Conclusions: We report differences in expression of the BM molecules, laminin and collagen type IV, in normal and degenerative cartilaginous tissues from adult humans and goats. In degenerative tissues laminin is depleted from the pericellular matrix before collagen type IV. The findings may inform future studies of the processes underlying cartilage degeneration and the functional

  4. Distribution of Basement Membrane Molecules, Laminin and Collagen Type IV, in Normal and Degenerated Cartilage Tissues.

    PubMed

    Foldager, Casper Bindzus; Toh, Wei Seong; Gomoll, Andreas H; Olsen, Bjørn Reino; Spector, Myron

    2014-04-01

    The objective of the present study was to investigate the presence and distribution of 2 basement membrane (BM) molecules, laminin and collagen type IV, in healthy and degenerative cartilage tissues. Normal and degenerated tissues were obtained from goats and humans, including articular knee cartilage, the intervertebral disc, and meniscus. Normal tissue was also obtained from patella-tibial enthesis in goats. Immunohistochemical analysis was performed using anti-laminin and anti-collagen type IV antibodies. Human and goat skin were used as positive controls. The percentage of cells displaying the pericellular presence of the protein was graded semiquantitatively. When present, laminin and collagen type IV were exclusively found in the pericellular matrix, and in a discrete layer on the articulating surface of normal articular cartilage. In normal articular (hyaline) cartilage in the human and goat, the proteins were found co-localized pericellularly. In contrast, in human osteoarthritic articular cartilage, collagen type IV but not laminin was found in the pericellular region. Nonpathological fibrocartilaginous tissues from the goat, including the menisci and the enthesis, were also positive for both laminin and collagen type IV pericellularly. In degenerated fibrocartilage, including intervertebral disc, as in degenerated hyaline cartilage only collagen type IV was found pericellularly around chondrocytes but with less intense staining than in non-degenerated tissue. In calcified cartilage, some cells were positive for laminin but not type IV collagen. We report differences in expression of the BM molecules, laminin and collagen type IV, in normal and degenerative cartilaginous tissues from adult humans and goats. In degenerative tissues laminin is depleted from the pericellular matrix before collagen type IV. The findings may inform future studies of the processes underlying cartilage degeneration and the functional roles of these 2 extracellular matrix proteins

  5. Low dose native type II collagen prevents pain in a rat osteoarthritis model

    PubMed Central

    2013-01-01

    Background Osteoarthritis is the most widespread joint-affecting disease. Patients with osteoarthritis experience pain and impaired mobility resulting in marked reduction of quality of life. A progressive cartilage loss is responsible of an evolving disease difficult to treat. The characteristic of chronicity determines the need of new active disease modifying drugs. Aim of the present research is to evaluate the role of low doses of native type II collagen in the rat model of osteoarthritis induced by sodium monoiodoacetate (MIA). Methods 1, 3 and 10 mg kg-1 porcine native type II collagen were daily per os administered for 13 days starting from the day of MIA intra-articular injection. Results On day 14, collagen-treated rats showed a significant prevention of pain threshold alterations induced by MIA. Evaluation were performed on paws using mechanical noxious (Paw pressure test) or non-noxious (Electronic Von Frey test) stimuli, and a decrease of articular pain was directly measured on the damaged joint (PAM test). The efficacy of collagen in reducing pain was as higher as the dose was lowered. Moreover, a reduced postural unbalance, measured as hind limb weight bearing alterations (Incapacitance test), and a general improvement of motor activity (Animex test) were observed. Finally, the decrease of plasma and urine levels of CTX-II (Cross Linked C-Telopeptide of Type II Collagen), a biomarker of cartilage degradation, suggests a collagen-dependent decrease of structural joint damage. Conclusions These results describe the preclinical efficacy of low dosages of native type II collagen as pain reliever by a mechanism that involves a protective effect on cartilage. PMID:23915264

  6. Gallium nitrate ameliorates type II collagen-induced arthritis in mice.

    PubMed

    Choi, Jae-Hyeog; Lee, Jong-Hwan; Roh, Kug-Hwan; Seo, Su-Kil; Choi, Il-Whan; Park, Sae-Gwang; Lim, Jun-Goo; Lee, Won-Jin; Kim, Myoung-Hun; Cho, Kwang-rae; Kim, Young-Jae

    2014-05-01

    Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease. Gallium nitrate has been reported to reserve immunosuppressive activities. Therefore, we assessed the therapeutic effects of gallium nitrate in the mouse model of developed type II collagen-induced arthritis (CIA). CIA was induced by bovine type II collagen with Complete Freund's adjuvant. CIA mice were intraperitoneally treated from day 36 to day 49 after immunization with 3.5mg/kg/day, 7mg/kg/day gallium nitrate or vehicle. Gallium nitrate ameliorated the progression of mice with CIA. The clinical symptoms of collagen-induced arthritis did not progress after treatment with gallium nitrate. Gallium nitrate inhibited the increase of CD4(+) T cell populations (p<0.05) and also inhibited the type II collagen-specific IgG2a-isotype autoantibodies (p<0.05). Gallium nitrate reduced the serum levels of TNF-α, IL-6 and IFN-γ (p<0.05) and the mRNA expression levels of these cytokine and MMPs (MMP2 and MMP9) in joint tissues. Western blotting of members of the NF-κB signaling pathway revealed that gallium nitrate inhibits the activation of NF-κB by blocking IκB degradation. These data suggest that gallium nitrate is a potential therapeutic agent for autoimmune inflammatory arthritis through its inhibition of the NF-κB pathway, and these results may help to elucidate gallium nitrate-mediated mechanisms of immunosuppression in patients with RA. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. 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.

    PubMed

    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.

  8. Type I collagen aging impairs discoidin domain receptor 2-mediated tumor cell growth suppression

    PubMed Central

    Saby, Charles; Buache, Emilie; Brassart-Pasco, Sylvie; El Btaouri, Hassan; Courageot, Marie-Pierre; Van Gulick, Laurence; Garnotel, Roselyne; Jeannesson, Pierre; Morjani, Hamid

    2016-01-01

    Tumor cells are confronted to a type I collagen rich environment which regulates cell proliferation and invasion. Biological aging has been associated with structural changes of type I collagen. Here, we address the effect of collagen aging on cell proliferation in a three-dimensional context (3D). We provide evidence for an inhibitory effect of adult collagen, but not of the old one, on proliferation of human fibrosarcoma HT-1080 cells. This effect involves both the activation of the tyrosine kinase Discoidin Domain Receptor 2 (DDR2) and the tyrosine phosphatase SHP-2. DDR2 and SHP-2 were less activated in old collagen. DDR2 inhibition decreased SHP-2 phosphorylation in adult collagen and increased cell proliferation to a level similar to that observed in old collagen. In the presence of old collagen, a high level of JAK2 and ERK1/2 phosphorylation was observed while expression of the cell cycle negative regulator p21CIP1 was decreased. Inhibition of DDR2 kinase function also led to an increase in ERK1/2 phosphorylation and a decrease in p21CIP1 expression. Similar signaling profile was observed when DDR2 was inhibited in adult collagen. Altogether, these data suggest that biological collagen aging could increase tumor cell proliferation by reducingthe activation of the key matrix sensor DDR2. PMID:27121132

  9. Target-Specific Delivery of an Antibody That Blocks the Formation of Collagen Deposits in Skin and Lung.

    PubMed

    Fertala, Jolanta; Romero, Freddy; Summer, Ross; Fertala, Andrzej

    2017-10-01

    Regardless of the cause of organ fibrosis, its main unwanted consequence is the formation of collagen fibril-rich deposits that hamper the structure and function of affected tissues. Although many strategies have been proposed for the treatment of fibrotic diseases, no therapy has been developed, which can effectively block the formation of collagen fibril deposits. With this in mind, we recently developed an antibody-based therapy to block key interactions that drive collagen molecules into fibrils. In this study, we analyzed target specificity, which is a main parameter that defines the safe use of all antibody-based therapies in humans. We hypothesized that, regardless of the route of administration, our antibody would preferentially bind to free collagen molecules synthesized at the sites of fibrosis and have minimal off-target interactions when applied in various tissues. To test this hypothesis, we used two experimental models of organ fibrosis: (1) a keloid model, in which antibody constructs were directly implanted under the skin of nude mice and (2) an experimental model of pulmonary fibrosis, in which our antibody was administered systemically by intravenous injection. Following administration, we studied the distribution of our antibody within target and off-target sites as well as analyzed its effects on fibrotic tissue formation. We found that local and systemic application of our antibody had high specificity for targeting collagen fibrillogenesis and also appeared safe and therapeutically effective. In summary, this study provides the basis for further testing our antifibrotic antibody in a broad range of disease conditions and suggests that this treatment approach will be effective if delivered by local or systemic administration.

  10. A new method for meniscus repair using type I collagen scaffold and infrapatellar fat pad.

    PubMed

    Oda, Shuhei; Otsuki, Shuhei; Kurokawa, Yoshitaka; Hoshiyama, Yoshiaki; Nakajima, Mikio; Neo, Masashi

    2015-05-01

    The aim of this study was to investigate a new method for meniscal repair by combinative transplantation with type I collagen scaffold and infrapatellar fat pad. Two-mm cylindrical defects at the anterior part of bilateral medial menisci were prepared in nine Japanese white rabbits. The 18 knees were equally divided into three groups: I, no treatment; II, collagen scaffold transplantation; and III, collagen scaffold and infrapatellar fat pad transplantation. Another three rabbits (six knees) underwent sham surgery and served as controls. Rabbits were sacrificed at eight weeks after transplantation. Surface area of the medial meniscus was evaluated using macrophotographs. Ishida score for meniscal regeneration was used for assessment. To evaluate the composition of regenerated tissue, immunohistochemistry was analyzed with anti-type I and anti-type II collagen antibodies, and anti-Ki67 antibody. To investigate the effects of collagen scaffold on human meniscus, cells were isolated from human meniscus and infrapatellar fat pad, and cultured with collagen scaffold for three weeks. After that, gene expression was evaluated by using quantitative real-time polymerase chain reaction. In group I, the meniscus shrank anterior to posterior, and the surface area was significantly less than that of normal meniscus. However, the surface area was maintained in group III. Ishida score and Ki67-positive cell ratio in group III were significantly higher than that in any other group, and staining with type I and type II collagen was similar to that of the control. Expression of matrix metalloproteinase was significantly lower in cocultures of collagen scaffold, meniscus cell, and infrapatellar fat pad cell than in monocultured meniscus cell, and expression of interleukin-1β was not increased. This new method for meniscal repair by combinative transplantation with type I collagen scaffold and infrapatellar fat pad showed meniscal regeneration and potential for suppressing

  11. Collagen structural microheterogeneity and a possible role for glycosylated hydroxylysine in type I collagen

    PubMed Central

    Yamauchi, Mitsuo; Noyes, Claudia; Kuboki, Yoshinori; Mechanic, Gerald L.

    1982-01-01

    A three-chained peptide from type I collagen, crosslinked by hydroxyaldolhistidine, has been isolated from a tryptic digest of 5 M guanidine·HCl-insoluble bovine skin collagen (a small but as yet unknown percentage of the total collagen in whole skin). OsO4/NaIO4 specifically cleaved the crosslink at its double bond into a two-chained crosslink peptide and a single peptide. The sequence of the two-chained peptide containing the bifunctional crosslink was determined after amino acid analysis of the separated peptides. The crosslink consists of an aldehyde derived from hydroxylysine-87 in the aldehyde-containing cyanogen bromide fragment α1CB5ald and an aldehyde derived from the lysine in the COOH-terminal nonhelical region of the α1CB6ald fragment. The α1CB6ald portion of the peptide exhibited structural microheterogeneity, containing the inverted sequence Ala-Lys-His instead of the normal sequence Lys-Ala-His. This indicates that another structural gene exists for α1(I) chain. The original three-chained peptide did not contain any glycosylated hydroxylysine or glycosylated hydroxyaldolhistidine. The lack of glycosylation of hydroxylysine-87 in α1CB5, which is usually glycosylated, allowed formation of the aldehyde, and this, coupled with the sequence inversion, may have allowed formation of the nonreducible crosslink hydroxyaldolhistidine. We suggest that the role of glycosylation, a posttranslational modification, of specific hydroxylysine residues is to prevent their oxidative deamination to aldehydes, thereby precluding formation of complex stable crosslinks. Complex crosslinks would decrease the rate of collagen turnover. The decrease, with time, would increase the population of stable crosslinked collagen molecules, which would eventually accumulate with age. PMID:6961443

  12. An update on the constitutive relation of ligament tissues with the effects of collagen types.

    PubMed

    Wan, Chao; Hao, Zhixiu; Tong, Lingying; Lin, Jianhao; Li, Zhichang; Wen, Shizhu

    2015-10-01

    The musculoskeletal ligament is a kind of multiscale composite material with collagen fibers embedded in a ground matrix. As the major constituent in ligaments to bear external loads, collagens are composed mainly of two collagen contents with different mechanical properties, i.e., types I and III collagen. The constitutive relation of ligaments plays a critical role in the stability and normal function of human joints. However, collagen types have not been distinguished in the previous constitutive relations. In this paper a constitutive relation for ligament tissues was modified based on the previous constitutive relation by considering the effects of collagen types. Both the collagen contents and the mechanical properties of sixteen ligament specimens from four cadaveric human knee joints were measured for determining their material coefficients in the constitutive relation. The mechanical behaviors of ligaments were obtained from both the uniaxial tensile and simple shear tests. A linear regression between joint kinematic results from in vitro and in silico experiments was made to validate the accuracy of this constitutive relation. The high correlation coefficient (R(2)=0.93) and significance (P<0.0001) of the regression equation revealed that this modified constitutive relation of ligaments was accurate to be used in studying joint biomechanics. Another finite element analysis with collagen contents changing demonstrated that the effect of variations in collagen ratios on both joint kinematics and ligament biomechanics could be simulated by this constitutive relation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Determination of three-dimensional molecular orientation of type-I collagen by circularly-polarized second harmonic generation imaging

    NASA Astrophysics Data System (ADS)

    Zhuo, Guan-Yu; Hung, Wei-Han; Kao, Fu-Jen

    2017-04-01

    The content of collagen is up to 30% existing in mammals. It supports the main component of connective tissues such as skin, ligament, and cartilage. Among various types of collagen, type-I collagen is of the most abundance and has been broadly studied due to the importance in bioscience. Second harmonic generation (SHG) microscopy is an effective tool used to study the collagen organization without labeling. In this study, we used circular polarization instead of linear polarization to retrieve three-dimensional (3D) molecular orientation of type-I collagen with only two cross polarized SHG images without acquiring an image stack of varying polarization.

  14. Freeze-dried, cross-linked bovine type I collagen: analysis of properties.

    PubMed

    Hyder, P R; Dowell, P; Singh, G; Dolby, A E

    1992-03-01

    This study was undertaken to assess the physical and biological properties of freeze-dried cross-linked bovine type I collagen and to assess its potential for use in the guided tissue regeneration method of treatment of periodontal disease in human adult subjects. The modulus of elasticity, swelling ratio, and biodegradation rate were investigated. The collagen sponge was implanted subdermally into Sprague-Dawley rats and a histological study carried out at 2, 7, 21, 35, and 49 days post implantation. Growth of human gingival and periodontal ligament derived fibroblasts on collagen sponge was assessed, as well as the effect of bovine collagen supernatants upon gingival and periodontal fibroblast cultures. The physical properties of the collagen sponge were consistent with good handling qualities and, therefore, it was appropriate for use at a surgical site. The histological study demonstrated a reduction in thickness of the collagen at 21 days; at 35 days there was a hazy appearance of the collagen remnants; and at 49 days the graft material had been completely replaced with fibrous tissues. The in vitro response of human gingival and periodontal fibroblasts to bovine collagen showed that, after 21 days, confluent fibroblast growth was observed around and underneath the sponge. The effect of bovine collagen supernatants upon fibroblasts demonstrated an apparent proliferative effect of the supernatant with both gingival and periodontal ligament fibroblasts. However, the non-parametric Friedman test revealed no significant differences between dilutions or time points. The overall findings provide encouraging evidence of the safety of freeze-dried cross-linked bovine collagen sponge in the surgical treatment of periodontal disease.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Identification of Type VI Collagen Synthesizing Cells in Human Diabetic Glomerulosclerosis Using Renal Biopsy Sections

    PubMed Central

    Razzaque, Mohammed Shawkat; Koji, Takehiko; Harada, Takashi; Taguchi, Takashi

    1997-01-01

    Although the role of extracellular matrices in the development of glomerulosclerosis has been discussed widely, the cellular origin of type VI collagen in diabetic nephropathy (DN) has remained relatively unexplored. This study reports the distribution and cellular origin of type VI collagen in DN. Type VI collagen‐specific oligonucleotide probes and monoclonal antibody were used to assess the relative expression of mRNA for \\alpha1 (VI) chain and its translated protein in paraffin‐embedded renal biopsy sections of DN. By immunohistochemistry, compared to the control, increased deposition of type VI collagen was noted in the diffuse and nodular lesions of diabetic glomeruli. For cellular localization of type VI collagen mRNA, paraffin‐embedded renal sections of the control and DN were hybridized in situ with digoxigenin (Dig)‐labeled antisense oligo‐DNA probe complementary to a part of \\alpha1 (VI) mRNA. In comparison to the control kidney sections, increased numbers of intraglomerular cells (both mesangial and epithelial cells) were positive for α1 (VI) mRNA in renal biopsy sections of DN. From the results, we conclude that overexpression of type VI collagen by intraglomerular cells with its increased deposition might significantly contribute to the glomerulosclerosis found in DN. PMID:9497854

  16. Distribution of collagens type V and VI in the normal human alveolar mucosa: an immunoelectronmicroscopic study using ultrathin frozen sections.

    PubMed

    Rabanus, J P; Gelderblom, H R; Schuppan, D; Becker, J

    1991-05-01

    The ultrastructural localization of collagens type V and VI in normal human gingival mucosa was investigated by immunoelectron microscopy. Twenty biopsies were fixed in dimethylsuberimidate and shock-frozen in slush nitrogen. Collagen type V was mainly located to meshworks of uniform nonstriated microfibrils of 12 to 20 nm width, which preferentially appeared in larger spaces between cross-striated major collagen fibrils. Occasionally single microfibrils of collagen type V fanned out from the ends of major collagen fibrils, which may indicate a role as a core fibril. Collagen type V was not found in the subepithelial basement membrane and the immediately adjacent stroma. Collagen type VI was detected in a loose reticular network of unbanded microfilaments that were morphologically distinguishable by knoblike protrusions every 100-110 nm. These microfilaments were found in the vicinity, but not as an intrinsic component, of the subepithelial basement membrane. Single filaments of collagen type VI filaments appeared to form bridges between neighboring cross-striated major collagen fibrils, suggesting an interconnecting role for this collagen type. The method presented appears to be excellently suited to study the normal and pathological supramolecular organization of the oral extracellular matrix.

  17. Distribution of Type I Collagen Morphologies in Bone: Relation to Estrogen Depletion

    PubMed Central

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

    2009-01-01

    Bone is an amazing material evolved by nature to elegantly balance structural and metabolic needs in the body. Bone health is an integral part of overall health, but our lack of understanding of the ultrastructure of healthy bone precludes us from knowing how disease may impact nanoscale properties in this biological material. Here, we show that quantitative assessments of a distribution of Type I collagen fibril morphologies can be made using atomic force microscopy (AFM). We demonstrate that normal bone contains a distribution of collagen fibril morphologies and that changes in this distribution can be directly related to disease state. Specifically, by monitoring changes in the collagen fibril distribution of sham-operated and estrogen-depleted sheep, we have shown the ability to detect estrogen-deficiency-induced changes in Type I collagen in bone. This discovery provides new insight into the ultrastructure of bone as a tissue and the role of material structure in bone disease. The observation offers the possibility of a much-needed in vitro procedure to complement the current methods used to diagnose osteoporosis and other bone disease. PMID:19932773

  18. RNA protein interactions governing expression of the most abundant protein in human body, type I collagen.

    PubMed

    Stefanovic, Branko

    2013-01-01

    Type I collagen is the most abundant protein in human body. The protein turns over slowly and its replacement synthesis is low. However, in wound healing or in pathological fibrosis the cells can increase production of type I collagen several hundred fold. This increase is predominantly due to posttranscriptional regulation, including increased half-life of collagen messenger RNAs (mRNAs) and their increased translatability. Type I collagen is composed of two α1 and one α2 polypeptides that fold into a triple helix. This stoichiometry is strictly regulated to prevent detrimental synthesis of α1 homotrimers. Collagen polypeptides are co-translationally modified and the rate of modifications is in dynamic equilibrium with the rate of folding, suggesting coordinated translation of collagen α1(I) and α2(I) polypeptides. Collagen α1(I) mRNA has in the 3' untranslated region (UTR) a C-rich sequence that binds protein αCP, this binding stabilizes the mRNA in collagen producing cells. In the 5' UTR both collagen mRNAs have a conserved stem-loop (5' SL) structure. The 5' SL is critical for high collagen expression, knock in mice with disruption of the 5' SL are resistant to liver fibrosis. the 5' SL binds protein LARP6 with strict sequence specificity and high affinity. LARP6 recruits RNA helicase A to facilitate translation initiation and associates collagen mRNAs with vimentin and nonmuscle myosin filaments. Binding to vimentin stabilizes collagen mRNAs, while nonmuscle myosin regulates coordinated translation of α1(I) and α2(I) mRNAs. When nonmuscle myosin filaments are disrupted the cells secrete only α1 homotrimers. Thus, the mechanism governing high collagen expression involves two RNA binding proteins and development of cytoskeletal filaments. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Anti-Type VII Collagen Antibodies Are Identified in a Subpopulation of Bullous Pemphigoid Patients With Relapse

    PubMed Central

    Giusti, Delphine; Gatouillat, Grégory; Le Jan, Sébastien; Plée, Julie; Bernard, Philippe; Antonicelli, Frank; Pham, Bach-Nga

    2018-01-01

    Bullous pemphigoid (BP) is an autoimmune bullous skin disease characterized by anti-BP180 and anti-BP230 autoantibodies (AAbs). Mucous membrane involvement is an uncommon clinical feature of BP which may evoke epidermolysis bullosa acquisita, another skin autoimmune disease characterized by anti-type VII collagen AAbs. We therefore evaluated the presence of anti-type VII collagen AAbs in the serum of BP patients with and without mucosal lesions at time of diagnosis and under therapy. Anti-BP180, anti-BP230, and anti-type VII collagen AAbs were measured by ELISA in the serum of unselected patients fulfilling clinical and histo/immunopathological BP criteria at baseline (n = 71) and at time of relapse (n = 24). At baseline, anti-type VII collagen AAbs were detected in 2 out of 24 patients with BP presenting with mucosal involvement, but not in patients without mucosal lesions (n = 47). At the time of relapse, 10 out of 24 BP patients either displayed a significant induction or increase of concentrations of anti-type VII collagen AAbs (P < 0.01), independently of mucosal involvement. Those 10 relapsing BP patients were also characterized by a sustained high concentration of anti-BP180 AAb, whereas the serum anti-BP230 AAb concentrations did not vary in BP patients with relapse according to the presence of anti-type VII collagen AAbs. Thus, our study showed that anti-type VII collagen along with anti-BP180 AAbs detection stratified BP patients at time of relapse, illustrating a still dysregulated immune response that could reflect a potential epitope spreading mechanism in those BP patients. PMID:29619029

  20. Evaluating adhesion reduction efficacy of type I/III collagen membrane and collagen-GAG resorbable matrix in primary flexor tendon repair in a chicken model.

    PubMed

    Turner, John B; Corazzini, Rubina L; Butler, Timothy J; Garlick, David S; Rinker, Brian D

    2015-09-01

    Reduction of peritendinous adhesions after injury and repair has been the subject of extensive prior investigation. The application of a circumferential barrier at the repair site may limit the quantity of peritendinous adhesions while preserving the tendon's innate ability to heal. The authors compare the effectiveness of a type I/III collagen membrane and a collagen-glycosaminoglycan (GAG) resorbable matrix in reducing tendon adhesions in an experimental chicken model of a "zone II" tendon laceration and repair. In Leghorn chickens, flexor tendons were sharply divided using a scalpel and underwent repair in a standard fashion (54 total repairs). The sites were treated with a type I/III collagen membrane, collagen-GAG resorbable matrix, or saline in a randomized fashion. After 3 weeks, qualitative and semiquantitative histological analysis was performed to evaluate the "extent of peritendinous adhesions" and "nature of tendon healing." The data was evaluated with chi-square analysis and unpaired Student's t test. For both collagen materials, there was a statistically significant improvement in the degree of both extent of peritendinous adhesions and nature of tendon healing relative to the control group. There was no significant difference seen between the two materials. There was one tendon rupture observed in each treatment group. Surgical handling characteristics were subjectively favored for type I/III collagen membrane over the collagen-GAG resorbable matrix. The ideal method of reducing clinically significant tendon adhesions after injury remains elusive. Both materials in this study demonstrate promise in reducing tendon adhesions after flexor tendon repair without impeding tendon healing in this model.

  1. Nanoscale Morphology of Type I Collagen is Altered in the Brtl Mouse Model of Osteogenesis Imperfecta

    PubMed Central

    Wallace, Joseph M.; Orr, Bradford G.; Marini, Joan C.; Banaszak Holl, Mark M.

    2010-01-01

    Bone has a complex hierarchical structure that has evolved to serve structural and metabolic roles in the body. Due to the complexity of bone structure and the number of diseases which affect the ultrastructural constituents of bone, it is important to develop quantitative methods to assess bone nanoscale properties. Autosomal dominant Osteogenesis Imperfecta results predominantly from glycine substitutions (80%) and splice site mutations (20%) in the genes encoding the α1 or α2 chains of Type I collagen. Genotype-phenotype correlations using over 830 collagen mutations have revealed that lethal mutations are located in regions crucial for collagen-ligand binding in the matrix. However, few of these correlations have been extended to collagen structure in bone. Here, an atomic force microscopy-based approach was used to image and quantitatively analyze the D-periodic spacing of Type I collagen fibrils in femora from heterozygous (Brtl/+) mice (α1(I)G349C), compared to wild type (WT) littermates. This disease system has a well-defined change in the col1α1 allele, leading to a well characterized alteration in collagen protein structure, which are directly related to altered Type I collagen nanoscale morphology, as measured by the D-periodic spacing. In Brtl/+ bone, the D-periodic spacing shows significantly greater variability on average and along the length of the bone compared to WT, although the average spacing was unchanged. Brtl/+ bone also had a significant difference in the population distribution of collagen D-period spacings. These changes may be due to the mutant collagen structure, or to the heterogeneity of collagen monomers in the Brtl/+ matrix. These observations at the nanoscale level provide insight into the structural basis for changes present in bone composition, geometry and mechanical integrity in Brtl/+ bones. Further studies are necessary to link these morphological observations to nanoscale mechanical integrity. PMID:20696252

  2. Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro

    NASA Astrophysics Data System (ADS)

    Tamaddon, M.; Burrows, M.; Ferreira, S. A.; Dazzi, F.; Apperley, J. F.; Bradshaw, A.; Brand, D. D.; Czernuszka, J.; Gentleman, E.

    2017-03-01

    Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS - which was released from scaffolds quickly - significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA.

  3. In Situ Imaging of Tissue Remodeling with Collagen Hybridizing Peptides

    PubMed Central

    2017-01-01

    Collagen, the major structural component of nearly all mammalian tissues, undergoes extensive proteolytic remodeling during developmental states and a variety of life-threatening diseases such as cancer, myocardial infarction, and fibrosis. While degraded collagen could be an important marker of tissue damage, it is difficult to detect and target using conventional tools. Here, we show that a designed peptide (collagen hybridizing peptide: CHP), which specifically hybridizes to the degraded, unfolded collagen chains, can be used to image degraded collagen and inform tissue remodeling activity in various tissues: labeled with 5-carboxyfluorescein and biotin, CHPs enabled direct localization and quantification of collagen degradation in isolated tissues within pathologic states ranging from osteoarthritis and myocardial infarction to glomerulonephritis and pulmonary fibrosis, as well as in normal tissues during developmental programs associated with embryonic bone formation and skin aging. The results indicate the general correlation between the level of collagen remodeling and the amount of denatured collagen in tissue and show that the CHP probes can be used across species and collagen types, providing a versatile tool for not only pathology and developmental biology research but also histology-based disease diagnosis, staging, and therapeutic screening. This study lays the foundation for further testing CHP as a targeting moiety for theranostic delivery in various animal models. PMID:28877431

  4. Type I collagen gel protects murine fibrosarcoma L929 cells from TNFα-induced cell death

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Hong-Ju; He, Wen-Qi; Chen, Ling

    Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however,more » were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death. - Highlights: • Collagen gel culture changed the morphology of L929 cells. • L929 cell cultured on collagen gel were resistant to TNFα-induced cell death. • Collagen gel culture inhibited TNFα-induced autophagy in L929 cells.« less

  5. Chondrogenic properties of collagen type XI, a component of cartilage extracellular matrix.

    PubMed

    Li, Ang; Wei, Yiyong; Hung, Clark; Vunjak-Novakovic, Gordana

    2018-08-01

    Cartilage extracellular matrix (ECM) has been used for promoting tissue engineering. However, the exact effects of ECM on chondrogenesis and the acting mechanisms are not well understood. In this study, we investigated the chondrogenic effects of cartilage ECM on human mesenchymal stem cells (MSCs) and identified the contributing molecular components. To this end, a preparation of articular cartilage ECM was supplemented to pellets of chondrogenically differentiating MSCs, pellets of human chondrocytes, and bovine articular cartilage explants to evaluate the effects on cell proliferation and the production of cartilaginous matrix. Selective enzymatic digestion and screening of ECM components were conducted to identify matrix molecules with chondrogenic properties. Cartilage ECM promoted MSC proliferation, production of cartilaginous matrix, and maturity of chondrogenic differentiation, and inhibited the hypertrophic differentiation of MSC-derived chondrocytes. Selective digestion of ECM components revealed a contributory role of collagens in promoting chondrogenesis. The screening of various collagen subtypes revealed strong chondrogenic effect of collagen type XI. Finally, collagen XI was found to promote production and inhibit degradation of cartilage matrix in human articular chondrocyte pellets and bovine articular cartilage explants. Our results indicate that cartilage ECM promotes chondrogenesis and inhibits hypertrophic differentiation in MSCs. Collagen type XI is the ECM component that has the strongest effects on enhancing the production and inhibiting the degradation of cartilage matrix. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Bacopa monniera (L.) wettst inhibits type II collagen-induced arthritis in rats.

    PubMed

    Viji, V; Kavitha, S K; Helen, A

    2010-09-01

    Bacopa monniera (L.) Wettst is an Ayurvedic herb with antirheumatic potential. This study investigated the therapeutic efficacy of Bacopa monniera in treating rheumatoid arthritis using a type II collagen-induced arthritis rat model. Arthritis was induced in male Wistar rats by immunization with bovine type II collagen in complete Freund's adjuvant. Bacopa monniera extract (BME) was administered after the development of arthritis from day 14 onwards. The total duration of experiment was 60 days. Paw swelling, arthritic index, inflammatory mediators such as cyclooxygenase, lipoxygenase, myeloperoxidase and serum anti-collagen IgG and IgM levels were analysed in control and experimental rats. Arthritic induction significantly increased paw edema and other classical signs of arthritis coupled to upregulation of inflammatory mediators such as cyclooxygenase, lipoxygenase, neutrophil infiltration and increased anti-collagen IgM and IgG levels in serum. BME significantly inhibited the footpad swelling and arthritic symptoms. BME was effective in inhibiting cyclooxygenase and lipoxygenase activities in arthritic rats. Decreased neutrophil infiltration was evident from decreased myeloperoxidase activity and histopathological data where an improvement in joint architecture was also observed. Serum anti-collagen IgM and IgG levels were consistently decreased. Thus the study demonstrates the potential antiarthritic effect of Bacopa monniera for treating arthritis which might confer its antirheumatic activity. Copyright 2010 John Wiley & Sons, Ltd.

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

    PubMed

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

    2016-01-21

    Here, a growth-factor-integrated natural extracellular matrix of type I collagen is presented that induces angiogenesis. The developed matrix adapts type I collagen nanofibers integrated with synthetic colloidal particles of recombinant bacteriophages that display vascular endothelial growth factor (VEGF). The integration is achieved during or after gelation of the type I collagen and the matrix enables spatial delivery of VEGF into a desired region. Endothelial cells that contact the VEGF are found to invade into the matrix to form tube-like structures both in vitro and in vivo, proving the angiogenic potential of the matrix. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Proportion of collagen type II in the extracellular matrix promotes the differentiation of human adipose-derived mesenchymal stem cells into nucleus pulposus cells.

    PubMed

    Tao, Yiqing; Zhou, Xiaopeng; Liu, Dongyu; Li, Hao; Liang, Chengzhen; Li, Fangcai; Chen, Qixin

    2016-01-01

    During degeneration process, the catabolism of collagen type II and anabolism of collagen type I in nucleus pulposus (NP) may influence the bioactivity of transplanted cells. Human adipose-derived mesenchymal stem cells (hADMSCs) were cultured as a micromass or in a series of gradual proportion hydrogels of a mix of collagen types I and II. Cell proliferation and cytotoxicity were detected using CCK-8 and LDH assays respectively. The expression of differentiation-related genes and proteins, including SOX9, aggrecan, collagen type I, and collagen type II, was examined using RT-qPCR and Western blotting. Novel phenotypic genes were also detected by RT-qPCR and western blotting. Alcian blue and dimethylmethylene blue assays were used to investigate sulfate proteoglycan expression, and PI3K/AKT, MAPK/ERK, and Smad signaling pathways were examined by Western blotting. The results showed collagen hydrogels have good biocompatibility, and cell proliferation increased after collagen type II treatment. Expressions of SOX9, aggrecan, and collagen type II were increased in a collagen type II dependent manner. Sulfate proteoglycan synthesis increased in proportion to collagen type II concentration. Only hADMSCs highly expressed NP cell marker KRT19 in collagen type II culture. Additionally, phosphorylated Smad3, which is associated with phosphorylated ERK, was increased after collagen type II-stimulation. The concentration and type of collagen affect hADMSC differentiation into NP cells. Collagen type II significantly ameliorates hADMSC differentiation into NP cells and promotes extracellular matrix synthesis. Therefore, anabolism of collagen type I and catabolism of type II may attenuate the differentiation and biosynthesis of transplanted stem cells. © 2016 International Union of Biochemistry and Molecular Biology.

  9. Second Harmonic Generation Confocal Microscopy of Collagen Type I from Rat Tendon Cryosections

    PubMed Central

    Theodossiou, Theodossis A.; Thrasivoulou, Christopher; Ekwobi, Chidi; Becker, David L.

    2006-01-01

    We performed second harmonic generation (SHG) imaging of collagen in rat-tendon cryosections, using femtosecond laser scanning confocal microscopy, both in backscattering and transmission geometries. SHG transmission images of collagen fibers were spatially resolved due to a coherent, directional SHG component. This effect was enhanced with the use of an index-matching fluid (ni = 1.52). The average SHG intensity oscillated with wavelength in the backscattered geometry (isotropic SHG component), whereas the spectral profile was consistent with quasi-phase-matching conditions in transmission geometry (forward propagating, coherent SHG component) around 440 nm (λp = 880 nm). Collagen type I from bovine Achilles tendon was imaged for SHG in the backscattered geometry and its first-order effective nonlinear coefficient was determined (\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\vert}d_{{\\mathrm{eff}}}{\\vert}\\approx 0.085({\\pm}0.025){\\times}10^{-12}{\\mathrm{mV}}^{-1}\\end{equation*}\\end{document}) by comparison to samples of inorganic materials with known effective nonlinear coefficients (LiNbO3 and LiIO3). The SHG spectral response of collagen type I from bovine Achilles tendon matched that of the rat-tendon cryosections in backscattered geometry. Collagen types I, II, and VI powders (nonfibrous) did not show any detectable SHG, indicating a lack of noncentrosymmetric crystalline structure at the molecular level. The various stages of collagen thermal denaturation were investigated in rat-tendon cryosections using SHG and bright-field imaging. Thermal denaturation resulted in the gradual destruction of the SHG signal. PMID:17130233

  10. Type VII collagen is enriched in the enamel organic matrix associated with the dentin-enamel junction of mature human teeth.

    PubMed

    McGuire, Jacob D; Walker, Mary P; Mousa, Ahmad; Wang, Yong; Gorski, Jeff P

    2014-06-01

    The inner enamel region of erupted teeth is known to exhibit higher fracture toughness and crack growth resistance than bulk phase enamel. However, an explanation for this behavior has been hampered by the lack of compositional information for the residual enamel organic matrix. Since enamel-forming ameloblasts are known to express type VII collagen and type VII collagen null mice display abnormal amelogenesis, the aim of this study was to determine whether type VII collagen is a component of the enamel organic matrix at the dentin-enamel junction (DEJ) of mature human teeth. Immunofluorescent confocal microscopy of demineralized tooth sections localized type VII collagen to the organic matrix surrounding individual enamel rods near the DEJ. Morphologically, immunoreactive type VII collagen helical-bundles resembled the gnarled-pattern of enamel rods detected by Coomassie Blue staining. Western blotting of whole crown or enamel matrix extracts also identified characteristic Mr=280 and 230 kDa type VII dimeric forms, which resolved into 75 and 25 kDa bands upon reduction. As expected, the collagenous domain of type VII collagen was resistant to pepsin digestion, but was susceptible to purified bacterial collagenase. These results demonstrate the inner enamel organic matrix in mature teeth contains macromolecular type VII collagen. Based on its physical association with the DEJ and its well-appreciated capacity to complex with other collagens, we hypothesize that enamel embedded type VII collagen fibrils may contribute not only to the structural resilience of enamel, but may also play a role in bonding enamel to dentin. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Type VII Collagen is Enriched in the Enamel Organic Matrix Associated with the Dentin-Enamel Junction of Mature Human Teeth

    PubMed Central

    McGuire, Jacob D.; Walker, Mary P.; Mousa, Ahmad; Wang, Yong; Gorski, Jeff P.

    2014-01-01

    The inner enamel region of erupted teeth is known to exhibit higher fracture toughness and crack growth resistance than bulk phase enamel. However, an explanation for this behavior has been hampered by the lack of compositional information for the residual enamel organic matrix. Since enamel-forming ameloblasts are known to express type VII collagen and type VII collagen null mice display abnormal amelogenesis, the aim of this study was to determine whether type VII collagen is a component of the enamel organic matrix at the dentin-enamel junction (DEJ) of mature human teeth. Immunofluorescent confocal microscopy of demineralized tooth sections localized type VII collagen to the organic matrix surrounding individual enamel rods near the DEJ. Morphologically, immunoreactive type VII collagen helical-bundles resembled the gnarled-pattern of enamel rods detected by Coomassie Blue staining. Western blotting of whole crown or enamel matrix extracts also identified characteristic Mr=280 and 230 kDa type VII dimeric forms, which resolved into 75 and 25 kDa bands upon reduction. As expected, the collagenous domain of type VII collagen was resistant to pepsin digestion, but was susceptible to purified bacterial collagenase. These results demonstrate the inner enamel organic matrix in mature teeth contains macromolecular type VII collagen. Based on its physical association with the DEJ and its well-appreciated capacity to complex with other collagens, we hypothesize that enamel embedded type VII collagen fibrils may contribute not only to the structural resilience of enamel, but may also play a role in bonding enamel to dentin. PMID:24594343

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

    PubMed Central

    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

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

    PubMed

    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.

  14. Effects of immobilization and whole-body vibration on rat serum Type I collagen turnover.

    PubMed

    Dönmez, Gürhan; Doral, Mahmut Nedim; Suljevic, Şenay; Sargon, Mustafa Fevzi; Bilgili, Hasan; Demirel, Haydar Ali

    2016-08-01

    The aim of this study was to investigate the effects of short-term, high-magnitude whole-body vibration (WBV) on serum type I collagen turnover in immobilized rats. Thirty Wistar albino rats were randomly divided into the following 5 groups: immobilization (IS), immobilization + remobilization (IR), immobilization + WBV (IV), control (C), and WBV control (CV). Immobilization was achieved by casting from the crista iliaca anterior superior to the lower part of the foot for 2 weeks. The applied WBV protocol involved a frequency of 45 Hz and amplitude of 3 mm for 7 days starting a day after the end of the immobilization period. Serum type I collagen turnover markers were measured by using ELISA kits. Serum NH2-terminal propeptide of type I collagen (PINP) levels were significantly lower in the immobilization groups (p < 0.02) compared with the control groups. Although WBV improved PINP levels in the control groups, there were no differences in PINP levels among the immobilization groups. Similarly, serum COOH-terminal telopeptide of type I collagen (CTX) levels were higher in the WBV controls than their own controls (p < 0,05). Immobilization led to deterioration of tendon tissue, as observed by histopathological analysis with a transmission electron microscope. Although 1 week of WBV had a positive effect on type I collagen turnover in controls, it is not an efficient method for repairing tissue damage in the early stage following immobilization. Copyright © 2016 Turkish Association of Orthopaedics and Traumatology. Production and hosting by Elsevier B.V. All rights reserved.

  15. Imaging Prostate Cancer Microenvironment by Collagen Hybridization

    DTIC Science & Technology

    2016-10-01

    affinity to denatured collagens and collagens undergoing remodeling which simulate the microenvironment of metastatic tumors. We will focus on previously...specifically target digested collagens with unfolded and partially denatured collagen triple helices. 2. Demonstration of ex vivo and in vivo targeting...invasive prostate cancer due to the absence of non-specific affinity and high propensity to hybridize with denatured collagen strand (Aim 1). We

  16. [BIOCOMPATIBILITY OF POLY-LACTIDE-CO-GLYCOLIDE/COLLAGEN TYPE I SCAFFOLD WITH RAT VAGINAL EPITHELIAL CELLS].

    PubMed

    Li, Yachai; Huang, Xianghua; Zhang, Mingle; Li, Yanan; Chen, Yexing; Jia, Jingfei

    2015-09-01

    To explore the biocompatibility of the poly-lactide-co-glycolide (PLGA)/collagen type I scaffold with rat vaginal epithelial cells, and the feasibility of using PLGA/collagen type I as scaffold to reconstruct vagina by the tissue engineering. PLGA/collagen type I scaffold was prepared with PLGA covered polylysine and collagen type I. The vaginal epithelial cells of Sprague Dawley rat of 10-12 weeks old were cultured by enzyme digestion method. The vaginal epithelial cells of passage 2 were cultured in the leaching liquor of scaffold for 48 hours to detect its cytotoxicity by MTT. The vaginal epithelial cells were inoculated on the PLGA/collagen type I scaffold (experimental group) and PLGA scaffold (control group) to calculate the cell adhesion rate. Epithelial cells-scaffold complexes were implanted subcutaneously on the rat back. At 2, 4, and 8 weeks after implantation, the epithelial cells-scaffold complexes were harvested to observe the cell growth by HE staining and immunohistochemical analysis. The epithelial cells-scaffold complexes were transplanted to reconstruct vagina in 6 rats with vaginal defect. After 3 and 6 months, the vaginal length was measured and the appearance was observed. The neovagina tissues were harvested for histological evaluation after 6 months. The epithelial cells grew and proliferated well in the leaching liquor of PLGA/collagen type I scaffold, and the cytotoxicity was at grade 1. The cell adhesion rate on the PLGA/collagen type I scaffold was 71.8%±9.2%, which significantly higher than that on the PLGA scaffold (63.4%±5.7%) (t=2.195, P=0.005). The epithelial cells could grow and adhere to the PLGA/collagen type I scaffolds. At 2 weeks after implanted subcutaneously, the epithelial cells grew and proliferated in the pores of scaffolds, and the fibroblasts were observed. At 4 weeks, 1-3 layers epithelium formed on the surface of scaffold. At 8 weeks, the epithelial cells increased and arranged regularly, which formed the membrane

  17. Age-related modifications of type I collagen impair DDR1-induced apoptosis in non-invasive breast carcinoma cells.

    PubMed

    Charles, Saby; Hassan, Rammal; Kevin, Magnien; Emilie, Buache; Sylvie, Brassart-Pasco; Laurence, Van-Gulick; Pierre, Jeannesson; Erik, Maquoi; Hamid, Morjani

    2018-05-07

    Type I collagen and DDR1 axis has been described to decrease cell proliferation and to initiate apoptosis in non-invasive breast carcinoma in three-dimensional cell culture matrices. Moreover, MT1-MMP down-regulates these effects. Here, we address the effect of type I collagen aging and MT1-MMP expression on cell proliferation suppression and induced-apoptosis in non-invasive MCF-7 and ZR-75-1 breast carcinoma. We provide evidence for a decrease in cell growth and an increase in apoptosis in the presence of adult collagen when compared to old collagen. This effect involves a differential activation of DDR1, as evidenced by a higher DDR1 phosphorylation level in adult collagen. In adult collagen, inhibition of DDR1 expression and kinase function induced an increase in cell growth to a level similar to that observed in old collagen. The impact of aging on the sensitivity of collagen to MT1-MMP has been reported recently. We used the MT1-MMP expression strategy to verify whether, by degrading adult type I collagen, it could lead to the same phenotype observed in old collagen 3D matrix. MT1-MMP overexpression abrogated the proliferation suppression and induced-apoptosis effects only in the presence of adult collagen. This suggests that differential collagen degradation by MT1-MMP induced a structural disorganization of adult collagen and inhibits DDR1 activation. This could in turn impair DDR1-induced cell growth suppression and apoptosis. Taken together, our data suggest that modifications of collagen structural organization, due to aging, contribute to the loss of the growth suppression and induced apoptosis effect of collagen in luminal breast carcinoma. MT1-MMP-dependent degradation and aging of collagen have no additive effects on these processes.

  18. Cross-linking of type I collagen with microbial transglutaminase: identification of cross-linking sites.

    PubMed

    Stachel, Ines; Schwarzenbolz, Uwe; Henle, Thomas; Meyer, Michael

    2010-03-08

    Collagen is a popular biomaterial. To deal with its lack of thermal stability and its weak resistance to proteolytic degradation, collagen-based materials are stabilized via different cross-linking procedures. Regarding the potential toxicity of residual cross-linking agents, enzyme-mediated cross-linking would provide an alternative and nontoxic method for collagen stabilization. The results of this study show that type I collagen is a substrate for mTG. However, epsilon-(gamma-glutamyl)lysine cross-links are only incorporated at elevated temperatures when the protein is partially or completely denatured. A maximum number of 5.4 cross-links per collagen monomer were found for heat-denatured collagen. Labeling with the primary amine monodansylcadaverine revealed that at least half of the cross-links are located within the triple helical region of the collagen molecule. Because the triple helix is highly ordered in its native state, this finding might explain why the glutamine residues are inaccessible for mTG under nondenaturing conditions.

  19. Jellyfish collagen matrices conserve the chondrogenic phenotype in two- and three-dimensional collagen matrices.

    PubMed

    Sewing, Judith; Klinger, Matthias; Notbohm, Holger

    2017-03-01

    Cartilage is a tissue with a very low capability of self-repair and the search for suitable materials supporting the chondrogenic phenotype and thus avoiding fibrotic dedifferentiation for matrix-associated chondrocyte transplantation (MACI) is ongoing. Jellyfish collagen was thought to be a suitable material mainly because of its good availability and easy handling. Collagen was extracted from jellyfish Rhopilema esculentum and the spreading of porcine chondrocytes on two (2D) and three dimensional (3D) collagen matrices examined in comparison with vertebrate collagens, placenta collagen and a commercially available matrix from porcine collagen type I (Optimaix®). In 2D, most chondrocytes kept their round shape on jellyfish collagen and vertebrate collagen type II compared with vertebrate collagen type I. This was also confirmed in 3D experiments, where chondrocytes preserved their phenotype on jellyfish collagen, as indicated by high collagen II/(II + I) ratios (≥54 % and ~92 % collagen type II in mRNA and protein, respectively) and no proliferation during 28 days of cultivation. These observations were discussed with a view to potential structural differences of jellyfish collagen, which might influence the integrin-mediated adhesion mechanisms of vertebrate cells on jellyfish collagen. This probably results from a lack of integrin-binding sites and the existence of an alternative binding mechanism such that cells kept their round shape on jellyfish collagen, preventing chondrocytes from dedifferentiation. Thus, collagen from R. esculentum is a very suitable and promising material for cartilage tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  20. Identification of type IV collagen exposure as a molecular imaging target for early detection of thoracic aortic dissection

    PubMed Central

    Xu, Ke; Xu, Chen; Zhang, Yanzhenzi; Qi, Feiran; Yu, Bingran; Li, Ping; Jia, Lixin; Li, Yulin; Xu, Fu-jian; Du, Jie

    2018-01-01

    Thoracic aortic dissection (TAD) is an aggressive and life-threatening vascular disease and there is no effective means of early diagnosis of dissection. Type IV collagen (Col-IV) is a major component of the sub-endothelial basement membrane, which is initially exposed followed by endothelial injury as early-stage event of TAD. So, we want to build a noninvasive diagnostic method to detect early dissection by identifying the exposed Col-IV via MRI. Methods: Col-IV-targeted magnetic resonance/ fluorescence dual probe (Col-IV-DOTA-Gd-rhodamine B; CDR) was synthesized by amide reaction and coordination reaction. Flow cytometry analysis was used to evaluate the cell viability of SMC treated with CDR and fluorescence assays were used to assess the Col-IV targeting ability of CDR in vitro. We then examined the sensitivity and specificity of CDR at different stages of TAD via MRI and bioluminescence imaging in vivo. Results: The localization of Col-IV (under the intima) was observed by histology images. CDR bound specifically to Col-IV-expressing vascular smooth muscle cells and BAPN-induced dissected aorta. The CDR signal was co-detected by magnetic resonance imaging (MRI) and bioluminescence imaging as early as 2 weeks after BAPN administration (pre-dissection stage). The ability to detect rupture of dissected aorta was indicated by a strong normalized signal enhancement (NSE) in vivo. Moreover, NSE was negatively correlated with the time of dissection rupture after BAPN administration (r2 = 0.8482). Conclusion: As confirmed by in vivo studies, the CDR can identify the exposed Col-IV in degenerated aorta to monitor the progress of aortic dissection from the early stage to the rupture via MRI. Thus, CDR-enhanced MRI proposes a potential method for dissection screening, and for monitoring disease progression and therapeutic response. PMID:29290819

  1. Alterations in biosynthetic accumulation of collagen types I and III during growth and morphogenesis of embryonic mouse salivary glands

    NASA Technical Reports Server (NTRS)

    Hardman, P.; Spooner, B. S.

    1992-01-01

    We examined the biosynthetic patterns of interstitial collagens in mouse embryonic submandibular and sublingual glands cultured in vitro. Rudiments explanted on day 13 of gestation and cultured for 24, 48, and 72 h all synthesized collagen types I, III, and V. However, while the total incorporation of label into collagenous proteins did not change over the three-day culture period, the rate of accumulation of newly synthesized types I and III did change. At 24 h, the ratio of newly synthesized collagen types I:III was approximately 2, whereas at 72 h, the ratio was approximately 5. These data suggest that collagen types I and III may be important in initiation of branching in this organ, but that type I may become dominant in the later stages of development and in maintenance of the adult organ.

  2. P3h3-null and Sc65-null Mice Phenocopy the Collagen Lysine Under-hydroxylation and Cross-linking Abnormality of Ehlers-Danlos Syndrome Type VIA.

    PubMed

    Hudson, David M; Weis, MaryAnn; Rai, Jyoti; Joeng, Kyu Sang; Dimori, Milena; Lee, Brendan H; Morello, Roy; Eyre, David R

    2017-03-03

    Tandem mass spectrometry was applied to tissues from targeted mutant mouse models to explore the collagen substrate specificities of individual members of the prolyl 3-hydroxylase (P3H) gene family. Previous studies revealed that P3h1 preferentially 3-hydroxylates proline at a single site in collagen type I chains, whereas P3h2 is responsible for 3-hydroxylating multiple proline sites in collagen types I, II, IV, and V. In screening for collagen substrate sites for the remaining members of the vertebrate P3H family, P3h3 and Sc65 knock-out mice revealed a common lysine under-hydroxylation effect at helical domain cross-linking sites in skin, bone, tendon, aorta, and cornea. No effect on prolyl 3-hydroxylation was evident on screening the spectrum of known 3-hydroxyproline sites from all major tissue collagen types. However, collagen type I extracted from both Sc65 -/- and P3h3 -/- skin revealed the same abnormal chain pattern on SDS-PAGE with an overabundance of a γ 112 cross-linked trimer. The latter proved to be from native molecules that had intramolecular aldol cross-links at each end. The lysine under-hydroxylation was shown to alter the divalent aldimine cross-link chemistry of mutant skin collagen. Furthermore, the ratio of mature HP/LP cross-links in bone of both P3h3 -/- and Sc65 -/- mice was reversed compared with wild type, consistent with the level of lysine under-hydroxylation seen in individual chains at cross-linking sites. The effect on cross-linking lysines was quantitatively very similar to that previously observed in EDS VIA human and Plod1 -/- mouse tissues, suggesting that P3H3 and/or SC65 mutations may cause as yet undefined EDS variants. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Transient expression of collagen type II at epitheliomesenchymal interfaces during morphogenesis of the cartilaginous neurocranium.

    PubMed

    Thorogood, P; Bee, J; von der Mark, K

    1986-08-01

    In the avian embryo a matrix-mediated tissue interaction between retinal pigmented epithelium and neural crest-derived periocular mesenchyme leads to the differentiation of (scleral) cartilage. The composition of the extracellular matrix at the interface between these two tissues has been examined immunohistochemically, both during and after the interaction has taken place. Of the matrix components studied (fibronectin, laminin, and collagen types I, II, IV, and V) only collagen type II displayed a dramatic change in distribution between the two stages. During the interaction, at stage 15, type II was present in the extracellular compartment basal to the epithelium. After completion of the interaction, collagen type II was no longer detectable at the interface even though it was readily detectable in the vitreous humor, cornea, and perinotochordal sheath, and subsequently will be expressed by the chondrogenic tissue itself as overt differentiation commences. These results suggest that collagen type II might be causally involved in this particular epitheliomesenchymal interaction. Examination of the spatial and temporal patterns of collagen type II expression elsewhere in the developing craniofacial complex revealed a hitherto unreported pattern of distribution. In addition to its predictable locations (i.e., cornea, vitreous, and perinotochordal sheath) it was found to be present at certain other sites, for example, at the basal surfaces of some neuroepithelia. These additional locations are all known to be sites of chondrogenesis-promoting tissue interactions which result in the formation of the elements of the cartilaginous neurocranium (e.g., otic vesicle). Furthermore this spatial distribution exhibits a changing temporal pattern in that it is detectable at the time that the interactions are known to be taking place, but subsequently is no longer detectable by the immunohistochemical means employed. This definable pattern of transient collagen type II

  4. Panax ginseng induces human Type I collagen synthesis through activation of Smad signaling.

    PubMed

    Lee, Jongsung; Jung, Eunsun; Lee, Jiyoung; Huh, Sungran; Kim, Jieun; Park, Mijung; So, Jungwoon; Ham, Younggeun; Jung, Kwangseon; Hyun, Chang-Gu; Kim, Yeong Shik; Park, Deokhoon

    2007-01-03

    Skin aging appears to be principally related to a decrease in levels of Type I collagen, the primary component of the dermal layer of skin. It is important to introduce an efficient agent for effective management of skin aging; this agent should have the fewest possible side effects and the greatest wrinkle-reducing effect. In the course of screening collagen production-promoting agents, we obtained Panax ginseng C.A. Meyer. This study was designed to investigate the possible collagen production-promoting activities of Panax ginseng C.A. Meyer root extract (PGRE) in human dermal fibroblast cells. As a first step to this end, human COL1A2 promoter luciferase assay was performed in human dermal fibroblast cells. In this assay, PGRE activated human COL1A2 promoter activity in a concentration-dependent manner. Human Type I procollagen synthesis was also induced by PGRE. These results suggest that PGRE promotes collagen production in human dermal fibroblast cells. Additionally, we have attempted to characterize the mechanism of action of PGRE in Type I procollagen synthesis. PGRE was found to induce the phosphorylation of Smad2, an important transcription factor in the production of Type I procollagen. When applied topically in a human skin primary irritation test, PGRE did not induce any adverse reactions. Therefore, based on these results, we suggest the possibility that PGRE may be considered as an attractive, wrinkle-reducing candidate for topical application.

  5. Type II Collagen and Gelatin from Silvertip Shark (Carcharhinus albimarginatus) Cartilage: Isolation, Purification, Physicochemical and Antioxidant Properties

    PubMed Central

    Jeevithan, Elango; Bao, Bin; Bu, Yongshi; Zhou, Yu; Zhao, Qingbo; Wu, Wenhui

    2014-01-01

    Type II acid soluble collagen (CIIA), pepsin soluble collagen (CIIP) and type II gelatin (GII) were isolated from silvertip shark (Carcharhinus albimarginatus) cartilage and examined for their physicochemical and antioxidant properties. GII had a higher hydroxyproline content (173 mg/g) than the collagens and cartilage. CIIA, CIIP and GII were composed of two identical α1 and β chains and were characterized as type II. Amino acid analysis of CIIA, CIIP and GII indicated imino acid contents of 150, 156 and 153 amino acid residues per 1000 residues, respectively. Differing Fourier transform infrared (FTIR) spectra of CIIA, CIIP and GII were observed, which suggested that the isolation process affected the secondary structure and molecular order of collagen, particularly the triple-helical structure. The denaturation temperature of GII (32.5 °C) was higher than that of CIIA and CIIP. The antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl radicals and the reducing power of CIIP was greater than that of CIIA and GII. SEM microstructure of the collagens depicted a porous, fibrillary and multi-layered structure. Accordingly, the physicochemical and antioxidant properties of type II collagens (CIIA, CIIP) and GII isolated from shark cartilage were found to be suitable for biomedical applications. PMID:24979271

  6. Immunohistochemical distribution of collagens type I, III, IV and VI, of undulin and of tenascin in oral fibrous hyperplasia.

    PubMed

    Becker, J; Schuppan, D; Müller, S

    1993-11-01

    The distribution of collagens type I, IV and VI, of procollagen type III, of undulin and of tenascin was studied in 10 lesions which were clinically and histologically diagnosed as localized oral fibrous hyperplasias. The immunohistochemical distribution of these proteins was similar to that observed for normal oral mucosa. Undulin showed a pattern of parallel fibers throughout. Collagen type VI was pronounced in the subepithelial connective tissue, whereas the collagen fiber bundles were equally reactive for collagens type I and III. Tenascin was observed close to the subepithelial basement membrane and in proximity to collagen fiber bundles in the upper connective tissue. The present findings indicate that oral fibrous hyperplasias that are probably caused by inflammation or chronic irritation show the differentiated and ordered pattern of extracellular matrix proteins characteristic of normal oral mucosa.

  7. Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion

    PubMed Central

    Mazur, Anna; Holthoff, Emily; Vadali, Shanthi; Kelly, Thomas; Post, Steven R.

    2016-01-01

    Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM). These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP), a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204). Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages. PMID:26934296

  8. Preparation and characterization of injectable fibrillar type I collagen and evaluation for pseudoaneurysm treatment in a pig model.

    PubMed

    Geutjes, Paul J; van der Vliet, J Adam; Faraj, Kaeuis A; de Vries, Noes; van Moerkerk, Herman T B; Wismans, Ronnie G; Hendriks, Thijs; Daamen, Willeke F; van Kuppevelt, Toin H

    2010-11-01

    Despite the efficacy of collagen in femoral artery pseudoaneurysm treatment, as reported in one patient study, its use has not yet gained wide acceptance in clinical practice. In this particular study, the collagen was not described in detail. To further investigate the potential of collagen preparations, we prepared and characterized highly purified injectable fibrillar type I collagen and evaluated its use for femoral artery pseudoaneurysm (PSA) treatment in vivo using a pig model. Purified fibrillar type I collagen was characterized using electron microscopy. The effect of three different sterilization procedures, ie, hydrogen peroxide gas plasma (H2O2), ethylene oxide gas (EtO), and gamma irradiation, was studied on both SDS-PAGE and platelet aggregation. Different collagen injectables were prepared (3%, 4%, and 5%) and tested using an injection force test applying a 21-gauge needle. To evaluate the network characteristics of the injectable collagen, the collagen was suspended in phosphate buffered saline (PBS) at 37°C and studied both macroscopically and electron microscopically. To determine whether the collagen induced hemostasis in vivo, a pig PSA model was used applying a 4% EtO sterilized collagen injectable, and evaluation by angiography and routine histology. Electron microscopy of the purified type I collagen revealed intact fibrils with a distinct striated pattern and a length<300 μm. Both SDS-PAGE and platelet aggregation analysis of the sterilized collagen indicated no major differences between EtO and H2O2 sterilization, although gamma-irradiated collagen showed degradation products. Both 3% and 4% (w/v) collagen suspensions were acceptable with respect to the force used (<50 N). The 4% suspension was selected as the preferred injectable collagen, which formed a dense network under physiologic conditions. Testing the collagen in vivo (n=5), the angiograms revealed that the PSA partly or completely coagulated. Histology confirmed the network

  9. IgE antibody to fish gelatin (type I collagen) in patients with fish allergy.

    PubMed

    Sakaguchi, M; Toda, M; Ebihara, T; Irie, S; Hori, H; Imai, A; Yanagida, M; Miyazawa, H; Ohsuna, H; Ikezawa, Z; Inouye, S

    2000-09-01

    Most children with anaphylaxis to measles, mumps, and rubella vaccines had shown sensitivity to bovine gelatin that was included in the vaccines. Recently, it was found that bovine type I collagen, which is the main content in the gelatin, is a major allergen in bovine gelatin allergy. Fish meat and skin also contain type I collagen. The present study was designed to investigate IgE antibody to fish gelatin in children with fish allergy. Serum samples were taken from patients in 3 groups: (1) 10 patients with fish allergy and specific IgE to fish meat; (2) two patients with allergies to both fish meat and bovine gelatin and specific IgE to fish meat and bovine gelatin; and (3) 15 patients with atopic dermatitis and specific IgE to fish meat. Various fish gelatins (type I collagen) were prepared from fish skin. IgE antibody to fish gelatin was analyzed by using ELISA and immunoblotting. Of 10 patients with fish allergy, 3 had specific IgE to fish gelatin. Of two patients with fish allergy and bovine gelatin allergy, all had specific IgE to fish gelatin. Of 15 patients with atopic dermatitis and specific IgE to fish meat, 5 had specific IgE to fish gelatin. Furthermore, IgE from pooled serum of the patients reacted with both the alpha1 and alpha2 chains of fish type I collagen in immunoblots. There is cross-reactivity among gelatins from various fishes, but there is little cross-reactivity between fish and bovine gelatins. Some fish-sensitive patients possessed IgE antibody to fish gelatin. Fish gelatin (type I collagen) might be an allergen in subjects with fish allergy.

  10. Flash photolysis and pulse radiolysis studies on collagen Type I in acetic acid solution.

    PubMed

    Sionkowska, Alina

    2006-07-03

    An investigation of the photochemical properties of collagen Type I in acetic acid solution was carried out using nanosecond laser irradiation. The transient spectra of collagen solution excited at 266 nm show two bands. One of them with maximum at 295 nm and the second one with maximum at 400 nm. The peak at 400 nm is assigned to tyrosyl radicals. The first peak of the transient absorption spectra at 295 nm is probably due to photoionisation producing collagen radical cation. The transient for collagen solution in acetic acid at 640 nm was not observed. It is evidence that there is no hydrated electron in the irradiated collagen solution. The reactions of hydrated electrons and (*)OH radicals with collagen have been studied by pulse radiolysis. In the absorption spectra of products resulting from the reaction of collagen with e(aq)(-) no characteristic maximum absorption in UV and visible light region has been observed. In the absorption spectra of products resulting from the reaction of the hydroxyl radicals with collagen two bands have been observed. The first one at 320 nm and the second one at 405 nm. Reaction of (*)OH radicals with tyrosine residues in collagen chains gives rise to Tyr phenoxyl radicals (absorption at 400 nm).

  11. A Quantitative Study of the Relationship between the Distribution of Different Types of Collagen and the Mechanical Behavior of Rabbit Medial Collateral Ligaments

    PubMed Central

    Wan, Chao; Hao, Zhixiu; Wen, Shizhu; Leng, Huijie

    2014-01-01

    The mechanical properties of ligaments are key contributors to the stability and function of musculoskeletal joints. Ligaments are generally composed of ground substance, collagen (mainly type I and III collagen), and minimal elastin fibers. However, no consensus has been reached about whether the distribution of different types of collagen correlates with the mechanical behaviors of ligaments. The main objective of this study was to determine whether the collagen type distribution is correlated with the mechanical properties of ligaments. Using axial tensile tests and picrosirius red staining-polarization observations, the mechanical behaviors and the ratios of the various types of collagen were investigated for twenty-four rabbit medial collateral ligaments from twenty-four rabbits of different ages, respectively. One-way analysis of variance was used in the comparison of the Young's modulus in the linear region of the stress-strain curves and the ratios of type I and III collagen for the specimens (the mid-substance specimens of the ligaments) with different ages. A multiple linear regression was performed using the collagen contents (the ratios of type I and III collagen) and the Young's modulus of the specimens. During the maturation of the ligaments, the type I collagen content increased, and the type III collagen content decreased. A significant and strong correlation () was identified by multiple linear regression between the collagen contents (i.e., the ratios of type I and type III collagen) and the mechanical properties of the specimens. The collagen content of ligaments might provide a new perspective for evaluating the linear modulus of global stress-strain curves for ligaments and open a new door for studying the mechanical behaviors and functions of connective tissues. PMID:25062068

  12. Suppression of murine collagen-induced arthritis by targeted apoptosis of synovial neovasculature

    PubMed Central

    Gerlag, Danielle M; Borges, Eric; Tak, Paul P; Ellerby, H Michael; Bredesen, Dale E; Pasqualini, Renata; Ruoslahti, Erkki; Firestein, Gary S

    2001-01-01

    Because angiogenesis plays a major role in the perpetuation of inflammatory arthritis, we explored a method for selectively targeting and destroying new synovial blood vessels. Mice with collagen-induced arthritis were injected intravenously with phage expressing an RGD motif. In addition, the RGD peptide (RGD-4C) was covalently linked to a proapoptotic heptapeptide dimer, D(KLAKLAK)2, and was systemically administered to mice with collagen-induced arthritis. A phage displaying an RGD-containing cyclic peptide (RGD-4C) that binds selectively to the αvβ3 and αvβ5 integrins accumulated in inflamed synovium but not in normal synovium. Homing of RGD-4C phage to inflamed synovium was inhibited by co-administration of soluble RGD-4C. Intravenous injections of the RGD-4C–D(KLAKLAK)2 chimeric peptide significantly decreased clinical arthritis and increased apoptosis of synovial blood vessels, whereas treatment with vehicle or uncoupled mixture of the RGD-4C and the untargeted proapoptotic peptide had no effect. Targeted apoptosis of synovial neovasculature can induce apoptosis and suppress clinical arthritis. This form of therapy has potential utility in the treatment of inflammatory arthritis. PMID:11714389

  13. Tooth agenesis in osteogenesis imperfecta related to mutations in the collagen type I genes.

    PubMed

    Malmgren, B; Andersson, K; Lindahl, K; Kindmark, A; Grigelioniene, G; Zachariadis, V; Dahllöf, G; Åström, E

    2017-01-01

    Osteogenesis imperfecta (OI) is a heterogeneous group of disorders of connective tissue, mainly caused by mutations in the collagen type I genes (COL1A1 and COL1A2). Tooth agenesis is a common feature of OI. We investigated the association between tooth agenesis and collagen type I mutations in individuals with OI. In this cohort study, 128 unrelated individuals with OI were included. Panoramic radiographs were analyzed regarding dentinogenesis imperfecta (DGI) and congenitally missing teeth. The collagen I genes were sequenced in all individuals, and in 25, multiplex ligation-dependent probe amplification was performed. Mutations in the COL1A1 and COL1A2 genes were found in 104 of 128 individuals. Tooth agenesis was diagnosed in 17% (hypodontia 11%, oligodontia 6%) and was more frequent in those with DGI (P = 0.016), and in those with OI type III, 47%, compared to those with OI types I, 12% (P = 0.003), and IV, 13% (P = 0.017). Seventy-five percent of the individuals with oligodontia (≥6 missing teeth) had qualitative mutations, but there was no association with OI type, gender, or presence of DGI. The prevalence of tooth agenesis is high (17%) in individuals with OI, and OI caused by a qualitative collagen I mutation is associated with oligodontia. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Mesenchymal Stem Cells Sense Three Dimensional Type I Collagen through Discoidin Domain Receptor 1.

    PubMed

    Lund, A W; Stegemann, J P; Plopper, G E

    2009-01-01

    The extracellular matrix provides structural and organizational cues for tissue development and defines and maintains cellular phenotype during cell fate determination. Multipotent mesenchymal stem cells use this matrix to tightly regulate the balance between their differentiation potential and self-renewal in the native niche. When understood, the mechanisms that govern cell-matrix crosstalk during differentiation will allow for efficient engineering of natural and synthetic matrices to specifically direct and maintain stem cell phenotype. This work identifies the discoidin domain receptor 1 (DDR1), a collagen activated receptor tyrosine kinase, as a potential link through which stem cells sense and respond to the 3D organization of their extracellular matrix microenvironment. DDR1 is dependent upon both the structure and proteolytic state of its collagen ligand and is specifically expressed and localized in three dimensional type I collagen culture. Inhibition of DDR1 expression results in decreased osteogenic potential, increased cell spreading, stress fiber formation and ERK1/2 phosphorylation. Additionally, loss of DDR1 activity alters the cell-mediated organization of the naïve type I collagen matrix. Taken together, these results demonstrate a role for DDR1 in the stem cell response to and interaction with three dimensional type I collagen. Dynamic changes in cell shape in 3D culture and the tuning of the local ECM microstructure, directs crosstalk between DDR1 and two dimensional mechanisms of osteogenesis that can alter their traditional roles.

  15. High pressure Raman study of type-I collagen.

    PubMed

    Paschou, Amalia Maria; Katsikini, Maria; Christofilos, Dimitrios; Arvanitidis, John; Ves, Sotirios

    2018-05-18

    The high pressure response of type-I collagen from bovine Achilles tendon is investigated with micro-Raman spectroscopy. Fluorinert ™ and methanol-ethanol mixtures were used as pressure transmitting media (PTM) in a diamond anvil cell. The Raman spectrum of collagen is dominated by three bands centred at approximately 1450, 1660 and 2930 cm -1 , attributed to C-H deformation, C=O stretching of the peptide bond (amide-I band) and C-H stretching modes, respectively. Upon pressure increase, using Fluorinert ™ as PTM, a shift towards higher frequencies of the C-H stretching and deformation peaks is observed. Contrary, the amide-I band peaks are shifted to lower frequencies with moderate pressure slopes. On the other hand, by using the alcohol mixture as PTM, the amide-I band exhibits more pronounced C=O bond softening, deduced from the shift to lower frequencies, suggesting a strengthening of the hydrogen bonds between glycine and proline residues of different collagen chains due to the presence of the polar alcohol molecules. Furthermore, some of the peaks exhibit abrupt changes in their pressure slopes at approximately 2 GPa, implying a variation in the compressibility of the collagen fibres. This could be attributed to a pitch change from 10/3 to 7/2, sliding of the tropocollagen molecules, twisting variation at the molecular level and/or elimination of the D-gaps induced by kink compression. All spectral changes are reversible upon pressure release, which indicates that denaturation has not taken place. Finally, a minor lipid phase contamination was detected in some sample spots. Its pressure response is also monitored. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  16. Zebrafish Collagen Type I: Molecular and Biochemical Characterization of the Major Structural Protein in Bone and Skin

    PubMed Central

    Gistelinck, C.; Gioia, R.; Gagliardi, A.; Tonelli, F.; Marchese, L.; Bianchi, L.; Landi, C.; Bini, L.; Huysseune, A.; Witten, P. E.; Staes, A.; Gevaert, K.; De Rocker, N.; Menten, B.; Malfait, F.; Leikin, S.; Carra, S.; Tenni, R.; Rossi, A.; De Paepe, A.; Coucke, P.; Willaert, A.; Forlino, A.

    2016-01-01

    Over the last years the zebrafish imposed itself as a powerful model to study skeletal diseases, but a limit to its use is the poor characterization of collagen type I, the most abundant protein in bone and skin. In tetrapods collagen type I is a trimer mainly composed of two α1 chains and one α2 chain, encoded by COL1A1 and COL1A2 genes, respectively. In contrast, in zebrafish three type I collagen genes exist, col1a1a, col1a1b and col1a2 coding for α1(I), α3(I) and α2(I) chains. During embryonic and larval development the three collagen type I genes showed a similar spatio-temporal expression pattern, indicating their co-regulation and interdependence at these stages. In both embryonic and adult tissues, the presence of the three α(I) chains was demonstrated, although in embryos α1(I) was present in two distinct glycosylated states, suggesting a developmental-specific collagen composition. Even though in adult bone, skin and scales equal amounts of α1(I), α3(I) and α2(I) chains are present, the presented data suggest a tissue-specific stoichiometry and/or post-translational modification status for collagen type I. In conclusion, this data will be useful to properly interpret results and insights gained from zebrafish models of skeletal diseases. PMID:26876635

  17. Extracellular Collagen Promotes Interleukin-1β-Induced Urokinase-Type Plasminogen Activator Production by Human Corneal Fibroblasts.

    PubMed

    Sugioka, Koji; Kodama-Takahashi, Aya; Yoshida, Koji; Aomatsu, Keiichi; Okada, Kiyotaka; Nishida, Teruo; Shimomura, Yoshikazu

    2017-03-01

    Keratocytes maintain homeostasis of the corneal stroma through synthesis, secretion, and degradation of collagen fibrils of the extracellular matrix. Given that these cells are essentially embedded in a collagen matrix, keratocyte-collagen interactions may play a key role in regulation of the expression or activation of enzymes responsible for matrix degradation including urokinase-type plasminogen activator (uPA), plasmin, and matrix metalloproteinases (MMPs). We examined the effect of extracellular collagen on the production of uPA by corneal fibroblasts (activated keratocytes) stimulated with the proinflammatory cytokine interleukin-1β (IL-1β). Human corneal fibroblasts were cultured either on plastic or in a three-dimensional gel of type I collagen. Plasminogen activators were detected by fibrin zymography, whereas the IL-1 receptor (IL-1R) and MMPs were detected by immunoblot analysis. Collagen degradation by corneal fibroblasts was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. Collagen and IL-1β synergistically increased the synthesis and secretion of uPA in corneal fibroblasts. Collagen also upregulated IL-1R expression in the cells in a concentration-dependent manner. The conversion of extracellular plasminogen to plasmin, as well as the plasminogen-dependent activation of MMP-1 and MMP-3 and degradation of collagen apparent in three-dimensional cultures of corneal fibroblasts exposed to IL-1β, were all abolished by a selective uPA inhibitor. Collagen and IL-1β cooperate to upregulate uPA production by corneal fibroblasts. Furthermore, IL-1β-induced collagen degradation by these cells appears to be strictly dependent on uPA expression and mediated by a uPA-plasmin-MMP pathway.

  18. Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist inhibits collagen synthesis in human hypertrophic scar fibroblasts by targeting Smad3 via miR-145

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhu, Hua-Yu; Li, Chao; Zheng, Zhao

    The transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ) functions to regulate cell differentiation and lipid metabolism. Recently, its agonist has been documented to regulate extracellular matrix production in human dermal fibroblasts. This study explored the underlying molecular mechanisms and gene interactions in hypertrophic scar fibroblasts (HSFBs) in vitro. HSFBs were cultured and treated with or without PPAR-γ agonist or antagonist for gene expression. Bioinformatical analysis predicted that miR-145 could target Smad3 expression. Luciferase assay was used to confirm such an interaction. The data showed that PPAR-γ agonist troglitazone suppressed expression of Smad3 and Col1 in HSFBs. PPAR-γ agonist induced miR-145 at themore » gene transcriptional level, which in turn inhibited Smad3 expression and Col1 level in HSFBs. Furthermore, ELISA data showed that Col1 level in HSFBs was controlled by a feedback regulation mechanism involved in PPAR-γ agonist and antagonist-regulated expression of miR-145 and Smad3 in HSFBs. These findings indicate that PPAR-γ-miR-145-Smad3 axis plays a role in regulation of collagen synthesis in HSFBs. - Highlights: • PPAR-γ agonist inhibits collagen synthesis in HSFBs. • Smad3 and type I collagen expression are decreased by PPAR-γ agonist. • miR-145 expression is increased by PPAR-γ agonist in HSFBs. • Increased miR-145 inhibits collagen synthesis by targeting Smad3. • miR-145 regulates collagen synthesis.« less

  19. Comparative study on the antioxidant activity of peptides from pearl oyster ( Pinctada martensii) mantle type V collagen and tilapia ( Oreochromis niloticus) scale type I collagen

    NASA Astrophysics Data System (ADS)

    Xia, Guanghua; Zhang, Xueying; Dong, Zhenghua; Shen, Xuanri

    2017-12-01

    In this study, Pearl oyster mantle type V collagen (POMC) and tilapia scale type I collagen (TSC) were extracted and hydrolyzed by various proteases in order to obtain peptides. The antioxidant activity of the peptides was investigated by DPPH, hydroxyl radical scavenging experiments and a dynamic digestion model in vitro. The results show that there are significant differences in amino acid composition between POMC and TSC. The collagen peptides obtained from pearl oyster mantle (POMCP) by treating with alkaline protease exhibited higher antioxidant activity than that from tilapia scale (TSCP) treated with papaya protease, and both of them showed greater DPPH and hydroxyl radical scavenging activity than other peptides. After being separated via Sephadex G-25 chromatography, the M1 fraction isolated from POMCP, and the S1 fraction from TSCP with which both had higher molecular weights showed the strongest antioxidant activity than other fractions, and the M1 fraction exhibited stronger antioxidant activity than the S1 fraction in scavenging free-radicals and protecting cells from the oxidation damage. Furthermore, after treating the dynamic digestion system model in vitro, the DPPH and hydroxyl radical scavenging activity of the M1 fraction increased slightly. These results suggest that POMCP exhibits stronger antioxidant activity than TSCP, which means that PMOP may be a good candidate to be a potential natural antioxidant in the food-processing industry.

  20. A quantitative study of the relationship between the distribution of different types of collagen and the mechanical behavior of rabbit medial collateral ligaments.

    PubMed

    Wan, Chao; Hao, Zhixiu; Wen, Shizhu; Leng, Huijie

    2014-01-01

    The mechanical properties of ligaments are key contributors to the stability and function of musculoskeletal joints. Ligaments are generally composed of ground substance, collagen (mainly type I and III collagen), and minimal elastin fibers. However, no consensus has been reached about whether the distribution of different types of collagen correlates with the mechanical behaviors of ligaments. The main objective of this study was to determine whether the collagen type distribution is correlated with the mechanical properties of ligaments. Using axial tensile tests and picrosirius red staining-polarization observations, the mechanical behaviors and the ratios of the various types of collagen were investigated for twenty-four rabbit medial collateral ligaments from twenty-four rabbits of different ages, respectively. One-way analysis of variance was used in the comparison of the Young's modulus in the linear region of the stress-strain curves and the ratios of type I and III collagen for the specimens (the mid-substance specimens of the ligaments) with different ages. A multiple linear regression was performed using the collagen contents (the ratios of type I and III collagen) and the Young's modulus of the specimens. During the maturation of the ligaments, the type I collagen content increased, and the type III collagen content decreased. A significant and strong correlation (R2 = 0.839, P < 0.05) was identified by multiple linear regression between the collagen contents (i.e., the ratios of type I and type III collagen) and the mechanical properties of the specimens. The collagen content of ligaments might provide a new perspective for evaluating the linear modulus of global stress-strain curves for ligaments and open a new door for studying the mechanical behaviors and functions of connective tissues.

  1. Eccentric rehabilitation exercise increases peritendinous type I collagen synthesis in humans with Achilles tendinosis.

    PubMed

    Langberg, H; Ellingsgaard, H; Madsen, T; Jansson, J; Magnusson, S P; Aagaard, P; Kjaer, M

    2007-02-01

    It has been shown that 12 weeks of eccentric heavy resistance training can reduce pain in runners suffering from chronic Achilles tendinosis, but the mechanism behind the effectiveness of this treatment is unknown. The present study investigates the local effect of an eccentric training regime on elite soccer players suffering from chronic Achilles tendinosis on the turnover of the peritendinous connective tissue. Twelve elite male soccer players, of whom six suffered from unilateral tendinosis and six were healthy controls, participated in this study. All participants performed 12 weeks of heavy-resistance eccentric training apart from their regular training and soccer activity. Before and after the training period the tissue concentration of indicators of collagen turnover was measured by the use of the microdialysis technique. After training, collagen synthesis was increased in the initially injured tendon (n=6; carboxyterminal propeptide of type I collagen (PICP): pre 3.9+/-2.5 microg/L to post 19.7+/-5.4 microg/L, P<0.05). The collagen synthesis was unchanged in healthy tendons in response to training (n=6; PICP: pre 8.3+/-5.2 microg/L to post 11.5+/-5.0 microg/L, P>0.05). Collagen degradation, measured as carboxyterminal telopeptide region of type I collagen (ICTP), was not affected by training neither in the injured nor in the healthy tendons. The clinical effect of the 12 weeks of eccentric training was determined by using a standardized loading procedure of the Achilles tendons showing a decrease in pain in all the chronic injured tendons (VAS before 44+/-9, after 13+/-9; P<0.05), and all subjects were back playing soccer following the eccentric training regime. The present study demonstrates that chronically injured Achilles tendons respond to 12 weeks of eccentric training by increasing collagen synthesis rate. In contrast, the collagen metabolism in healthy control tendons seems not to be affected by eccentric training. These findings could indicate a

  2. Enigmatic insight into collagen

    PubMed Central

    Deshmukh, Shrutal Narendra; Dive, Alka M; Moharil, Rohit; Munde, Prashant

    2016-01-01

    Collagen is a unique, triple helical molecule which forms the major part of extracellular matrix. It is the most abundant protein in the human body, representing 30% of its dry weight. It is the fibrous structural protein that makes up the white fibers (collagen fibers) of skin, tendons, bones, cartilage and all other connective tissues. Collagens are not only essential for the mechanical resistance and resilience of multicellular organisms, but are also signaling molecules defining cellular shape and behavior. The human body has at least 16 types of collagen, but the most prominent types are I, II and III. Collagens are produced by several cell types and are distinguishable by their molecular compositions, morphologic characteristics, distribution, functions and pathogenesis. This is the major fibrous glycoprotein present in the extracellular matrix and in connective tissue and helps in maintaining the structural integrity of these tissues. It has a triple helical structure. Various studies have proved that mutations that modify folding of the triple helix result in identifiable genetic disorders. Collagen diseases share certain similarities with autoimmune diseases, because autoantibodies specific to each collagen disease are produced. Therefore, this review highlights the role of collagen in normal health and also the disorders associated with structural and functional defects in collagen. PMID:27601823

  3. Physics of soft hyaluronic acid-collagen type II double network gels

    NASA Astrophysics Data System (ADS)

    Morozova, Svetlana; Muthukumar, Murugappan

    2015-03-01

    Many biological hydrogels are made up of multiple interpenetrating, charged components. We study the swelling, elastic diffusion, mechanical, and optical behaviors of 100 mol% ionizable hyaluronic acid (HA) and collagen type II fiber networks. Dilute, 0.05-0.5 wt% hyaluronic acid networks are extremely sensitive to solution salt concentration, but are stable at pH above 2. When swelled in 0.1M NaCl, single-network hyaluronic acid gels follow scaling laws relevant to high salt semidilute solutions; the elastic shear modulus G' and diffusion constant D scale with the volume fraction ϕ as G' ~ϕ 9 / 4 and D ~ϕ 3 / 4 , respectively. With the addition of a collagen fiber network, we find that the hyaluronic acid network swells to suspend the rigid collagen fibers, providing extra strength to the hydrogel. Results on swelling equilibria, elasticity, and collective diffusion on these double network hydrogels will be presented.

  4. Urinary type IV collagen excretion predicts subsequent declining renal function in type 2 diabetic patients with proteinuria.

    PubMed

    Katavetin, Pisut; Katavetin, Paravee; Susantitaphong, Paweena; Townamchai, Natavudh; Tiranathanagul, Khajohn; Tungsanga, Kriang; Eiam-Ong, Somchai

    2010-08-01

    Baseline urinary type IV collagen excretion was negatively correlated with the subsequent GFR change (r(s)=-0.39, p=0.04) in our cohort of 30 type 2 diabetic patients with proteinuria. Therefore, it could be used to predict subsequent declining renal function in type 2 diabetic patients with proteinuria. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  5. Kidney diseases caused by glomerular basement membrane type IV collagen defects in dogs.

    PubMed

    Lees, George E

    2013-01-01

    To review the pathogenesis, as well as the clinical and pathologic features of canine glomerular diseases caused by genetic type IV collagen defects. Original studies and review articles from human and veterinary medical fields. Presence in glomerular basement membranes (GBM) of a network composed of α3.α4.α5 heterotrimers of type IV collagen is required to maintain structure and function of glomerular capillary walls. Hereditary nephropathy (HN) is the most commonly used name for kidney diseases that occur in dogs due to genetic type IV collagen abnormalities. To date, 4 different collagen IV gene mutations have been identified in dogs with HN; 2 are COL4A5 mutations that cause X-linked HN (XL-HN), and 2 are COL4A4 mutations that cause autosomal recessive HN (AR-HN). Affected males with XL-HN and affected males and females with AR-HN develop juvenile-onset kidney disease manifested by proteinuria typically starting at 3-6 months of age and followed by progressive kidney disease leading to terminal failure usually at 6-24 months of age. Carrier female dogs with XL-HN also develop proteinuria starting at 3-6 months of age, but progressive disease causing kidney failure is uncommon until they are >5 years old. The distinctive pathologic lesions of HN are extensive multilaminar splitting and thickening of the GBM, as demonstrated by electron microscopy, and abnormal type IV collagen α-chain content of basement membranes, as demonstrated by immunolabeling. Identification of the underlying gene mutations has permitted genetic testing and selective breeding practices that currently are minimizing HN in breeds known to be at risk. Canine HN is a rare disease that should be considered whenever a dog exhibits a juvenile-onset kidney disease characterized partly by proteinuria, but highly specialized methods are required to pursue a definitive diagnosis. © Veterinary Emergency and Critical Care Society 2013.

  6. Quantitative analysis of the synthesis and secretion of type VII collagen in cultured human dermal fibroblasts with a sensitive sandwich enzyme-linked immunoassay.

    PubMed

    Amano, Satoshi; Ogura, Yuki; Akutsu, Nobuko; Nishiyama, Toshio

    2007-02-01

    Type VII collagen is the major component of anchoring fibrils in the epidermal basement membrane. Its expression has been analyzed by immunostaining or Northern blotting, but rarely at the protein level. In this study, we have quantitatively examined the effects of ascorbic acid and various cytokines/growth factors on the protein synthesis and secretion of type VII collagen by human dermal fibroblasts in culture, using a developed, highly sensitive sandwich enzyme-linked immunoassay with two kinds of specific monoclonal antibodies against the non-collagenous domain-1. Ascorbic acid and its derivative induced a twofold increase in type VII collagen synthesis, and markedly increased the secretion of type VII collagen into the medium when compared with the control culture. This effect was not influenced by the presence of transforming growth factor-beta1 (TGF-beta1). The synthesis of type VII collagen was elevated by TGF-beta1, platelet-derived growth factor, tumor necrosis factor-alpha, and interleukin-1beta, but not by TGF-alpha. Thus, our data indicate that the synthesis and secretion of type VII collagen in human dermal fibroblasts are regulated by ascorbate and the enhancement of type VII collagen gene expression by cytokines/growth factors is accompanied with elevated production of type VII collagen at the protein level.

  7. Gene expression profile of collagen types, osteopontin in the tympanic membrane of patients with tympanosclerosis.

    PubMed

    Sakowicz-Burkiewicz, Monika; Kuczkowski, Jerzy; Przybyła, Tomasz; Grdeń, Marzena; Starzyńska, Anna; Pawełczyk, Tadeusz

    2017-09-01

    Tympanosclerosis is a pathological process involving the middle ear. The hallmark of this disease is the formation of calcium deposits. In the submucosal layer, as well as in the right layer of the tympanic membrane, the calcium deposits result in a significant increase in the activity of fibroblasts and deposition of collagen fibers. The aim of our study was to examine the expression level of genes encoding collagen type I, II, III and IV (COL1A1, COL2A1, COL3A1, COL4A1) and osteopontin (SPP1) in the tympanic membrane of patients with tympanosclerosis. The total RNA was isolated from middle ear tissues with tympanosclerosis, received from 25 patients and from 19 normal tympanic membranes. The gene expression level was determined by real-time RT-PCR. The gene expression levels were correlated with clinical Tos classification of tympanosclerosis. We observed that in the tympanic membrane of patients with tympanosclerosis, the expression of type I collagen is decreased, while the expression of type II and IV collagen and osteopontin is increased. Moreover, mRNA levels of the investigated genes strongly correlated with the clinical stages of tympanosclerosis. The strong correlations between the expression of type I, II, IV collagen and osteopontin and the clinical stage of tympanosclerosis indicate the involvement of these proteins in excessive fibrosis and pathological remodeling of the tympanic membrane. In the future, a treatment aiming to modulate these gene expressions and/or regulation of the degradation of their protein products could be used as a new medical approach for patients with tympanosclerosis.

  8. Changes in gravity inhibit lymphocyte locomotion through type I collagen

    NASA Technical Reports Server (NTRS)

    Pellis, N. R.; Goodwin, T. J.; Risin, D.; McIntyre, B. W.; Pizzini, R. P.; Cooper, D.; Baker, T. L.; Spaulding, G. F.

    1997-01-01

    Immunity relies on the circulation of lymphocytes through many different tissues including blood vessels, lymphatic channels, and lymphoid organs. The ability of lymphocytes to traverse the interstitium in both nonlymphoid and lymphoid tissues can be determined in vitro by assaying their capacity to locomote through Type I collagen. In an attempt to characterize potential causes of microgravity-induced immunosuppression, we investigated the effects of simulated microgravity on human lymphocyte function in vitro using a specialized rotating-wall vessel culture system developed at the Johnson Space Center. This very low shear culture system randomizes gravitational vectors and provides an in vitro approximation of microgravity. In the randomized gravity of the rotating-wall vessel culture system, peripheral blood lymphocytes did not locomote through Type I collagen, whereas static cultures supported normal movement. Although cells remained viable during the entire culture period, peripheral blood lymphocytes transferred to unit gravity (static culture) after 6 h in the rotating-wall vessel culture system were slow to recover and locomote into collagen matrix. After 72 h in the rotating-wall vessel culture system and an additional 72 h in static culture, peripheral blood lymphocytes did not recover their ability to locomote. Loss of locomotory activity in rotating-wall vessel cultures appears to be related to changes in the activation state of the lymphocytes and the expression of adhesion molecules. Culture in the rotating-wall vessel system blunted the ability of peripheral blood lymphocytes to respond to polyclonal activation with phytohemagglutinin. Locomotory response remained intact when peripheral blood lymphocytes were activated by anti-CD3 antibody and interleukin-2 prior to introduction into the rotating-wall vessel culture system. Thus, in addition to the systemic stress factors that may affect immunity, isolated lymphocytes respond to gravitational changes

  9. In Vitro Expression of the Extracellular Matrix Components Aggrecan, Collagen Types I and II by Articular Cartilage-Derived Chondrocytes.

    PubMed

    Schneevoigt, J; Fabian, C; Leovsky, C; Seeger, J; Bahramsoltani, M

    2017-02-01

    The extracellular matrix (ECM) of hyaline cartilage is perfectly suited to transmit articular pressure load to the subchondral bone. Pressure is transferred by a high amount of aggrecan-based proteoglycans and collagen type II fibres in particular. After any injury, the hyaline cartilage is replaced by fibrocartilage, which is low in proteoglycans and contains collagen type I predominantly. Until now, long-term results of therapeutic procedures including cell-based therapies like autologous chondrocyte transplantation (ACT) lead to a replacement tissue meeting the composition of fibrocartilage. Therefore, it is of particular interest to discover how and to what extent isolation and in vitro cultivation of chondrocytes affect the cells and their expression of ECM components. Hyaline cartilage-derived chondrocytes were cultivated in vitro and observed microscopically over a time period of 35 days. The expression of collagen type I, collagen type II and aggrecan was analysed using RT-qPCR and Western blot at several days of cultivation. Chondrocytes presented a longitudinal shape for the entire cultivation period. While expression of collagen type I prevailed within the first days, only prolonged cultivation led to an increase in collagen type II and aggrecan expression. The results indicate that chondrocyte isolation and in vitro cultivation lead to a dedifferentiation at least to the stage of chondroprogenitor cells. © 2016 Blackwell Verlag GmbH.

  10. The Collagen Family

    PubMed Central

    Ricard-Blum, Sylvie

    2011-01-01

    Collagens are the most abundant proteins in mammals. The collagen family comprises 28 members that contain at least one triple-helical domain. Collagens are deposited in the extracellular matrix where most of them form supramolecular assemblies. Four collagens are type II membrane proteins that also exist in a soluble form released from the cell surface by shedding. Collagens play structural roles and contribute to mechanical properties, organization, and shape of tissues. They interact with cells via several receptor families and regulate their proliferation, migration, and differentiation. Some collagens have a restricted tissue distribution and hence specific biological functions. PMID:21421911

  11. Collagens--structure, function, and biosynthesis.

    PubMed

    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.

  12. Long-term type 1 diabetes alters the deposition of collagens and proteoglycans in the early pregnant myometrium of mice.

    PubMed

    Favaro, Rodolfo R; Raspantini, Priscila R; Salgado, Renato M; Fortes, Zuleica B; Zorn, Telma M T

    2015-04-01

    We have previously shown that long-term type 1 diabetes affects the structural organization, contractile apparatus and extracellular matrix (ECM) of the myometrium during early pregnancy in mice. This study aimed to identify which myometrial ECM components are affected by diabetes, including fibril-forming collagen types I, III and V, as well as proteoglycans, decorin, lumican, fibromodulin and biglycan. Alloxan-induced type 1 diabetic female mice were divided into subgroups D1 and D2, formed by females that bred 90-100 and 100-110 days after diabetes induction, respectively. The deposition of ECM components in the myometrium was evaluated by immunohistochemistry/immunofluorescence. The subgroup D1 showed decreased deposition of collagen types I and III in the external muscle layer (EML) and decreased collagen types III and V in the internal muscle layer (IML). Collagen types I and III were decreased in both muscle layers of the subgroup D2. In addition, increased deposition of collagen types I and III and lumican as well as decreased collagen type V were observed in the connective tissue between muscle layers of D2. Lumican was decreased in the EML of the subgroups D1 and D2. Fibromodulin was repressed in the IML and EML of both D1 and D2. In contrast, decorin deposition diminished only in muscle layers of D2. No changes were noticed for biglycan. Subgroups D1 and D2 showed distinct stages of progression of diabetic complications in the myometrium, characterized by both common and specific sets of changes in the ECM composition.

  13. Evidence for an extensive collagen type III/VI proximal domain in the rat femur. I. Diminution with ovariectomy.

    PubMed

    Luther, F; Saino, H; Carter, D H; Aaron, J E

    2003-06-01

    Collagenous proteins other than Type I have received little attention in hypogonadal bone loss. Using femora from 25 young (2.5 months) and older (11 months) control and ovariectomized adult rats killed 1-4 months postoperation, cancellous atrophy was histologically confirmed, and the immunolocalization of collagen Type III was examined. This occurred as numerous immunofluorescent Sharpey-like fibers, 5-25 microm thick, regularly associated with collagen Type VI, which ramified the femoral cortex. Sequential transverse cryosections enabled the mapping of the fibers in three-dimensions, demonstrating that they constituted an extensive subperiosteal domain which may be a lasting legacy of early skeletal development. Fiber density was greatest in the trochanters and femoral neck. The domain tapered distally and was apparently anchored into the mid-shaft by intracortical cartilaginous islands, staining for collagen Type VI (as well as Type II and fibronectin). Ovariectomy caused disconnection of the fibers and reduced the proximal domain of both young and older animals, previously positive areas of the cortex becoming negative. It is concluded that collagen Type III/VI occupies a substantial, discrete domain in the rat proximal femur as a complex extension of the periosteum. Diminution of this cortical domain with trabecular atrophy suggests that it has a proactive or reactive role in determining bone mass and strength by facilitating musculoskeletal exchange in a form that is disengaged by ovariectomy.

  14. Ameloblasts express type I collagen during amelogenesis.

    PubMed

    Assaraf-Weill, N; Gasse, B; Silvent, J; Bardet, C; Sire, J Y; Davit-Béal, T

    2014-05-01

    Enamel and enameloid, the highly mineralized tooth-covering tissues in living vertebrates, are different in their matrix composition. Enamel, a unique product of ameloblasts, principally contains enamel matrix proteins (EMPs), while enameloid possesses collagen fibrils and probably receives contributions from both odontoblasts and ameloblasts. Here we focused on type I collagen (COL1A1) and amelogenin (AMEL) gene expression during enameloid and enamel formation throughout ontogeny in the caudate amphibian, Pleurodeles waltl. In this model, pre-metamorphic teeth possess enameloid and enamel, while post-metamorphic teeth possess enamel only. In first-generation teeth, qPCR and in situ hybridization (ISH) on sections revealed that ameloblasts weakly expressed AMEL during late-stage enameloid formation, while expression strongly increased during enamel deposition. Using ISH, we identified COL1A1 transcripts in ameloblasts and odontoblasts during enameloid formation. COL1A1 expression in ameloblasts gradually decreased and was no longer detected after metamorphosis. The transition from enameloid-rich to enamel-rich teeth could be related to a switch in ameloblast activity from COL1A1 to AMEL synthesis. P. waltl therefore appears to be an appropriate animal model for the study of the processes involved during enameloid-to-enamel transition, especially because similar events probably occurred in various lineages during vertebrate evolution.

  15. Anisotropic Shape-Memory Alginate Scaffolds Functionalized with Either Type I or Type II Collagen for Cartilage Tissue Engineering.

    PubMed

    Almeida, Henrique V; Sathy, Binulal N; Dudurych, Ivan; Buckley, Conor T; O'Brien, Fergal J; Kelly, Daniel J

    2017-01-01

    Regenerating articular cartilage and fibrocartilaginous tissue such as the meniscus is still a challenge in orthopedic medicine. While a range of different scaffolds have been developed for joint repair, none have facilitated the development of a tissue that mimics the complexity of soft tissues such as articular cartilage. Furthermore, many of these scaffolds are not designed to function in mechanically challenging joint environments. The overall goal of this study was to develop a porous, biomimetic, shape-memory alginate scaffold for directing cartilage regeneration. To this end, a scaffold was designed with architectural cues to guide cellular and neo-tissue alignment, which was additionally functionalized with a range of extracellular matrix cues to direct stem cell differentiation toward the chondrogenic lineage. Shape-memory properties were introduced by covalent cross-linking alginate using carbodiimide chemistry, while the architecture of the scaffold was modified using a directional freezing technique. Introducing such an aligned pore structure was found to improve the mechanical properties of the scaffold, and promoted higher levels of sulfated glycosaminoglycans (sGAG) and collagen deposition compared to an isotropic (nonaligned) pore geometry when seeded with adult human stem cells. Functionalization with collagen improved stem cell recruitment into the scaffold and facilitated more homogenous cartilage tissue deposition throughout the construct. Incorporating type II collagen into the scaffolds led to greater cell proliferation, higher sGAG and collagen accumulation, and the development of a stiffer tissue compared to scaffolds functionalized with type I collagen. The results of this study demonstrate how both scaffold architecture and composition can be tailored in a shape-memory alginate scaffold to direct stem cell differentiation and support the development of complex cartilaginous tissues.

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

    PubMed

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

    2015-02-01

    Skin aging appears to be principally attributed to a decrease in type I collagen level and the regeneration ability of dermal fibroblasts. We hypothesized that vegetable peptones promote cell proliferation and production of type I collagen in human dermal fibroblasts. Therefore, we investigated the effects of vegetable peptones on cell proliferation and type I collagen production and their possible mechanisms in human dermal fibroblasts. Vegetable peptones significantly promoted cell proliferation in a concentration-dependent manner. In addition, the human luciferase type I collagen α2 promoter and type I procollagen synthesis assays showed that the vegetable peptones induced type I procollagen production by activating the type I collagen α2 promoter. Moreover, the vegetable peptones activated p90 ribosomal s6 kinase, which was mediated by activating the Raf-p44/42 mitogen-activated protein kinase signaling pathway. Furthermore, the vegetable peptone-induced increase in cell proliferation and type I collagen production decreased upon treatment with the ERK inhibitor PD98059. Taken together, these findings suggest that increased proliferation of human dermal fibroblasts and enhanced production of type I collagen by vegetable peptones occur primarily by inducing the p90 ribosomal s6 kinase-CCAAT/enhancer binding protein β phosphorylation pathway, which is mediated by activating Raf-ERK signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Persistence of collagen type II-specific T-cell clones in the synovial membrane of a patient with rheumatoid arthritis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Londei, M.; Savill, C.M.; Verhoef, A.

    Rheumatoid arthritis is an autoimmune disease characterized by T-cell infiltration of the synovium of joints. Analysis of the phenotype and antigen specificity of the infiltrating cells may thus provide insight into the pathogenesis of rheumatoid arthritis. T cells were cloned with interleukin 2, a procedure that selects for in vivo-activated cells. All clones had the CD4 CDW29 phenotype. Their antigen specificity was tested by using a panel of candidate joint autoantigens. Four of 17 reacted against autologous blood mononuclear cells. Two clones proliferated in response to collagen type II. After 21 months, another set of clones was derived from synovialmore » tissue of the same joint. One of eight clones tested showed a strong proliferative response against collagen type II. The uncloned synovial T cells of a third operation from another joint also responded to collagen type II. The persistence of collagen type II-specific T cells in active rheumatoid joints over a period of 3 years suggests that collagen type II could be one of the autoantigens involved in perpetuating the inflammatory process in rheumatoid arthritis.« less

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

    PubMed Central

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

    2015-01-01

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

  19. Endoplasmic reticulum stress in chondrodysplasias caused by mutations in collagen types II and X.

    PubMed

    Gawron, Katarzyna

    2016-11-01

    The endoplasmic reticulum is primarily recognized as the site of synthesis and folding of secreted, membrane-bound, and some organelle-targeted proteins. An imbalance between the load of unfolded proteins and the processing capacity in endoplasmic reticulum leads to the accumulation of unfolded or misfolded proteins and endoplasmic reticulum stress, which is a hallmark of a number of storage diseases, including neurodegenerative diseases, a number of metabolic diseases, and cancer. Moreover, its contribution as a novel mechanistic paradigm in genetic skeletal diseases associated with abnormalities of the growth plates and dwarfism is considered. In this review, I discuss the mechanistic significance of endoplasmic reticulum stress, abnormal folding, and intracellular retention of mutant collagen types II and X in certain variants of skeletal chondrodysplasia.

  20. Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis.

    PubMed

    McKleroy, William; Lee, Ting-Hein; Atabai, Kamran

    2013-06-01

    Pulmonary fibrosis is a vexing clinical problem with no proven therapeutic options. In the normal lung there is continuous collagen synthesis and collagen degradation, and these two processes are precisely balanced to maintain normal tissue architecture. With lung injury there is an increase in the rate of both collagen production and collagen degradation. The increase in collagen degradation is critical in preventing the formation of permanent scar tissue each time the lung is exposed to injury. In pulmonary fibrosis, collagen degradation does not keep pace with collagen production, resulting in extracellular accumulation of fibrillar collagen. Collagen degradation occurs through both extracellular and intracellular pathways. The extracellular pathway involves cleavage of collagen fibrils by proteolytic enzyme including the metalloproteinases. The less-well-described intracellular pathway involves binding and uptake of collagen fragments by fibroblasts and macrophages for lysosomal degradation. The relationship between these two pathways and their relevance to the development of fibrosis is complex. Fibrosis in the lung, liver, and skin has been associated with an impaired degradative environment. Much of the current scientific effort in fibrosis is focused on understanding the pathways that regulate increased collagen production. However, recent reports suggest an important role for collagen turnover and degradation in regulating the severity of tissue fibrosis. The objective of this review is to evaluate the roles of the extracellular and intracellular collagen degradation pathways in the development of fibrosis and to examine whether pulmonary fibrosis can be viewed as a disease of impaired matrix degradation rather than a disease of increased matrix production.

  1. Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis

    PubMed Central

    McKleroy, William; Lee, Ting-Hein

    2013-01-01

    Pulmonary fibrosis is a vexing clinical problem with no proven therapeutic options. In the normal lung there is continuous collagen synthesis and collagen degradation, and these two processes are precisely balanced to maintain normal tissue architecture. With lung injury there is an increase in the rate of both collagen production and collagen degradation. The increase in collagen degradation is critical in preventing the formation of permanent scar tissue each time the lung is exposed to injury. In pulmonary fibrosis, collagen degradation does not keep pace with collagen production, resulting in extracellular accumulation of fibrillar collagen. Collagen degradation occurs through both extracellular and intracellular pathways. The extracellular pathway involves cleavage of collagen fibrils by proteolytic enzyme including the metalloproteinases. The less-well-described intracellular pathway involves binding and uptake of collagen fragments by fibroblasts and macrophages for lysosomal degradation. The relationship between these two pathways and their relevance to the development of fibrosis is complex. Fibrosis in the lung, liver, and skin has been associated with an impaired degradative environment. Much of the current scientific effort in fibrosis is focused on understanding the pathways that regulate increased collagen production. However, recent reports suggest an important role for collagen turnover and degradation in regulating the severity of tissue fibrosis. The objective of this review is to evaluate the roles of the extracellular and intracellular collagen degradation pathways in the development of fibrosis and to examine whether pulmonary fibrosis can be viewed as a disease of impaired matrix degradation rather than a disease of increased matrix production. PMID:23564511

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

    PubMed Central

    2012-01-01

    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. PMID:22694981

  3. Role of the recombinant protein of the platelet receptor for type I collagen in the release of nitric oxide during platelet aggregation.

    PubMed

    Chiang, T M; Wang, Y B; Kang, E S

    2000-12-01

    Nitric oxide plays an important role in platelet function and platelets possess the endothelial isoform of nitric oxide synthase. Several reports have indicated that nitric oxide is released upon exposure of platelets to collagen. We have reported that a non-integrin platelet protein of 65 kDa is a receptor for type I collagen. By direct measurement of NO release from washed human platelets suspended in Tyrode buffer with a ISO-NO Mark II, World Precision Instruments, Sarasota, FL, USA, p30 sensor, type I collagen, but not ADP and epinephrine, induces the release of NO in a time-dependent manner. The production of NO is inhibited either by preincubation of type I collagen with the platelet type I collagen receptor recombinant protein or by preincubation of platelets with the antibody to the receptor protein, the anti-65 antibody. However, preincubation of platelets with anti-P-selectin and anti-glycoprotein IIb/IIIa did not affect the release of NO by platelets. These results suggest that the 65 kDa platelet receptor for type I collagen is specifically linked to the generation of NO, and that the 65 kDa platelet receptor for type I collagen plays an important new role in platelet function.

  4. Stretching human mesenchymal stromal cells on stiffness-customized collagen type I generates a smooth muscle marker profile without growth factor addition

    NASA Astrophysics Data System (ADS)

    Rothdiener, Miriam; Hegemann, Miriam; Uynuk-Ool, Tatiana; Walters, Brandan; Papugy, Piruntha; Nguyen, Phong; Claus, Valentin; Seeger, Tanja; Stoeckle, Ulrich; Boehme, Karen A.; Aicher, Wilhelm K.; Stegemann, Jan P.; Hart, Melanie L.; Kurz, Bodo; Klein, Gerd; Rolauffs, Bernd

    2016-10-01

    Using matrix elasticity and cyclic stretch have been investigated for inducing mesenchymal stromal cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage but not in combination. We hypothesized that combining lineage-specific stiffness with cyclic stretch would result in a significantly increased expression of SMC markers, compared to non-stretched controls. First, we generated dense collagen type I sheets by mechanically compressing collagen hydrogels. Atomic force microscopy revealed a nanoscale stiffness range known to support myogenic differentiation. Further characterization revealed viscoelasticity and stable biomechanical properties under cyclic stretch with >99% viable adherent human MSC. MSCs on collagen sheets demonstrated a significantly increased mRNA but not protein expression of SMC markers, compared to on culture flasks. However, cyclic stretch of MSCs on collagen sheets significantly increased both mRNA and protein expression of α-smooth muscle actin, transgelin, and calponin versus plastic and non-stretched sheets. Thus, lineage-specific stiffness and cyclic stretch can be applied together for inducing MSC differentiation towards SMCs without the addition of recombinant growth factors or other soluble factors. This represents a novel stimulation method for modulating the phenotype of MSCs towards SMCs that could easily be incorporated into currently available methodologies to obtain a more targeted control of MSC phenotype.

  5. IgE reactivity to alpha1 and alpha2 chains of bovine type 1 collagen in children with bovine gelatin allergy.

    PubMed

    Sakaguchi, M; Hori, H; Hattori, S; Irie, S; Imai, A; Yanagida, M; Miyazawa, H; Toda, M; Inouye, S

    1999-09-01

    Anaphylactic reactions to measles, mumps, and rubella vaccines, including gelatin as a stabilizer, have been reported. It had been found that most of these reactions to live vaccines are caused by the bovine gelatin included in these vaccines. Gelatin mainly includes denatured type I collagen, which consists of alpha1 and alpha2 chains. The current study was designed to investigate the IgE reactivity to alpha1 and alpha2 chains of bovine type I collagen in gelatin-sensitive children. Serum samples were taken from 10 children who had anaphylaxis to the vaccines and high levels of specific IgE to bovine gelatin. Bovine type I collagen was isolated from bovine skin and then separated to alpha1 and alpha2 chains by column chromatography. IgE reactivity to denatured type I collagen and its alpha1 and alpha2 chains was analyzed by immunoblotting, ELISA, and histamine release from the mast cells passive sensitized with IgE antibodies in pooled serum of the children. All children had specific IgE to bovine type I collagen. Furthermore, IgE antibodies in their sera reacted with the alpha;2 chain but not with the alpha1 chain. Similarly, the mast cells sensitized with pooled sera in the children showed alpha2 chain-specific histamine release but not alpha1 chain-specific histamine release. In gelatin allergy denatured bovine type I collagen is a major allergen and IgE-binding sites exist in the alpha2 chain of type I collagen.

  6. Suppression of hedgehog signaling regulates hepatic stellate cell activation and collagen secretion.

    PubMed

    Li, Tao; Leng, Xi-Sheng; Zhu, Ji-Ye; Wang, Gang

    2015-01-01

    Hepatic stellate cells (HSCs) play an important role in liver fibrosis. This study investigates the expression of hedgehog in HSC and the role of hedgehog signaling on activation and collagen secretion of HSC. Liver ex vivo perfusion with collagenase IV and density gradient centrifugation were used to isolate HSC. Expression of hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 in HSC were detected by RT-PCR. Hedgehog siRNA vectors targeting Ihh, Smo and Gli2 were constructed and transfected into HSC respectively. Suppression of hedgehog signaling were detected by SYBR Green fluorescence quantitative RT-PCR. Effects of hedgehog signaling inhibition on HSC activation and collagen I secretion were analyzed. Hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 were expressed in HSC. siRNA vectors targeting Ihh, Smo and Gli2 were successfully constructed and decreased target gene expression. Suppression of hedgehog signaling significantly decreased the expression of α-SMA in HSC (P<0.01). Collagen type I secretion of HSC were also significantly decreased (P<0.01). In summary, HSC activation and collagen secretion can be regulated by hedgehog signaling. Hedgehog may play a role in the pathogenesis of liver fibrosis.

  7. Urinary type IV collagen is related to left ventricular diastolic function and brain natriuretic peptide in hypertensive patients with prediabetes.

    PubMed

    Iida, Masato; Yamamoto, Mitsuru; Ishiguro, Yuko S; Yamazaki, Masatoshi; Ueda, Norihiro; Honjo, Haruo; Kamiya, Kaichirou

    2014-01-01

    Urinary type IV collagen is an early biomarker of diabetic nephropathy. Concomitant prediabetes (the early stage of diabetes) was associated with left ventricular (LV) diastolic dysfunction and increased brain natriuretic peptide (BNP) in hypertensive patients. We hypothesized that urinary type IV collagen may be related to these cardiac dysfunctions. We studied hypertensive patients with early prediabetes (HbA1c <5.7% and fasting glucose >110, n=18), those with prediabetes (HbA1c 5.7-6.4, n=98), and those with diabetes (HbA1c>6.5 or on diabetes medications, n=92). The participants underwent echocardiography to assess left atrial volume/body surface area (BSA) and the ratio of early mitral flow velocity to mitral annular velocity (E/e'). Left ventricular diastolic dysfunction (LVDD) was defined if patients had E/e'≥15, or E/e'=9-14 accompanied by left atrial volume/BSA≥32ml/mm(2). Urinary samples were collected for type IV collagen and albumin, and blood samples were taken for BNP and HbA1c. Urinary type IV collagen and albumin increased in parallel with the deterioration of glycemic status. In hypertensive patients with prediabetes, subjects with LVDD had higher levels of BNP and urinary type IV collagen than those without LVDD. In contrast, in hypertensive patients with diabetes, subjects with LVDD had higher urinary albumin and BNP than those without LVDD. Urinary type IV collagen correlated positively with BNP in hypertensive patients with prediabetes, whereas it correlated with HbA1c in those with diabetes. In hypertensive patients with prediabetes, urinary type IV collagen was associated with LV diastolic dysfunction and BNP. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Irradiation Alters MMP-2/TIMP-2 System and Collagen Type IV Degradation in Brain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Won Hee; Warrington, Junie P.; Sonntag, William E.

    Purpose: Blood-brain barrier (BBB) disruption is one of the major consequences of radiation-induced normal tissue injury in the central nervous system. We examined the effects of whole-brain irradiation on matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) and extracellular matrix (ECM) degradation in the brain. Methods and Materials: Animals received either whole-brain irradiation (a single dose of 10 Gy {gamma}-rays or a fractionated dose of 40 Gy {gamma}-rays, total) or sham-irradiation and were maintained for 4, 8, and 24 h following irradiation. mRNA expression levels of MMPs and TIMPs in the brain were analyzed by real-time reverse transcriptase-polymerase chain reaction (PCR).more » The functional activity of MMPs was measured by in situ zymography, and degradation of ECM was visualized by collagen type IV immunofluorescent staining. Results: A significant increase in mRNA expression levels of MMP-2, MMP-9, and TIMP-1 was observed in irradiated brains compared to that in sham-irradiated controls. In situ zymography revealed a strong gelatinolytic activity in the brain 24 h postirradiation, and the enhanced gelatinolytic activity mediated by irradiation was significantly attenuated in the presence of anti-MMP-2 antibody. A significant reduction in collagen type IV immunoreactivity was also detected in the brain at 24 h after irradiation. In contrast, the levels of collagen type IV were not significantly changed at 4 and 8 h after irradiation compared with the sham-irradiated controls. Conclusions: The present study demonstrates for the first time that radiation induces an imbalance between MMP-2 and TIMP-2 levels and suggests that degradation of collagen type IV, a major ECM component of BBB basement membrane, may have a role in the pathogenesis of brain injury.« less

  9. Prevalence and relevance of antibodies to type-I and -II collagen in synovial fluid of dogs with cranial cruciate ligament damage.

    PubMed

    de Rooster, H; Cox, E; van Bree, H

    2000-11-01

    To measure and compare synovial fluid antibody titers to type-I and -II collagen in stifle joints with instability caused by complete or partial cranial cruciate ligament (CCL) rupture and joints with osteoarthrosis secondary to other pathologic changes in dogs. 82 dogs with diseased stifle joints. Synovial fluid samples were collected from 7 dogs with clinically normal stifles (control group) and 82 dogs with diseased joints (50 stifle joints with complete rupture of the CCL, 20 with partial damage of the CCL, and 12 joints with radiographic signs of osteoarthritis secondary to other arthropathies). Synovial fluid samples were tested for autoantibodies to type-I and -II collagen by an ELISA. In dogs with complete and partial CCL rupture, synovial fluid antibody titers to type-I and -II collagen were significantly increased, compared with control dogs. Forty-eight percent (24/50) of samples from dogs with complete CCL rupture and 35% (7/20) of samples from dogs with partial CCL rupture had antibody titers to type-I collagen that were greater than the mean plus 2 standard deviations of the control group titers. Synovial fluid antibody titers to type-II collagen were high in 40% of the dogs with partial or (8/20) complete (20/50) CCL rupture. Dogs with osteoarthrosis secondary to other pathologic changes had significantly increased synovial fluid antibodies to type-I and -II collagen, compared with control dogs. Increases in autoantibodies to collagen in synovial fluid are not specific for the type of joint disorder. It is unlikely that the anticollagen antibodies play an active role in the initiation of weakening of the CCL.

  10. Role of 17 beta-estradiol on type IV collagen fibers volumetric density in the basement membrane of bladder wall.

    PubMed

    de Fraga, Rogerio; Dambros, Miriam; Miyaoka, Ricardo; Riccetto, Cássio Luís Zanettini; Palma, Paulo César Rodrigues

    2007-10-01

    The authors quantified the type IV collagen fibers volumetric density in the basement membrane of bladder wall of ovariectomized rats with and without estradiol replacement. This study was conducted on 40 Wistar rats (3 months old) randomly divided in 4 groups: group 1, remained intact (control); group 2, submitted to bilateral oophorectomy and daily replacement 4 weeks later of 17 beta-estradiol for 12 weeks; group 3, sham operated and daily replacement 4 weeks later of sesame oil for 12 weeks; and group 4, submitted to bilateral oophorectomy and killed after 12 weeks. It was used in immunohistochemistry evaluation using type IV collagen polyclonal antibody to stain the fibers on paraffin rat bladder sections. The M-42 stereological grid system was used to analyze the fibers. Ovariectomy had an increase effect on the volumetric density of the type IV collagen fibers in the basement membrane of rat bladder wall. Estradiol replacement in castrated animals demonstrated a significative difference in the stereological parameters when compared to the castrated group without hormonal replacement. Surgical castration performed on rats induced an increasing volumetric density of type IV collagen fibers in the basement membrane of rats bladder wall and the estradiol treatment had a significant effect in keeping a low volumetric density of type IV collagen fibers in the basement membrane of rats bladder wall.

  11. Unique Conformation in a Natural Interruption Sequence of Type XIX Collagen Revealed by Its High-Resolution Crystal Structure.

    PubMed

    Xu, Tingting; Zhou, Cong-Zhao; Xiao, Jianxi; Liu, Jinsong

    2018-02-20

    Naturally occurring interruptions in nonfibrillar collagen play key roles in molecular flexibility, collagen degradation, and ligand binding. The structural feature of the interruption sequences and the molecular basis for their functions have not been well studied. Here, we focused on a G5G type natural interruption sequence G-POALO-G from human type XIX collagen, a homotrimer collagen, as this sequence possesses distinct properties compared with those of a pathological similar Gly mutation sequence in collagen mimic peptides. We determined the crystal structures of the host-guest peptide (GPO) 3 -GPOALO-(GPO) 4 to 1.03 Å resolution in two crystal forms. In these structures, the interruption zone brings localized disruptions to the triple helix and introduces a light 6-8° bend with the same directional preference to the whole molecule, which may correspond structurally to the first physiological kink site in type XIX collagen. Furthermore, at the G5G interruption site, the presence of Ala and Leu residues, both with free N-H groups, allows the formation of more direct and water-mediated interchain hydrogen bonds than in the related Gly → Ala structure. These could partly explain the difference in thermal stability between the different interruptions. In addition, our structures provide a detailed view of the dynamic property of such an interrupted zone with respect to hydrogen bonding topology, torsion angles, and helical parameters. Our results, for the first time, also identified the binding of zinc to the end of the triple helix. These findings will shed light on how the interruption sequence influences the conformation of the collagen molecule and provide a structural basis for further functional studies.

  12. Biomimetic Proteoglycan Interactions with Type I Collagen Investigated via 2D and 3D TEM

    NASA Astrophysics Data System (ADS)

    Moorehead, Carli

    Collagen is one of the leading components in extracellular matrix (ECM), providing durability, structural integrity, and functionality for many tissues. Regulation of collagen fibrillogenesis and degradation is important in the treatment of a number of diseases from orthopedic injuries to genetic deficiencies. Recently, novel, biocompatible, semi-synthetic biomimetic proteoglycans (BPGs) were developed, which consist of an enzymatically resistant synthetic polymer core and natural chondroitin sulfate bristles. It was demonstrated that BPGs affect type I collagen fibrillogenesis in vitro, as reflected by their impact delaying the kinetic formation of gels similar to native PGs. This indicates that the morphology of collagen scaffolds as well as endogenous ECM could also be modulated by these proteoglycan mimics. However, the imaging modality used previously, reflectance confocal microscopy, did not yield the resolution necessary to spatially localize BPGs within the collagen network or investigate the effect of BPGs on the quality of collagen fibrils produced in an in vitro fibrillogenesis model which is important for understanding the method of interaction. Consequently, a histological technique, electron tomography, was adapted and utilized to 3D image the nano-scale structures within this simplified tissue model. BPGs were found to aid in lateral growth and enhance fibril banding periodicity resulting in structures more closely resembling those in tissue, in addition to attaching to the collagen surface despite the lack of a protein core.

  13. PPAR-δ Agonist With Mesenchymal Stem Cells Induces Type II Collagen-Producing Chondrocytes in Human Arthritic Synovial Fluid.

    PubMed

    Heck, Bruce E; Park, Joshua J; Makani, Vishruti; Kim, Eun-Cheol; Kim, Dong Hyun

    2017-08-01

    Osteoarthritis (OA) is an inflammatory joint disease characterized by degeneration of articular cartilage within synovial joints. An estimated 27 million Americans suffer from OA, and the population is expected to reach 67 million in the United States by 2030. Thus, it is urgent to find an effective treatment for OA. Traditional OA treatments have no disease-modifying effect, while regenerative OA therapies such as autologous chondrocyte implantation show some promise. Nonetheless, current regenerative therapies do not overcome synovial inflammation that suppresses the differentiation of mesenchymal stem cells (MSCs) to chondrocytes and the expression of type II collagen, the major constituent of functional cartilage. We discovered a synergistic combination that overcame synovial inflammation to form type II collagen-producing chondrocytes. The combination consists of peroxisome proliferator-activated receptor (PPAR) δ agonist, human bone marrow (hBM)-derived MSCs, and hyaluronic acid (HA) gel. Interestingly, those individual components showed their own strong enhancing effects on chondrogenesis. GW0742, a PPAR-δ agonist, greatly enhanced MSC chondrogenesis and the expression of type II collagen and glycosaminoglycan (GAG) in hBM-MSC-derived chondrocytes. GW0742 also increased the expression of transforming growth factor β that enhances chondrogenesis and suppresses cartilage fibrillation, ossification, and inflammation. HA gel also increased MSC chondrogenesis and GAG production. However, neither GW0742 nor HA gel could enhance the formation of type II collagen-producing chondrocytes from hBM-MSCs within human OA synovial fluid. Our data demonstrated that the combination of hBM-MSCs, PPAR-δ agonist, and HA gel significantly enhanced the formation of type II collagen-producing chondrocytes within OA synovial fluid from 3 different donors. In other words, the novel combination of PPAR-δ agonist, hBM-MSCs, and HA gel can overcome synovial inflammation to form

  14. Differential expression of type X collagen in a mechanically active 3-D chondrocyte culture system: a quantitative study

    PubMed Central

    Yang, Xu; Vezeridis, Peter S; Nicholas, Brian; Crisco, Joseph J; Moore, Douglas C; Chen, Qian

    2006-01-01

    Objective Mechanical loading of cartilage influences chondrocyte metabolism and gene expression. The gene encoding type X collagen is expressed specifically by hypertrophic chondrocytes and up regulated during osteoarthritis. In this study we tested the hypothesis that the mechanical microenvironment resulting from higher levels of local strain in a three dimensional cell culture construct would lead to an increase in the expression of type X collagen mRNA by chondrocytes in those areas. Methods Hypertrophic chondrocytes were isolated from embryonic chick sterna and seeded onto rectangular Gelfoam sponges. Seeded sponges were subjected to various levels of cyclic uniaxial tensile strains at 1 Hz with the computer-controlled Bio-Stretch system. Strain distribution across the sponge was quantified by digital image analysis. After mechanical loading, sponges were cut and the end and center regions were separated according to construct strain distribution. Total RNA was extracted from the cells harvested from these regions, and real-time quantitative RT-PCR was performed to quantify mRNA levels for type X collagen and a housing-keeping gene 18S RNA. Results Chondrocytes distributed in high (9%) local strain areas produced more than two times type X collagen mRNA compared to the those under no load conditions, while chondrocytes located in low (2.5%) local strain areas had no appreciable difference in type X collagen mRNA production in comparison to non-loaded samples. Increasing local strains above 2.5%, either in the center or end regions of the sponge, resulted in increased expression of Col X mRNA by chondrocytes in that region. Conclusion These findings suggest that the threshold of chondrocyte sensitivity to inducing type X collagen mRNA production is more than 2.5% local strain, and that increased local strains above the threshold results in an increase of Col X mRNA expression. Such quantitative analysis has important implications for our understanding of

  15. Diagnostic efficiency of amylase and type IV collagen in predicting chronic pancreatitis.

    PubMed

    Das, Subir Kumar; Varadhan, Sowmya; Dhanya, L; Mukherjee, Sukhes; Mohana, S; Balakrishnan, V; Vasudevan, D M

    2009-01-01

    Chronic pancreatitis, an irreversible inflammatory disease of the pancreas, is associated with the replacement of the destroyed parenchyma by extended development of fibrosis. Despite marked progress in diagnostic tools, no consensus has been reached in diagnosis of chronic pancreatitis. In this study we examined the hematological and biochemical parameters among 40 chronic pancreatitis patients within 18 to 67 yrs. ESR level and ALP activity was elevated in 40% cases. Serum amylase activity increased in 32 patients and it showed significant correlation with ALP (r=0.458, p=0.003), CA-19.9 (r=0.556, p<0.001), and calcium level (r=-0.472, p=0.002). Type IV collagen level in chronic pancreatitis also elevated (164.4 ± 55.5 ng/ml) and showed negative significant correlation with calcium level (r= -0.505, p=0.001). However, no significant correlation was observed between amylase activity and type IV collagen (r=0.289, p= 0.07).

  16. Analysis of transcriptional isoforms of collagen types IX, II, and I in the developing avian cornea by competitive polymerase chain reaction.

    PubMed

    Fitch, J M; Gordon, M K; Gibney, E P; Linsenmayer, T F

    1995-01-01

    The genes for the alpha 1(IX), alpha 1(II), and alpha 2(I) collagen chains can give rise to different isoforms of mRNA, generated by alternative promotor usage [for alpha 1(IX) and alpha 2(I)] or alternative splicing [for alpha 1(II)]. In this study, we employed competitive reverse transcriptase PCR to quantitate the amounts of transcriptional isoforms for these genes in the embryonic avian cornea from its inception (about 3 1/2 days of development) to 11 days. In order to compare values at different time points, the results were normalized to those obtained for the "housekeeping" enzyme, glycerol-3-phosphate dehydrogenase (G3PDH). These values were compared to those obtained from other tissues (anterior optic cup and cartilage) that synthesize different combinations of the collagen isoforms. We found that, in the cornea, transcripts from the upstream promotor of alpha 1(IX) collagen (termed "long IX") were predominant at stage 18-20 (about 3 1/2 days), but then fell rapidly, and remained at a low level. By 5 days (just before stromal swelling) the major mRNA isoform of alpha 1(IX) was from the downstream promoter (termed "short IX"). The relative amount of transcript for the short form of type IX collagen rose to a peak at about 6 days of development, and then declined. Throughout this period, the predominant transcriptional isoform of the collagen type II gene was IIA (i.e., containing the alternatively spliced exon 2). This indicates that the molecules of type II collagen that are assembled into heterotypic fibrils with type I collagen possess, at least transiently, an amino-terminal globular domain similar to that found in collagen types I, III, and V. For type I, the "bone/tendon" mRNA isoform of the alpha 2(I) collagen gene was predominant; transcripts from the downstream promotor were at basal levels. In other tissues expressing collagen types IX and II, long IX was expressed predominantly with the IIA form in the anterior optic cup at stage 22/23; in 14 1

  17. Bone sialoprotein-collagen interaction promotes hydroxyapatite nucleation.

    PubMed

    Baht, Gurpreet S; Hunter, Graeme K; Goldberg, Harvey A

    2008-09-01

    In bone, hydroxyapatite (HA) crystals are deposited onto the type I collagen scaffold by a mechanism that has yet to be elucidated. Bone sialoprotein (BSP) is an acidic phosphoprotein that is expressed at high levels in mineralized tissues, capable of binding type I collagen, and nucleating HA. Both bone-extracted and recombinant BSP (rBSP) bind with equal affinity to collagen. The nature of the BSP-collagen interaction and its role in HA nucleation are not known. We have used a solid-phase binding assay and affinity chromatography to characterize the BSP-collagen interaction. rBSP-binding affinities of triple-helical and fibrillar type I collagen were similar (K(D) approximately 13 nM), while that of heat-denatured type I collagen was lower (K(D) approximately 44 nM), indicating the importance of triple-helical structure in binding BSP. Pepsin treatment of collagen had no effect on rBSP binding, demonstrating that the telopeptides of collagen are not involved. The majority of collagen-bound rBSP was eluted by acetonitrile, indicating that hydrophobic interactions are principally responsible for binding. Using an HA-nucleation assay, it was shown that rBSP is ten-fold more potent in reconstituted fibrillar collagen gels than in agarose gels. Nucleating potency of a non-collagen-binding, HA-nucleating peptide [rBSP(134-206)] showed no difference in the two gel systems. The work here shows that optimal binding of rBSP requires collagen to be in a native, triple-helical structure, does not require the telopeptides, and is stabilized by hydrophobic interactions. Upon binding to collagen, rBSP displays an increase in nucleation potency, implying a co-operative effect of BSP and collagen in mineral formation.

  18. A Trypanosoma cruzi-secreted 80 kDa proteinase with specificity for human collagen types I and IV.

    PubMed Central

    Santana, J M; Grellier, P; Schrével, J; Teixeira, A R

    1997-01-01

    Specific interactions between parasites and extracellular matrix components are an important mechanism in the dissemination of Chagas' disease. Binding of the extracellular matrix proteins to Trypanosoma cruzi receptors has been described as a significant step in this phenomenon. In this study, a specific proteinase activity was identified in cell-free extracts of amastigote, trypomastigote and epimastigote forms of T. cruzi using the collagenase fluorogenic substrate N-Suc-Gly-Pro-Leu-Gly-Pro-7-amido-4-methylcoumarin. Isolation of this activity was achieved by a four-step FPLC procedure. Optimal enzyme activity was found to occur at pH 8.0 and was associated with a single T. cruzi 80 kDa protein (Tc 80 proteinase) on SDS/PAGE under reducing conditions. An internal peptide sequence of Tc 80 proteinase was obtained (AGDNYTPPE), and no similarity was found to previously described proteinases of T. cruzi. This enzyme activity is strongly inhibited by HgCl2, tosyl-lysylchloromethane ('TLCK') p-chloromercuribenzoate and benzyloxycarbonyl-Phe-Ala-diazomethane. The purified enzyme was able to hydrolyse purified human [14C]collagen types I and IV at neutral pH, but not 14C-labelled BSA, rat laminin, rabbit IgG or small proteins such as insulin or cytochrome c. In addition, Tc 80 proteinase activity was found to be secreted by T. cruzi forms infective to mammalian cells. Furthermore we demonstrated that purified Tc 80 proteinase mediates native collagen type I hydrolysis in rat mesentery. This feature is compared with that of Clostridium histolyticum collagenase. These findings suggest that Tc 80 proteinase may facilitate T. cruzi host-cell infection by degrading the collagens of the extracellular matrix and could represent a good target for Chagas' disease chemotherapy. PMID:9224638

  19. Genetic and orthopedic aspects of collagen disorders.

    PubMed

    Carter, Erin M; Raggio, Cathleen L

    2009-02-01

    'Collagens' are a family of structurally related proteins that play a wide variety of roles in the extracellular matrix. To date, there are at least 29 known types of collagen. Accordingly, abnormality in the various collagens produces a large category of diseases with heterogeneous symptoms. This review presents genetic and orthopedic aspects of type II, IX, and XI collagen disorders. Although a diverse group of conditions, mutation of collagens affecting the articular cartilage typically produces an epiphyseal skeletal dysplasia phenotype. Often, the ocular or auditory systems or both are also involved. Treatment of these collagenopathies is symptomatic and individualized. Study of tissue from animal models allows examination of mutation effects on the abnormal protein structure and function. The collagen superfamily comprises an important structural protein in mammalian connective tissue. Mutation of collagens produces a wide variety of genetic disorders, and those mutations affecting types II, IX, and XI collagens produce an overlapping spectrum of skeletal dysplasias. Findings range from lethal to mild, depending on the mutation of the collagen gene and its subsequent effect on the structure and/or metabolism of the resultant procollagen and/or collagen protein and its function in the body.

  20. Concentration of mutations causing Schmid metaphyseal chondrodysplasia in the NC1 domain of type X collagen

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McIntosh, I.; Abbott, M.H.; Francomano, C.A.

    1994-09-01

    Schmid metaphyseal chondrodysplasia (SMCD, MIM 156500) is an autosomal dominant disorder of the osseous skeleton resulting in short stature, coxa vara and a waddling gait. Type X collagen is an extracellular matrix protein expressed exclusively by hypertrophic chondrocytes. We have previously identified four mutations in the type X collagen gene (COL10A1) in patients with SMCD. Each of these mutations, as well as another three reported by other investigators, are in the carboxy-terminal non-collagenous domain (NC1). Here, we present data for another three mutations each predicted to cause premature termination of translation within the NC1 domain. Two are nonsense mutations, Y628Xmore » and W651X, while the third is a frameshift resulting from the deletion of two nucleotides, 1856delCC. Each of these mutations occurred de novo, resulting in sporadic cases of SMCD. Four frameshift mutations have now been reported to initiate within 10bp of each other in the NC1 domain, namely 1865delC, 1856delCC, 1856del13 and 1866del10. These findings further support the hypothesis that SMCD is the result of the mutant type X collagen molecule being unable to participate in trimerization, although a dominant-negative model of disease pathogenesis has not been formally excluded.« less

  1. Molecular level detection and localization of mechanical damage in collagen enabled by collagen hybridizing peptides.

    PubMed

    Zitnay, Jared L; Li, Yang; Qin, Zhao; San, Boi Hoa; Depalle, Baptiste; Reese, Shawn P; Buehler, Markus J; Yu, S Michael; Weiss, Jeffrey A

    2017-03-22

    Mechanical injury to connective tissue causes changes in collagen structure and material behaviour, but the role and mechanisms of molecular damage have not been established. In the case of mechanical subfailure damage, no apparent macroscale damage can be detected, yet this damage initiates and potentiates in pathological processes. Here, we utilize collagen hybridizing peptide (CHP), which binds unfolded collagen by triple helix formation, to detect molecular level subfailure damage to collagen in mechanically stretched rat tail tendon fascicle. Our results directly reveal that collagen triple helix unfolding occurs during tensile loading of collagenous tissues and thus is an important damage mechanism. Steered molecular dynamics simulations suggest that a likely mechanism for triple helix unfolding is intermolecular shearing of collagen α-chains. Our results elucidate a probable molecular failure mechanism associated with subfailure injuries, and demonstrate the potential of CHP targeting for diagnosis, treatment and monitoring of tissue disease and injury.

  2. [The characteristics of type I, III collagen and LN in pulmonary fibrosis induced by uranium ore dust in rats].

    PubMed

    Hu, Ying-chun; Luo, Zhen-hua; Yuan, Xing-jiang; Yang, Li-ping; Wang, Shou-feng; Li, Guang-yue; He, Xing-peng

    2011-02-01

    To explore the characteristics of LN and type I, III collagen in pulmonary fibrosis induced by uranium ore dust in rats. 60 adult Wistar rats were divided randomly into two groups, control group (30 rats) and uranium ore dust group (30 rats). Non-exposed intratracheal instillation method was used. Uranium ore dust group was exposed 20 mg/ml uranium ore dust suspension 1ml per rat, meanwhile control group was exposed normal saline 1ml per rat. Post-exposed the 7, 14, 21, 30 and 60 d, 6 rats in each group were killed randomly, lung tissue were collected. The pathological changes in lung tissue were observed by microscope using HE staining, the collagen I and III in lungs were observed by polarizing microscope using Biebrich scarlet staining. The expression of LN protein in lung tissue was observed by immunohistochemistry-SP. During lung fibrosis, a large amount of the proliferated I and III collagen in lungs were observed. Post-exposure to uranium ore dust, the characteristics in proliferated collagen in lungs were type I collagen deposited in lung interstitium mainly in the early stage. The area percentage of collagen I and III was increased significantly at 7, 14, 21, 30 and 60d in the experimental group as compared with that in the control group (P < 0.05 or P < 0.01). The over expression of LN in the lung tissue were observed. The expression of LN was distributed in the lung tissue as thickening of the linear or cluster. The integral optical density of LN was increased significantly at 21, 30 and 60 d in the experimental group as compared with that in the control group (P < 0.05 or P < 0.01). After exposure to uranium ore dust, the characteristics in proliferated collagen in lungs are the type of I collagen deposited in lung interstitium mainly in the early stage, while the type of III collagen increase significantly at the later period. The overexpression of LN exists in the process of pulmonary fibrosis. It suggests that LN has a role effect in the process of

  3. In situ hybridization reveals that type I and III collagens are produced by pericytes in the anterior pituitary gland of rats.

    PubMed

    Fujiwara, Ken; Jindatip, Depicha; Kikuchi, Motoshi; Yashiro, Takashi

    2010-12-01

    Type I and III collagens widely occur in the rat anterior pituitary gland and are the main components of the extracellular matrix (ECM). Although ECM components possibly play an important role in the function of the anterior pituitary gland, little is known about collagen-producing cells. Type I collagen is a heterotrimer of two α1(I) chains (the product of the col1a1 gene) and one α2(I) chain (the product of the col1a2 gene). Type III collagen is a homotrimer of α1(III) chains (the product of the col3a1 gene). We used in situ hybridization with digoxigenin-labeled cRNA probes to examine the expression of col1a1, col1a2, and col3a1 mRNAs in the pituitary gland of adult rats. mRNA expression for these collagen genes was clearly observed, and cells expressing col1a1, col1a2, and col3a1 mRNA were located around capillaries in the gland. We also investigated the possible double-staining of collagen mRNA and pituitary hormones, S-100 protein (a marker of folliculo-stellate cells), or desmin (a marker of pericytes). Col1a1 and col3a1 mRNA were identified in desmin-immunopositive cells. Thus, only pericytes produce type I and III collagens in the rat anterior pituitary gland.

  4. Imaging Prostate Cancer Microenvironment by Collagen Hybridization

    DTIC Science & Technology

    2015-10-01

    expected to exhibit selective affinity to metastatic PCa tumors known to contain processed and denatured collagens. The motivating hypothesis is that the...CMP’s ability to bind to collagen/ denatured collagen can be used to image PCa in vivo as well as to determine the level of PCa malignancy. 15...targeted by antibodies (monoclonal antibody raised against denatured collagen); however antibodies have poor pharmacokinetics for in vivo imaging2. Recently

  5. Collagen as potential cell scaffolds for tissue engineering.

    PubMed

    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.

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

    PubMed

    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.

  7. De novo self-assembling collagen heterotrimers using explicit positive and negative design.

    PubMed

    Xu, Fei; Zhang, Lei; Koder, Ronald L; Nanda, Vikas

    2010-03-23

    We sought to computationally design model collagen peptides that specifically associate as heterotrimers. Computational design has been successfully applied to the creation of new protein folds and functions. Despite the high abundance of collagen and its key role in numerous biological processes, fibrous proteins have received little attention as computational design targets. Collagens are composed of three polypeptide chains that wind into triple helices. We developed a discrete computational model to design heterotrimer-forming collagen-like peptides. Stability and specificity of oligomerization were concurrently targeted using a combined positive and negative design approach. The sequences of three 30-residue peptides, A, B, and C, were optimized to favor charge-pair interactions in an ABC heterotrimer, while disfavoring the 26 competing oligomers (i.e., AAA, ABB, BCA). Peptides were synthesized and characterized for thermal stability and triple-helical structure by circular dichroism and NMR. A unique A:B:C-type species was not achieved. Negative design was partially successful, with only A + B and B + C competing mixtures formed. Analysis of computed versus experimental stabilities helps to clarify the role of electrostatics and secondary-structure propensities determining collagen stability and to provide important insight into how subsequent designs can be improved.

  8. Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies.

    PubMed

    Girkontaite, I; Frischholz, S; Lammi, P; Wagner, K; Swoboda, B; Aigner, T; Von der Mark, K

    1996-09-01

    For studies on processing and tissue distribution of type X collagen, monoclonal antibodies were prepared against human recombinant collagen type X (hrCol X) and tested by ELISA, immunoblotting and immunohistology. Forty-two clones were obtained which were grouped into four different subsets based on their reactivity against native and denatured hrCol X, pepsin-treated hrCol X, and the C-terminal NC-1 domain. Here we present results obtained with four monoclonal antibodies: Clone X 53, a representative of group I, binds with high affinity to both native and pepsin-digested hrCol X but with low affinity to the NC-1 dimer; monoclonal antibodies of group II and III recognized native and denatured hrCol X but not NC-1; antibodies of group II, but not III, reacted to some extent with pepsin treated hrCol X; one antibody (X 34) was obtained that reacted strongly with the isolated NC-1 dimer and native hrCol X but not with the NC-1 monomer or pepsin-digested hrCol X (group IV). Antibodies of all groups stained specifically the hypertrophic zone of fetal human epiphyseal cartilage. Mab X 53 stained the peri- and extracellular matrix of hypertrophic chondrocytes in the lower hypertrophic zone and in the calcified cartilage core in endochondral bone trabecules, while clone X 34 stained intracellularly and the pericellular matrix. All other tissues or cells of the epiphysis were negative. Antibody X 53 reacted also with canine, murine and guinea pig hypertrophic cartilage in tissue sections, but not with bovine or porcine type X collagen. In sections of osteoarthritic cartilage, clusters of hypertrophic chondrocytes in the deep zone were stained, confirming previous observations on enhanced chondrocyte hypertrophy and type X collagen expression in osteoarthritic articular cartilage.

  9. Failed Degradation of JunB Contributes to Overproduction of Type I Collagen and Development of Dermal Fibrosis in Patients With Systemic Sclerosis

    PubMed Central

    Ponticos, Markella; Papaioannou, Ioannis; Xu, Shiwen; Holmes, Alan M; Khan, Korsa; Denton, Christopher P; Bou-Gharios, George; Abraham, David J

    2015-01-01

    Objective The excessive deposition of extracellular matrix, including type I collagen, is a key aspect in the pathogenesis of connective tissue diseases such as systemic sclerosis (SSc; scleroderma). To further our understanding of the mechanisms governing the dysregulation of type I collagen production in SSc, we investigated the role of the activator protein 1 (AP-1) family of transcription factors in regulating COL1A2 transcription. Methods The expression and nuclear localization of AP-1 family members (c-Jun, JunB, JunD, Fra-1, Fra-2, and c-Fos) were examined by immunohistochemistry and Western blotting in dermal biopsy specimens and explanted skin fibroblasts from patients with diffuse cutaneous SSc and healthy controls. Gene activation was determined by assessing the interaction of transcription factors with the COL1A2 enhancer using transient transfection of reporter gene constructs, electrophoretic mobility shift assays, chromatin immunoprecipitation analysis, and RNA interference involving knockdown of individual AP-1 family members. Inhibition of fibroblast mammalian target of rapamycin (mTOR), Akt, and glycogen synthase kinase 3β (GSK-3β) signaling pathways was achieved using small-molecule pharmacologic inhibitors. Results Binding of JunB to the COL1A2 enhancer was observed, with its coalescence directed by activation of gene transcription through the proximal promoter. Knockdown of JunB reduced enhancer activation and COL1A2 expression in response to transforming growth factor β. In SSc dermal fibroblasts, increased mTOR/Akt signaling was associated with inactivation of GSK-3β, leading to blockade of JunB degradation and, thus, constitutively high expression of JunB. Conclusion In patients with SSc, the accumulation of JunB resulting from altered mTOR/Akt signaling and a failure of proteolytic degradation underpins the aberrant overexpression of type I collagen. These findings identify JunB as a potential target for antifibrotic therapy in SSc

  10. The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis

    PubMed Central

    Clarke, Cassie J.; Berg, Tracy J.; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L.; Vermeulen, Peter B.; Foo, Shane; Kostaras, Eleftherios; Jones, J. Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R.; Norman, Jim C.

    2016-01-01

    Summary Expression of the initiator methionine tRNA (tRNAiMet) is deregulated in cancer. Despite this fact, it is not currently known how tRNAiMet expression levels influence tumor progression. We have found that tRNAiMet expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAiMet in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAiMet contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAiMet gene (2+tRNAiMet mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAiMet mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAiMet mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAiMet significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAiMet-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAiMet-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAiMet mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAiMet levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. PMID:26948875

  11. The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis.

    PubMed

    Clarke, Cassie J; Berg, Tracy J; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L; Vermeulen, Peter B; Foo, Shane; Kostaras, Eleftherios; Jones, J Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R; Norman, Jim C

    2016-03-21

    Expression of the initiator methionine tRNA (tRNAi(Met)) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi(Met) expression levels influence tumor progression. We have found that tRNAi(Met) expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi(Met) in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi(Met) contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi(Met) gene (2+tRNAi(Met) mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi(Met) mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi(Met) mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi(Met) significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi(Met)-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi(Met)-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi(Met) mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi(Met) levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Hyaluronic acid increases tendon derived cell viability and collagen type I expression in vitro: Comparative study of four different Hyaluronic acid preparations by molecular weight.

    PubMed

    Osti, Leonardo; Berardocco, Martina; di Giacomo, Viviana; Di Bernardo, Graziella; Oliva, Francesco; Berardi, Anna C

    2015-10-06

    Hyaluronic Acid (HA) has been already approved by Food and Drug Administration (FDA) for osteoarthritis (OA), while its use in the treatment of tendinopathy is still debated. The aim of this study was to evaluate in human rotator cuff tendon derived cells the effects of four different HA on cell viability, proliferation, apoptosis and the expression of collagen type I and collagen type III. An in vitro model was developed on human tendon derived cells from rotator cuff tears to study the effects of four different HA preparations (Ps) (sodium hyaluronate MW: 500-730 KDa - Hyalgan®, 1000 kDa Artrosulfur HA®, 1600 KDa Hyalubrix® and 2200 KDa Synolis-VA®) at various concentrations. Tendon derived cells morphology were evaluated after 0, 7 and 14 d of culture. Viability, proliferation, apoptosis were evaluated after 0, 24 and 48 h of culture. The expression and deposition of collagen type I and collagen type III were evaluated after 1, 7 and 14 d of culture. All HAPs tested increased viability and proliferation, in dose dependent manner. HAPs already reduce apoptosis at 24 h compared to control cells (without HAPs). Furthermore, HAPs stimulated the synthesis of collagen type I in a dose dependent fashion over 14 d, without increase in collagen type III; moreover, in the presence of Synolis-VA® the expression and deposition of collagen type I was significantly higher as compare with the other HAPs. HAPs enhanced viability, proliferation and expression of collagen type I in tendon derived cells.

  13. Changes in content and synthesis of collagen types and proteoglycans in osteoarthritis of the knee joint and comparison of quantitative analysis with Photoshop-based image analysis.

    PubMed

    Lahm, Andreas; Mrosek, Eike; Spank, Heiko; Erggelet, Christoph; Kasch, Richard; Esser, Jan; Merk, Harry

    2010-04-01

    The different cartilage layers vary in synthesis of proteoglycan and of the distinct types of collagen with the predominant collagen Type II with its associated collagens, e.g. types IX and XI, produced by normal chondrocytes. It was demonstrated that proteoglycan decreases in degenerative tissue and a switch from collagen type II to type I occurs. The aim of this study was to evaluate the correlation of real-time (RT)-PCR and Photoshop-based image analysis in detecting such lesions and find new aspects about their distribution. We performed immunohistochemistry and histology with cartilage tissue samples from 20 patients suffering from osteoarthritis compared with 20 healthy biopsies. Furthermore, we quantified our results on the gene expression of collagen type I and II and aggrecan with the help of real-time (RT)-PCR. Proteoglycan content was measured colorimetrically. Using Adobe Photoshop the digitized images of histology and immunohistochemistry stains of collagen type I and II were stored on an external data storage device. The area occupied by any specific colour range can be specified and compared in a relative manner directly from the histogram using the "magic wand tool" in the select similar menu. In the image grow menu gray levels or luminosity (colour) of all pixels within the selected area, including mean, median and standard deviation, etc. are depicted. Statistical Analysis was performed using the t test. With the help of immunohistochemistry, RT-PCR and quantitative RT- PCR we found that not only collagen type II, but also collagen type I is synthesized by the cells of the diseased cartilage tissue, shown by increasing amounts of collagen type I mRNA especially in the later stages of osteoarthritis. A decrease of collagen type II is visible especially in the upper fibrillated area of the advanced osteoarthritic samples, which leads to an overall decrease. Analysis of proteoglycan showed a loss of the overall content and a quite uniform staining in

  14. Streptococcus pyogenes collagen type I-binding Cpa surface protein. Expression profile, binding characteristics, biological functions, and potential clinical impact.

    PubMed

    Kreikemeyer, Bernd; Nakata, Masanobu; Oehmcke, Sonja; Gschwendtner, Caroline; Normann, Jana; Podbielski, Andreas

    2005-09-30

    The Streptococcus pyogenes collagen type I-binding protein Cpa (collagen-binding protein of group A streptococci) expressed by 28 serotypes of group A streptococci has been extensively characterized at the gene and protein levels. Evidence for three distinct families of cpa genes was found, all of which shared a common sequence encoding a 60-amino acid domain that accounted for selective binding to type I collagen. Surface plasmon resonance-based affinity measurements and functional studies indicated that the expression of Cpa was consistent with an attachment role for bacteria to tissue containing collagen type I. A cpa mutant displayed a significantly decreased internalization rate when incubated with HEp-2 cells but had no effect on the host cell viability. By utilizing serum from patients with a positive titer for streptolysin/DNase antibody, an increased anti-Cpa antibody titer was noted for patients with a clinical history of arthritis or osteomyelitis. Taken together, these results suggest Cpa may be a relevant matrix adhesin contributing to the pathogenesis of S. pyogenes infection of bones and joints.

  15. Differential expression of human lysyl hydroxylase genes, lysine hydroxylation, and cross-linking of type I collagen during osteoblastic differentiation in vitro

    NASA Technical Reports Server (NTRS)

    Uzawa, K.; Grzesik, W. J.; Nishiura, T.; Kuznetsov, S. A.; Robey, P. G.; Brenner, D. A.; Yamauchi, M.

    1999-01-01

    The pattern of lysyl hydroxylation in the nontriple helical domains of collagen is critical in determining the cross-linking pathways that are tissue specific. We hypothesized that the tissue specificity of type I collagen cross-linking is, in part, due to the differential expression of lysyl hydroxylase genes (Procollagen-lysine,2-oxyglutarate,5-dioxygenase 1, 2, and 3 [PLOD1, PLOD2, and PLOD3]). In this study, we have examined the expression patterns of these three genes during the course of in vitro differentiation of human osteoprogenitor cells (bone marrow stromal cells [BMSCs]) and normal skin fibroblasts (NSFs). In addition, using the medium and cell layer/matrix fractions in these cultures, lysine hydroxylation of type I collagen alpha chains and collagen cross-linking chemistries have been characterized. High levels of PLOD1 and PLOD3 genes were expressed in both BMSCs and NSFs, and the expression levels did not change in the course of differentiation. In contrast to the PLOD1 and PLOD3 genes, both cell types showed low PLOD2 gene expression in undifferentiated and early differentiated conditions. However, fully differentiated BMSCs, but not NSFs, exhibited a significantly elevated level (6-fold increase) of PLOD2 mRNA. This increase coincided with the onset of matrix mineralization and with the increase in lysyl hydroxylation in the nontriple helical domains of alpha chains of type I collagen molecule. Furthermore, the collagen cross-links that are derived from the nontriple helical hydroxylysine-aldehyde were found only in fully differentiated BMSC cultures. The data suggests that PLOD2 expression is associated with lysine hydroxylation in the nontriple helical domains of collagen and, thus, could be partially responsible for the tissue-specific collagen cross-linking pattern.

  16. Transforming Growth Factor β1 Induces the Expression of Collagen Type I by DNA Methylation in Cardiac Fibroblasts

    PubMed Central

    Pan, Xiaodong; Chen, Zhongpu; Huang, Rong; Yao, Yuyu; Ma, Genshan

    2013-01-01

    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. PMID:23560091

  17. Immunohistochemical assessment of collagen types I, III, IV and VI in biopsy samples of the bovine uterine wall collected during the oestrous cycle.

    PubMed

    Boos, A

    2000-01-01

    Uterine biopsies were collected at cycle days 1 (oestrous), 8, 15 and 19 in six cows. Unfixed cryostat sections were used to immunolocalise collagen types I, III, IV and VI by an indirect FITC method. Collagen I was sparsely found in the endometrium where it formed a fine meshwork of thin fibres directly below the surface epithelium, clearly visible only at cycle days 8 and 15. Collagen III formed the bulk of connective tissue fibres and was arranged in fine aggregates within the superficial endometrial stroma, while in the deeper areas it consisted of many thick fibre bundles. Collagen IV was found in basement membranes underlying all endometrial epithelia. Furthermore, it surrounded smooth muscle cells of blood vessels. A few single fibrils also stained positively within the endometrial stroma, more numerous at cycle days 1 and 19 as compared to days 8 and 15. Collagen VI formed a mesh of fine and pericellularly situated fibrils within the endometrial stroma. The contribution of the collagen types studied to the connective tissue of caruncles, blood vessels, lymph follicles, and myometrium is also reported. The results of the present study indicate that the connective tissue of the bovine uterine wall is composed of different collagen types, which exhibit a characteristic distribution pattern each. The day of cycle may influence amounts and organisation of collagen types I and IV as demonstrated here at the light-microscopical level. Copyright 2000 S. Karger AG, Basel

  18. Deamidation of collagen.

    PubMed

    Hurtado, Pilar Perez; O'Connor, Peter B

    2012-03-20

    Collagen is the major component of skin, tendons, ligaments, teeth, and bones, it provides the framework that holds most multicellular animals together, and collagen type I constitutes the major fibrillar collagen of bone. Because of the complexity of collagen's structure, the study of post-translational modifications such as deamidation for this protein is challenging. Although there is no evidence of this protein being used for age assessment, it has been shown that deamidation of collagen is remarkably increased in old bones from mammals. Nonspectrometric methodologies have been used for the determination of the extent of deamidation as a measure of the amount of amide nitrogen released in ammonia as well as constant rates for deamidation of asparagine in collagen. In general, these methodologies required more sample and separation processes. To understand if collagen plays a significant role in the aging process of fossil materials, a simpler and more accurate method is needed to determine the extent of deamidation at the whole protein level. The present work shows a method to determine the extent of deamidation in collagen using Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) along with collisionally activated dissociation (CAD) and electron capture dissociation (ECD). The measured deamidation half-life for three different tryptic peptides from collagen (I) ranged from 2000 to 6000 s under high temperature conditions (∼62 °C) and pH 7.5.

  19. Biomimetic soluble collagen purified from bones.

    PubMed

    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.

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

    PubMed

    Fisher, Kevin E; Pop, Andreia; Koh, Wonshill; Anthis, Nicholas J; Saunders, W Brian; Davis, George E

    2006-12-08

    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. 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 (approximatly 700 microm 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 (approximately 125 microm vs. approximately 45 microm) and velocity of invasion (approximately 0.09 microm/min vs. approximately 0.03 microm/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

  1. Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.

    PubMed

    Cabral, Wayne A; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R; Chang, Weizhong; Perosky, Joseph E; Makareeva, Elena N; Mertz, Edward L; Leikin, Sergey; Tomer, Kenneth B; Kozloff, Kenneth M; Eyre, David R; Yamauchi, Mitsuo; Marini, Joan C

    2014-06-01

    Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Xianwei, E-mail: XWang2@UAMS.edu; Lu, Jingjun; Khaidakov, Magomed

    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 cardiacmore » 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

  3. Age Increases Monocyte Adhesion on Collagen

    NASA Astrophysics Data System (ADS)

    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.

  4. Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types

    PubMed Central

    Heinemeier, K M; Olesen, J L; Haddad, F; Langberg, H; Kjaer, M; Baldwin, K M; Schjerling, P

    2007-01-01

    Acute exercise induces collagen synthesis in both tendon and muscle, indicating an adaptive response in the connective tissue of the muscle–tendon unit. However, the mechanisms of this adaptation, potentially involving collagen-inducing growth factors (such as transforming growth factor-β-1 (TGF-β-1)), as well as enzymes related to collagen processing, are not clear. Furthermore, possible differential effects of specific contraction types on collagen regulation have not been investigated. Female Sprague–Dawley rats were subjected to 4 days of concentric, eccentric or isometric training (n = 7–9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve. RNA was extracted from medial gastrocnemius and Achilles tendon tissue 24 h after the last training bout, and mRNA levels for collagens I and III, TGF-β-1, connective tissue growth factor (CTGF), lysyl oxidase (LOX), metalloproteinases (MMP-2 and -9) and their inhibitors (TIMP-1 and 2) were measured by Northern blotting and/or real-time PCR. In tendon, expression of TGF-β-1 and collagens I and III (but not CTGF) increased in response to all types of training. Similarly, enzymes/factors involved in collagen processing were induced in tendon, especially LOX (up to 37-fold), which could indicate a loading-induced increase in cross-linking of tendon collagen. In skeletal muscle, a similar regulation of gene expression was observed, but in contrast to the tendon response, the effect of eccentric training was significantly greater than the effect of concentric training on the expression of several transcripts. In conclusion, the study supports an involvement of TGF-β-1 in loading-induced collagen synthesis in the muscle–tendon unit and importantly, it indicates that muscle tissue is more sensitive than tendon to the specific mechanical stimulus. PMID:17540706

  5. Expression of collagen and related growth factors in rat tendon and skeletal muscle in response to specific contraction types.

    PubMed

    Heinemeier, K M; Olesen, J L; Haddad, F; Langberg, H; Kjaer, M; Baldwin, K M; Schjerling, P

    2007-08-01

    Acute exercise induces collagen synthesis in both tendon and muscle, indicating an adaptive response in the connective tissue of the muscle-tendon unit. However, the mechanisms of this adaptation, potentially involving collagen-inducing growth factors (such as transforming growth factor-beta-1 (TGF-beta-1)), as well as enzymes related to collagen processing, are not clear. Furthermore, possible differential effects of specific contraction types on collagen regulation have not been investigated. Female Sprague-Dawley rats were subjected to 4 days of concentric, eccentric or isometric training (n = 7-9 per group) of the medial gastrocnemius, by stimulation of the sciatic nerve. RNA was extracted from medial gastrocnemius and Achilles tendon tissue 24 h after the last training bout, and mRNA levels for collagens I and III, TGF-beta-1, connective tissue growth factor (CTGF), lysyl oxidase (LOX), metalloproteinases (MMP-2 and -9) and their inhibitors (TIMP-1 and 2) were measured by Northern blotting and/or real-time PCR. In tendon, expression of TGF-beta-1 and collagens I and III (but not CTGF) increased in response to all types of training. Similarly, enzymes/factors involved in collagen processing were induced in tendon, especially LOX (up to 37-fold), which could indicate a loading-induced increase in cross-linking of tendon collagen. In skeletal muscle, a similar regulation of gene expression was observed, but in contrast to the tendon response, the effect of eccentric training was significantly greater than the effect of concentric training on the expression of several transcripts. In conclusion, the study supports an involvement of TGF-beta-1 in loading-induced collagen synthesis in the muscle-tendon unit and importantly, it indicates that muscle tissue is more sensitive than tendon to the specific mechanical stimulus.

  6. Collagen Accumulation in Osteosarcoma Cells lacking GLT25D1 Collagen Galactosyltransferase*

    PubMed Central

    Baumann, Stephan

    2016-01-01

    Collagen is post-translationally modified by prolyl and lysyl hydroxylation and subsequently by glycosylation of hydroxylysine. Despite the widespread occurrence of the glycan structure Glc(α1–2)Gal linked to hydroxylysine in animals, the functional significance of collagen glycosylation remains elusive. To address the role of glycosylation in collagen expression, folding, and secretion, we used the CRISPR/Cas9 system to inactivate the collagen galactosyltransferase GLT25D1 and GLT25D2 genes in osteosarcoma cells. Loss of GLT25D1 led to increased expression and intracellular accumulation of collagen type I, whereas loss of GLT25D2 had no effect on collagen secretion. Inactivation of the GLT25D1 gene resulted in a compensatory induction of GLT25D2 expression. Loss of GLT25D1 decreased collagen glycosylation by up to 60% but did not alter collagen folding and thermal stability. Whereas cells harboring individually inactivated GLT25D1 and GLT25D2 genes could be recovered and maintained in culture, cell clones with simultaneously inactive GLT25D1 and GLT25D2 genes could be not grown and studied, suggesting that a complete loss of collagen glycosylation impairs osteosarcoma cell proliferation and viability. PMID:27402836

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

    PubMed Central

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

    2012-01-01

    The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated. PMID:22512648

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

    PubMed

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

    2012-10-01

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

  9. Collagen: Biochemistry, biomechanics, biotechnology

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nimni, M.E.

    1988-01-01

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

  10. A Chemical Phosphorylation-inspired Design for Type I Collagen Biomimetic Remineralization

    PubMed Central

    Gu, Li-sha; Kim, Jongryul; Kim, Young Kyung; Liu, Yan; Dickens, Sabine H.; Pashley, David H.; Ling, Jun-qi; Tay, Franklin R.

    2010-01-01

    Objectives Type I collagen alone cannot initiate tissue mineralization. Sodium trimetaphosphate (STMP) is frequently employed as a chemical phosphorylating reagent in the food industry. This study examined the feasibility of using STMP as a functional analog of matrix phosphoproteins for biomimetic remineralization of resin-bonded dentin. Methods Equilibrium adsorption and desorption studies of STMP were performed using demineralized dentin powder (DDP). Interaction between STMP and DDP was examined using Fourier-transform infrared spectroscopy. Based on those results, a bio-inspired mineralization scheme was developed for chemical phosphorylation of acid-etched dentin with STMP, followed by infiltration of the STMP-treated collagen matrix with two etch-and-rinse adhesives. Resin-dentin interfaces were remineralized in a Portland cement-simulated body fluid system, with or without the use of polyacrylic acid (PAA) as a dual biomimetic analog. Remineralized resin-dentin interfaces were examined unstained using transmission electron microscopy. Results Analysis of saturation binding curves revealed the presence of irreversible phosphate group binding sites on the surface of the DDP. FT-IR provided additional evidence of chemical interaction between STMP and DDP, with increased in the peak intensities of the P=O and P–O–C stretching modes. Those peaks returned to their original intensities after alkaline phosphatase treatment. Evidence of intrafibrillar apatite formation could be seen in incompletely resin-infiltrated, STMP-phosphorylated collagen matrices only when PAA was present in the SBF. Significance These results reinforce the importance of PAA for sequestration of amorphous calcium phosphate nanoprecursors in the biomimetic remineralization scheme. They also highlight the role of STMP as a templating analog of dentin matrix phosphoproteins for inducing intrafibrillar remineralization of apatite nanocrystals within the collagen matrix of incompletely resin

  11. Tissue distribution and developmental expression of type XVI collagen in the mouse.

    PubMed

    Lai, C H; Chu, M L

    1996-04-01

    The expression of a recently identified collagen, alpha 1 (XVI), in adult mouse tissue and developing mouse embryo was examined by immunohistochemistry and in situ hybridization. A polyclonal antiserum was raised against a recombinant fusion protein, which contained a segment of 161 amino acids in the N-terminal noncollagenous domain of the human alpha 1 (XVI) collagen. Immunoprecipitation of metabolically labelled human or mouse fibroblast cell lysates with this antibody revealed a major, bacterial collagenase sensitive polypeptide of approximately 210 kDa. The size agrees with the prediction from the full-length cDNA. Immunofluorescence examination of adult mouse tissues using the affinity purified antibody revealed a rather broad distribution of the protein. The heart, kidney, intestine, ovary, testis, eye, arterial walls and smooth muscles all exhibited significant levels of expression, while the skeletal muscle, lung and brain showed very restricted and low signals. During development, no significant expression of the mRNA or protein was observed in embryo of day 8 of gestation, but strong signals was detected in placental trophoblasts. Expression in embryos was detectable first after day 11 of gestation with weak positive signals appearing in the heart. In later stages of development, stronger RNA hybridizations were observed in a variety of tissues, particularly in atrial and ventricular walls of the developing heart, spinal root neural fibers and skin. These data demonstrate that type XVI collagen represents another collagenous component widely distributed in the extracellular matrix and may contribute to the structural integrity of various tissues.

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

    NASA Technical Reports Server (NTRS)

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

    1991-01-01

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

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

    PubMed

    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.

  14. A Novel Functional Role of Collagen Glycosylation

    PubMed Central

    Jürgensen, Henrik J.; Madsen, Daniel H.; Ingvarsen, Signe; Melander, Maria C.; Gårdsvoll, Henrik; Patthy, Laszlo; Engelholm, Lars H.; Behrendt, Niels

    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. The molecular basis for this interaction is known to involve the fibronectin type II domain but nothing is known about the function of the lectin domains in this respect. In this study, we have investigated a possible role of the single active lectin domain of uPARAP/Endo180 in the interaction with collagens. By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate the endocytic efficiency of the receptor toward highly glycosylated collagens such as basement membrane collagen IV. Surprisingly, this property was not shared by the mannose receptor, which internalized glycosylated collagens independently of its lectin function. This role of modulating its uptake efficiency by a specific receptor is a previously unrecognized function of collagen glycosylation. PMID:21768090

  15. [The genetics of collagen diseases].

    PubMed

    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.

  16. Abnormal Type I Collagen Post-translational Modification and Crosslinking in a Cyclophilin B KO Mouse Model of Recessive Osteogenesis Imperfecta

    PubMed Central

    Cabral, Wayne A.; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R.; Chang, Weizhong; Perosky, Joseph E.; Makareeva, Elena N.; Mertz, Edward L.; Leikin, Sergey; Tomer, Kenneth B.; Kozloff, Kenneth M.; Eyre, David R.; Yamauchi, Mitsuo; Marini, Joan C.

    2014-01-01

    Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib−/− mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2–11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib−/− fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered

  17. A specific collagen type II gene (COL2A1) mutation presenting as spondyloperipheral dysplasia

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zabel, B.; Hilbert, K.; Spranger, J.

    1996-05-03

    We report on a patient with a skeletal dysplasia characterized by short stature, spondylo-epiphyseal involvement, and brachydactyly E-like changes. This condition has been described as spondyloperipheral dysplasia and the few published cases suggest autosomal dominant inheritance with considerable clinical variability. We found our sporadic case to be due to a collagen type II defect resulting from a specific COL2A1 mutation. This mutation is the first to be located at the C-terminal outside the helical domain of COL2A1. A frameshift as consequence of a 5 bp duplication in exon 51 leads to a stop codon. The resulting truncated C-propeptide region seemsmore » to affect helix formation and produces changes of chondrocyte morphology, collagen type II fibril structure and cartilage matrix composition. Our case with its distinct phenotype adds another chondrodysplasia to the clinical spectrum of type II collagenopathies. 16 refs., 4 figs.« less

  18. Collagen Accumulation in Osteosarcoma Cells lacking GLT25D1 Collagen Galactosyltransferase.

    PubMed

    Baumann, Stephan; Hennet, Thierry

    2016-08-26

    Collagen is post-translationally modified by prolyl and lysyl hydroxylation and subsequently by glycosylation of hydroxylysine. Despite the widespread occurrence of the glycan structure Glc(α1-2)Gal linked to hydroxylysine in animals, the functional significance of collagen glycosylation remains elusive. To address the role of glycosylation in collagen expression, folding, and secretion, we used the CRISPR/Cas9 system to inactivate the collagen galactosyltransferase GLT25D1 and GLT25D2 genes in osteosarcoma cells. Loss of GLT25D1 led to increased expression and intracellular accumulation of collagen type I, whereas loss of GLT25D2 had no effect on collagen secretion. Inactivation of the GLT25D1 gene resulted in a compensatory induction of GLT25D2 expression. Loss of GLT25D1 decreased collagen glycosylation by up to 60% but did not alter collagen folding and thermal stability. Whereas cells harboring individually inactivated GLT25D1 and GLT25D2 genes could be recovered and maintained in culture, cell clones with simultaneously inactive GLT25D1 and GLT25D2 genes could be not grown and studied, suggesting that a complete loss of collagen glycosylation impairs osteosarcoma cell proliferation and viability. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Membrane Type 1 Matrix Metalloproteinase Regulates Monocyte Migration and Collagen Destruction in Tuberculosis.

    PubMed

    Sathyamoorthy, Tarangini; Tezera, Liku B; Walker, Naomi F; Brilha, Sara; Saraiva, Luisa; Mauri, Francesco A; Wilkinson, Robert J; Friedland, Jon S; Elkington, Paul T

    2015-08-01

    Tuberculosis (TB) remains a global pandemic and drug resistance is rising. Multicellular granuloma formation is the pathological hallmark of Mycobacterium tuberculosis infection. The membrane type 1 matrix metalloproteinase (MT1-MMP or MMP-14) is a collagenase that is key in leukocyte migration and collagen destruction. In patients with TB, induced sputum MT1-MMP mRNA levels were increased 5.1-fold compared with matched controls and correlated positively with extent of lung infiltration on chest radiographs (r = 0.483; p < 0.05). M. tuberculosis infection of primary human monocytes increased MT1-MMP surface expression 31.7-fold and gene expression 24.5-fold. M. tuberculosis-infected monocytes degraded collagen matrix in an MT1-MMP-dependent manner, and MT1-MMP neutralization decreased collagen degradation by 73%. In human TB granulomas, MT1-MMP immunoreactivity was observed in macrophages throughout the granuloma. Monocyte-monocyte networks caused a 17.5-fold increase in MT1-MMP surface expression dependent on p38 MAPK and G protein-coupled receptor-dependent signaling. Monocytes migrating toward agarose beads impregnated with conditioned media from M. tuberculosis-infected monocytes expressed MT1-MMP. Neutralization of MT1-MMP activity decreased this M. tuberculosis network-dependent monocyte migration by 44%. Taken together, we demonstrate that MT1-MMP is central to two key elements of TB pathogenesis, causing collagen degradation and regulating monocyte migration. Copyright © 2015 The Authors.

  20. Controlled self assembly of collagen nanoparticle

    NASA Astrophysics Data System (ADS)

    Papi, Massimiliano; Palmieri, Valentina; Maulucci, Giuseppe; Arcovito, Giuseppe; Greco, Emanuela; Quintiliani, Gianluca; Fraziano, Maurizio; De Spirito, Marco

    2011-11-01

    In recent years carrier-mediated drug delivery has emerged as a powerful methodology for the treatment of various pathologies. The therapeutic index of traditional and novel drugs is enhanced via the increase of specificity due to targeting of drugs to a particular tissue, cell or intracellular compartment, the control over release kinetics, the protection of the active agent, or a combination of the above. Collagen is an important biomaterial in medical applications and ideal as protein-based drug delivery platform due to its special characteristics, such as biocompatibility, low toxicity, biodegradability, and weak antigenicity. While some many attempts have been made, further work is needed to produce fully biocompatible collagen hydrogels of desired size and able to release drugs on a specific target. In this article we propose a novel method to obtain spherical particles made of polymerized collagen surrounded by DMPC liposomes. The liposomes allow to control both the particles dimension and the gelling environment during the collagen polymerization. Furthermore, an optical based method to visualize and quantify each step of the proposed protocol is detailed and discussed.

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

    PubMed

    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.

  2. Rheology of heterotypic collagen networks.

    PubMed

    Piechocka, Izabela K; van Oosten, Anne S G; Breuls, Roel G M; Koenderink, Gijsje H

    2011-07-11

    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 the structure and rheology of networks of purified collagen I and V, combining fluorescence and atomic force microscopy, turbidimetry, and rheometry. We demonstrate that the network stiffness strongly decreases with increasing collagen V content, even though the network structure does not substantially change. We compare the rheological data with theoretical models for rigid polymers and find that the elasticity is dominated by nonaffine deformations. There is no analytical theory describing this regime, hampering a quantitative interpretation of the influence of collagen V. Our findings are relevant for understanding molecular origins of tissue biomechanics and for guiding rational design of collagenous biomaterials for biomedical applications.

  3. Imaging denatured collagen strands in vivo and ex vivo via photo-triggered hybridization of caged collagen mimetic peptides.

    PubMed

    Li, Yang; Foss, Catherine A; Pomper, Martin G; Yu, S Michael

    2014-01-31

    Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i) to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

  4. Lipo-PGE1 suppresses collagen production in human dermal fibroblasts via the ERK/Ets-1 signaling pathway.

    PubMed

    Yang, Yoolhee; Kim, Hee Jung; Woo, Kyong-Je; Cho, Daeho; Bang, Sa Ik

    2017-01-01

    Dysregulation of collagen production contributes to various pathological processes, including tissue fibrosis as well as impaired wound healing. Lipo-prostaglandin E1 (Lipo-PGE1), a lipid microsphere-incorporated prostaglandin E1, is used as a vasodilator for the treatment of peripheral vascular diseases. Lipo-PGE1 was recently shown to enhance human dermal fibroblast (HDF) migration and in vivo wound healing. No published study has characterized the role of Lipo-PGE1 in collagen regulation in HDFs. Here, we investigated the cellular signaling mechanism by which Lipo-PGE1 regulates collagen in HDFs. Collagen production was evaluated by the Sircol collagen assay, Western blot analysis of type I collagen and real time PCR. Unexpectedly, Lipo-PGE1 decreased mRNA expression of collagen 1A1, 1A2, and 3A1. Lipo-PGE1 markedly inhibited type I collagen and total soluble collagen production. In addition, Lipo-PGE1 inhibited transforming growth factor-β-induced collagen expression via Smad2 phosphorylation. To further investigate whether extracellular signal-regulated kinase (ERK)/Ets-1 signaling, a crucial pathway in collagen regulation, is involved in Lipo-PGE1-inhibited collagen production, cells were pretreated with an ERK-specific inhibitor, PD98059, prior to the addition of Lipo-PGE1. Lipo-PGE1-inhibited collagen mRNA expression and total soluble collagen production were recovered by pretreatment with PD98059. Moreover, Lipo-PGE1 directly induced the phosphorylation of ERK. Furthermore, silencing of Ets-1 recovered Lipo-PGE1-inhibited collagen production and PD98059 blocked Lipo-PGE1-enhanced Ets-1 expression. The present study reveals an important role for Lipo-PGE1 as a negative regulator of collagen gene expression and production via ERK/Ets-1 signaling. These results suggest that Lipo-PGE1 could potentially be a therapeutic target in diseases with deregulated collagen turnover.

  5. Mechanoregulation of human articular chondrocyte aggrecan and type II collagen expression by intermittent hydrostatic pressure in vitro.

    PubMed

    Ikenoue, Takashi; Trindade, Michael C D; Lee, Mel S; Lin, Eric Y; Schurman, David J; Goodman, Stuart B; Smith, R Lane

    2003-01-01

    This study addressed the hypothesis that duration and magnitude of applied intermittent hydrostatic pressure (IHP) are critical parameters in regulation of normal human articular chondrocyte aggrecan and type II collagen expression. Articular chondrocytes were isolated from knee cartilage and maintained as primary, high-density monolayer cultures. IHP was applied at magnitudes of 1, 5 and 10 MPa at 1 Hz for durations of either 4 h per day for one day (4 x 1) or 4 h per day for four days (4 x 4). Total cellular RNA was isolated and analyzed for aggrecan and type II collagen mRNA signal levels using specific primers and reverse transcription polymerase chain reaction (RT-PCR) nested with beta-actin primers as internal controls. With a 4x1 loading regimen, aggrecan mRNA signal levels increased 1.3- and 1.5-fold at 5 and 10 MPa, respectively, relative to beta-actin mRNA when compared to unloaded cultures. Changing the duration of loading to a 4x4 regimen increased aggrecan mRNA signal levels by 1.4-, 1.8- and 1.9-fold at loads of 1, 5 and 10 MPa, respectively. In contrast to the effects of IHP on aggrecan, type II collagen mRNA signal levels were only upregulated at loads of 5 and 10 MPa with the 4x4 loading regimen. Analysis of cell-associated protein by western blotting confirmed that IHP increased aggrecan and type II collagen in chondrocyte extracts. These data demonstrate that duration and magnitude of applied IHP differentially alter chondrocyte matrix protein expression. The results show that IHP provides an important stimulus for increasing cartilage matrix anabolism and may contribute to repair and regeneration of damaged or diseased cartilage.

  6. Engineering zonal cartilage through bioprinting collagen type II hydrogel constructs with biomimetic chondrocyte density gradient.

    PubMed

    Ren, Xiang; Wang, Fuyou; Chen, Cheng; Gong, Xiaoyuan; Yin, Li; Yang, Liu

    2016-07-20

    Cartilage tissue engineering is a promising approach for repairing and regenerating cartilage tissue. To date, attempts have been made to construct zonal cartilage that mimics the cartilaginous matrix in different zones. However, little attention has been paid to the chondrocyte density gradient within the articular cartilage. We hypothesized that the chondrocyte density gradient plays an important role in forming the zonal distribution of extracellular matrix (ECM). In this study, collagen type II hydrogel/chondrocyte constructs were fabricated using a bioprinter. Three groups were created according to the total cell seeding density in collagen type II pre-gel: Group A, 2 × 10(7) cells/mL; Group B, 1 × 10(7) cells/mL; and Group C, 0.5 × 10(7) cells/mL. Each group included two types of construct: one with a biomimetic chondrocyte density gradient and the other with a single cell density. The constructs were cultured in vitro and harvested at 0, 1, 2, and 3 weeks for cell viability testing, reverse-transcription quantitative PCR (RT-qPCR), biochemical assays, and histological analysis. We found that total ECM production was positively correlated with the total cell density in the early culture stage, that the cell density gradient distribution resulted in a gradient distribution of ECM, and that the chondrocytes' biosynthetic ability was affected by both the total cell density and the cell distribution pattern. Our results suggested that zonal engineered cartilage could be fabricated by bioprinting collagen type II hydrogel constructs with a biomimetic cell density gradient. Both the total cell density and the cell distribution pattern should be optimized to achieve synergistic biological effects.

  7. Perinatal collagen turnover markers in intrauterine growth restriction.

    PubMed

    Gourgiotis, Demetrios; Briana, Despina D; Georgiadis, Anestis; Boutsikou, Maria; Baka, Stavroula; Marmarinos, Antonios; Hassiakos, Demetrios; Malamitsi-Puchner, Ariadne

    2012-09-01

    To investigate bone and connective tissue collagen turnover in intrauterine growth restricted (IUGR) pregnancies, by determining circulating markers of type I collagen synthesis (carboxy-terminal propeptide of type I procollagen [PICP], representing bone formation) and degradation (cross-linked telopeptide of type I collagen [ICTP], representing bone resorption) as well as type III collagen synthesis (N-terminal propeptide of type-III procollagen [PIIINP], reflecting growth and tissue maturity). Plasma PICP, ICTP and PIIINP concentrations were measured in 40 mothers and their 20 asymmetric IUGR and 20 appropriate for gestational age (AGA) full-term fetuses and neonates on postnatal day 1-(N1) and 4-(N4). Fetal PICP, fetal and N4 ICTP, as well as fetal, N1 and N4 PIIINP concentrations were higher in the IUGR group (p ≤ 0.038, in all cases). In both groups, maternal PICP, ICTP and PIIINP concentrations were lower than fetal, N1 and N4 ones (p<0.001, in each case). Type I collagen turnover is enhanced in IUGR than AGA fetuses/neonates. Similarly, fetal/neonatal PIIINP concentrations are elevated in IUGR, probably due to stress, responsible for induction of tissue maturation, and/or to impaired excretory renal function, leading to reduced protein clearance. Fetal/neonatal PICP, ICTP and PIIINP concentrations are higher than maternal concentrations, possibly reflecting increased skeletal growth and collagen turnover in the former.

  8. Fibroblast activation protein is induced by inflammation and degrades type I collagen in thin-cap fibroatheromata

    PubMed Central

    Brokopp, Chad E.; Schoenauer, Roman; Richards, Peter; Bauer, Stefan; Lohmann, Christine; Emmert, Maximilian Y.; Weber, Benedikt; Winnik, Stephan; Aikawa, Elena; Graves, Kirk; Genoni, Michele; Vogt, Peter; Lüscher, Thomas F.; Renner, Christoph; Hoerstrup, Simon P.; Matter, Christian M.

    2011-01-01

    Aims Collagen degradation in atherosclerotic plaques with thin fibrous caps renders them more prone to rupture. Fibroblast activation protein (FAP) plays a role in arthritis and tumour formation through its collagenase activity. However, the significance of FAP in thin-cap human fibroatheromata remains unknown. Methods and results We detected enhanced FAP expression in type IV–V human aortic atheromata (n = 12), compared with type II–III lesions (n = 9; P < 0.01) and healthy aortae (n = 8; P < 0.01) by immunostaining and western blot analyses. Fibroblast activation protein was also increased in thin-cap (<65 µm) vs. thick-cap (≥65 µm) human coronary fibroatheromata (n = 12; P < 0.01). Fibroblast activation protein was expressed by human aortic smooth muscle cells (HASMC) as shown by colocalization on immunofluorescent aortic plaque stainings (n = 10; P < 0.01) and by flow cytometry in cell culture. Although macrophages did not express FAP, macrophage burden in human aortic plaques correlated with FAP expression (n = 12; R2= 0.763; P < 0.05). Enzyme-linked immunosorbent assays showed a time- and dose-dependent up-regulation of FAP in response to human tumour necrosis factor α (TNFα) in HASMC (n = 6; P < 0.01). Moreover, supernatants from peripheral blood-derived macrophages induced FAP expression in cultured HASMC (n = 6; P < 0.01), an effect abolished by blocking TNFα (n = 6; P < 0.01). Fibroblast activation protein associated with collagen-poor regions in human coronary fibrous caps and digested type I collagen and gelatin in vitro (n = 6; P < 0.01). Zymography revealed that FAP-mediated collagenase activity was neutralized by an antibody directed against the FAP catalytic domain both in HASMC (n = 6; P < 0.01) and in fibrous caps of atherosclerotic plaques (n = 10; P < 0.01). Conclusion Fibroblast activation protein expression in HASMC is induced by macrophage-derived TNFα. Fibroblast activation protein associates with thin-cap human coronary

  9. Collagen type I and III synthesis by Tenon's capsule fibroblasts in culture: individual patient characteristics and response to mitomycin C, 5-fluorouracil, and ascorbic acid.

    PubMed Central

    Gross, R L

    1999-01-01

    PURPOSE: This study was performed to better understand the differences between patients in specific components of wound healing as it may pertain to glaucoma filtration surgery, including the use of antimetabolites. METHODS: Human Tenon's capsule fibroblasts were obtained at the time of glaucoma filtering surgery and established in individual cell cultures from 35 glaucoma patients. The dose-response to 5-fluorouracil (5FU) and mitomycin C (MMC) was determined. The individual cell lines were exposed to the antimetabolites and ascorbic acid with measurement of collagen type I and III production by an ELISA-type dot blot assay. These results were then statistically compared to the individual patient characteristics including age, race, previous surgery and medications, and type of glaucoma. RESULTS: 5-FU had little effect on collagen type I and III production or protein synthesis. MMC had an inhibitory effect on collagen secretion and total protein synthesis with increasing concentration. Photomicrographs of the cells after each treatment condition revealed characteristic morphologic changes when compared to controls. There was a large range of collagen type I and III production with correlation between the amounts of each collagen type secreted in response to the antimetabolites. However, there was no correlation with accepted risk factors for filtration failure. CONCLUSION: These antimetabolites act similarly on different cell lines in a nonspecific manner. The results suggest that the increased risk of filtration failure due to age, race, diagnosis, and previous conjunctival surgery is not due to differences in secretion of collagen types I and III by Tenon's capsule fibroblasts. Images FIGURE 3 PMID:10703140

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

    PubMed Central

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

    2015-01-01

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

  11. The response to oestrogen deprivation of the cartilage collagen degradation marker, CTX-II, is unique compared with other markers of collagen turnover

    PubMed Central

    Bay-Jensen, Anne-Christine; Tabassi, Nadine CB; Sondergaard, Lene V; Andersen, Thomas L; Dagnaes-Hansen, Frederik; Garnero, Patrick; Kassem, Moustapha; Delaissé, Jean-Marie

    2009-01-01

    Introduction The urinary level of the type II collagen degradation marker CTX-II is increased in postmenopausal women and in ovariectomised rats, suggesting that oestrogen deprivation induces cartilage breakdown. Here we investigate whether this response to oestrogen is also true for other type II collagen turnover markers known to be affected in osteoarthritis, and whether it relates to its presence in specific areas of cartilage tissue. Methods The type II collagen degradation markers CTX-II and Helix-II were measured in the body fluids of premenopausal and postmenopausal women and in those of ovariectomised rats receiving oestrogen or not. Levels of PIIANP, a marker of type II collagen synthesis, were also measured in rats. Rat knee cartilage was analysed for immunoreactivity of CTX-II and PIIANP and for type II collagen expression. Results As expected, urinary levels of CTX-II are significantly increased in postmenopausal women and also in oestrogen-deprived rats, although only transiently. However, in neither case were these elevations paralleled by a significant increase of Helix-II levels and PIIANP levels did not change at any time. CTX-II immunoreactivity and collagen expression were detected in different cartilage areas. The upper zone is the area where CTX-II immunoreactivity and collagen expression best reflected the differences in urinary levels of CTX-II measured in response to oestrogen. However, correlations between urinary levels of CTX-II and tissue immunostainings in individual rats were not statistically significant. Conclusions We found only a small effect of oestrogen deprivation on cartilage. It was detected by CTX-II, but not by other type II collagen turnover markers typically affected in osteoarthritis. PMID:20527083

  12. A preliminary study of the effects of an extract of Ligularia fischeri leaves on type II collagen-induced arthritis in DBA/1J mice.

    PubMed

    Choi, Eun Mi; Kim, Young Ho

    2008-01-01

    The present study was undertaken to determine whether Ligularia fischeri leaf extract (LF) is efficacious against collagen-induced arthritis (CIA) in mice. DBA/1J mice were immunized with bovine type II collagen and treated with LF (100 and 200 mg/kg) for 49 days. Mice were assessed regularly for signs of arthritis and the levels of rheumatoid factor, anti-type II collagen antibody, cytokines, AST, ALT, and creatinine in serum were also examined after the animals were killed. The arthritis score and paw edema were markedly suppressed in the groups treated with LF. Moreover, levels of rheumatoid factor, anti-type II collagen antibody, tumor necrosis factor-alpha, interleukin (IL)-1, and IL-6 in sera were reduced by LF administration. These data suggest that L. fischeri might be effective for the treatment of inflammatory arthritis like human rheumatoid arthritis.

  13. Effects of collagen and collagen hydrolysate from jellyfish umbrella on histological and immunity changes of mice photoaging.

    PubMed

    Fan, Jian; Zhuang, Yongliang; Li, Bafang

    2013-01-17

    Jellyfish collagen (JC) was extracted from jellyfish umbrella and hydrolyzed to prepare jellyfish collagen hydrolysate (JCH). The effects of JC and JCH on UV-induced skin damage of mice were evaluated by the skin moisture, microscopic analyses of skin and immunity indexes. The skin moisture analyses showed that moisture retention ability of UV-induced mice skin was increased by JC and JCH. Further histological analysis showed that JC and JCH could repair the endogenous collagen and elastin protein fibers, and could maintain the natural ratio of type I to type III collagen. The immunity indexes showed that JC and JCH play a role in enhancing immunity of photoaging mice in vivo. JCH showed much higher protective ability than JC. These results suggest that JCH as a potential novel antiphotoaging agent from natural resources.

  14. Effects of Collagen and Collagen Hydrolysate from Jellyfish Umbrella on Histological and Immunity Changes of Mice Photoaging

    PubMed Central

    Fan, Jian; Zhuang, Yongliang; Li, Bafang

    2013-01-01

    Jellyfish collagen (JC) was extracted from jellyfish umbrella and hydrolyzed to prepare jellyfish collagen hydrolysate (JCH). The effects of JC and JCH on UV-induced skin damage of mice were evaluated by the skin moisture, microscopic analyses of skin and immunity indexes. The skin moisture analyses showed that moisture retention ability of UV-induced mice skin was increased by JC and JCH. Further histological analysis showed that JC and JCH could repair the endogenous collagen and elastin protein fibers, and could maintain the natural ratio of type I to type III collagen. The immunity indexes showed that JC and JCH play a role in enhancing immunity of photoaging mice in vivo. JCH showed much higher protective ability than JC. These results suggest that JCH as a potential novel antiphotoaging agent from natural resources. PMID:23344251

  15. Transdermal Delivery of Functional Collagen Via Polyvinylpyrrolidone Microneedles

    PubMed Central

    Sun, Wenchao; Inayathullah, Mohammed; Manoukian, Martin A. C.; Malkovskiy, Andrey V.; Manickam, Sathish; Marinkovich, M. Peter; Lane, Alfred T.; Tayebi, Lobat; Seifalian, Alexander M.; Rajadas, Jayakumar

    2017-01-01

    Collagen makes up a large proportion of the human body, particularly the skin. As the body ages, collagen content decreases, resulting in wrinkled skin and decreased wound healing capabilities. This paper presents a method of delivering type I collagen into porcine and human skin utilizing a polyvinylpyrrolidone microneedle delivery system. The microneedle patches were made with concentrations of 1, 2, 4, and 8% type I collagen (w/w). Microneedle structures and the distribution of collagen were characterized using scanning electron microscopy and confocal microscopy. Patches were then applied on the porcine and human skin, and their effectiveness was examined using fluorescence microscopy. The results illustrate that this microneedle delivery system is effective in delivering collagen I into the epidermis and dermis of porcine and human skin. Since the technique presented in this paper is quick, safe, effective and easy, it can be considered as a new collagen delivery method for cosmetic and therapeutic applications. PMID:26066056

  16. Misbalance in type III collagen formation/degradation as a novel serological biomarker for penetrating (Montreal B3) Crohn's disease.

    PubMed

    van Haaften, W T; Mortensen, J H; Karsdal, M A; Bay-Jensen, A C; Dijkstra, G; Olinga, P

    2017-07-01

    Misbalances in extracellular matrix turnover are key factors in the development of stricturing (Montreal B2) and penetrating (Montreal B3) Crohn's disease. To determine whether serological markers for collagen formation and degradation could serve as biomarkers for complications of Crohn's disease. Serum biomarkers for type I, III, V and VI collagen formation (P1NP, Pro-C3, Pro-C5, Pro-C6) and matrix metalloproteinase mediated degradation (C1M, C3M, C5M and C6M) were measured in a retrospective, single centre cohort of 112 patients with Crohn's disease in the terminal ileum (nonstricturing/nonpenetrating: n=40, stricturing: n=55, penetrating: n=17) and 24 healthy controls. Active inflammation was defined as CRP >5 mg/L. C3M and Pro-C5 levels were higher in penetrating vs nonpenetrating/nonstricturing and stricturing disease (33.6±5 vs 25.8±2.2 [P=.004] and 27.2±2.3 [P=.018] nmol/L C3M, 1262.7±259.4 vs 902.9±109.9 [P=.005] and 953.0±106.4 [P=.015] nmol/L Pro-C5). C1M (71.2±26.1 vs 46.2±6.2 nmol/L [P<.001]), C3M (31.6±3.9 vs 26.1±1.6 nmol/L [P=.002] and Pro-C5 levels (1171.7±171.5 vs 909.6±80.4 nmol/L [P=.002]) were higher in patients with active inflammation vs without active inflammation. Pro-C3/C3M-ratios were best to differentiate between penetrating vs nonstricturing/nonpenetrating and stricturing disease with area under the curves of 0.815±0.109 (P<.001) and 0.746±0.114 (P=.002) respectively. Serological biomarkers show that penetrating Crohn's disease is characterised by increased matrix metalloproteinase-9 degraded type III collagen and formation of type V collagen. Active inflammation in Crohn's disease is characterised by increased formation of type V collagen and increased matrix metalloproteinase mediated breakdown of type I, III collagen. Pro-C3/C3M ratios are superior in differentiating between penetrating Crohn's disease vs inflammatory and stricturing Crohn's disease. © 2017 The Authors. Alimentary Pharmacology and Therapeutics

  17. α3 Chains of type V collagen regulate breast tumour growth via glypican-1

    PubMed Central

    Huang, Guorui; Ge, Gaoxiang; Izzi, Valerio; Greenspan, Daniel S.

    2017-01-01

    Pericellular α3(V) collagen can affect the functioning of cells, such as adipocytes and pancreatic β cells. Here we show that α3(V) chains are an abundant product of normal mammary gland basal cells, and that α3(V) ablation in a mouse mammary tumour model inhibits mammary tumour progression by reducing the proliferative potential of tumour cells. These effects are shown to be primarily cell autonomous, from loss of α3(V) chains normally produced by tumour cells, in which they affect growth by enhancing the ability of cell surface proteoglycan glypican-1 to act as a co-receptor for FGF2. Thus, a mechanism is presented for microenvironmental influence on tumour growth. α3(V) chains are produced in both basal-like and luminal human breast tumours, and its expression levels are tightly coupled with those of glypican-1 across breast cancer types. Evidence indicates α3(V) chains as potential targets for inhibiting tumour growth and as markers of oncogenic transformation. PMID:28102194

  18. Collagen Scaffolds in Bone Sialoprotein-Mediated Bone Regeneration

    PubMed Central

    Kruger, Thomas E.; Miller, Andrew H.; Wang, Jinxi

    2013-01-01

    Decades of research in bioengineering have resulted in the development of many types of 3-dimentional (3D) scaffolds for use as drug delivery systems (DDS) and for tissue regeneration. Scaffolds may be comprised of different natural fibers and synthetic polymers as well as ceramics in order to exert the most beneficial attributes including biocompatibility, biodegradability, structural integrity, cell infiltration and attachment, and neovascularization. Type I collagen scaffolds meet most of these criteria. In addition, type I collagen binds integrins through RGD and non-RGD sites which facilitates cell migration, attachment, and proliferation. Type I collagen scaffolds can be used for bone tissue repair when they are coated with osteogenic proteins such as bone morphogenic protein (BMP) and bone sialoprotein (BSP). BSP, a small integrin-binding ligand N-linked glycoprotein (SIBLING), has osteogenic properties and plays an essential role in bone formation. BSP also mediates mineral deposition, binds type I collagen with high affinity, and binds αvβ 3 and αvβ 5 integrins which mediate cell signaling. This paper reviews the emerging evidence demonstrating the efficacy of BSP-collagen scaffolds in bone regeneration. PMID:23653530

  19. Collagen scaffolds in bone sialoprotein-mediated bone regeneration.

    PubMed

    Kruger, Thomas E; Miller, Andrew H; Wang, Jinxi

    2013-01-01

    Decades of research in bioengineering have resulted in the development of many types of 3-dimentional (3D) scaffolds for use as drug delivery systems (DDS) and for tissue regeneration. Scaffolds may be comprised of different natural fibers and synthetic polymers as well as ceramics in order to exert the most beneficial attributes including biocompatibility, biodegradability, structural integrity, cell infiltration and attachment, and neovascularization. Type I collagen scaffolds meet most of these criteria. In addition, type I collagen binds integrins through RGD and non-RGD sites which facilitates cell migration, attachment, and proliferation. Type I collagen scaffolds can be used for bone tissue repair when they are coated with osteogenic proteins such as bone morphogenic protein (BMP) and bone sialoprotein (BSP). BSP, a small integrin-binding ligand N-linked glycoprotein (SIBLING), has osteogenic properties and plays an essential role in bone formation. BSP also mediates mineral deposition, binds type I collagen with high affinity, and binds α v β 3 and α v β 5 integrins which mediate cell signaling. This paper reviews the emerging evidence demonstrating the efficacy of BSP-collagen scaffolds in bone regeneration.

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

    PubMed

    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.

  1. [Collagens: why such a structural complexity?].

    PubMed

    Borel, J P; Monboisse, J C

    1993-01-01

    The collagens are a family of extracellular fibrillar proteins, characterized by the presence of one or several domains termed "triple helix", that are made of three polypeptide chains folded around each other. They elicit a huge worldwide research activity, marked every year by the publishing of dozens of books and thousands of papers. This family is presently represented by more than 16 individualized types, all differing by their molecular structure and by the way helical and globular domains are arranged. In any case, however, at least one triple helical domain exists. It is formed by the association of three polypeptide chains, each of them containing a glycine every three residues and many proline or hydroxyproline residues, and attests for the belonging of the protein to the collagen group. These multiple molecular forms and their specific architecture raise questions that remain unsolved. Why is this triple helix structure adopted in the case of collagens? Is it because the simple alpha helix of protein cannot extend over more than a few nanometers and is not solid enough? Why not a double helix like that of DNA? It would probably not be rigid enough. Why are there many globular domains interspersed between fibrillar ones? Probably these domains are useful for the association of peptide chains in register prior to their folding, then they participate in the transport of the elementary molecules from the synthesizing cells to their final place in the connective tissue and, finally, they insert the molecules into their specific place inside the growing fibrils. Collagen fibres as they are evidenced by histological methods, for instance in tendons, are of complex structure. Most of their constituting sub-units are type I tropocollagen molecules but they also contain in their center a filament of type V collagen that seems to serve as a guide during their edification. On the surface of the fibres are molecules of type III collagen that limit the growth in

  2. Improving nanoparticle diffusion through tumor collagen matrix by photo-thermal gold nanorods

    NASA Astrophysics Data System (ADS)

    Raeesi, Vahid; Chan, Warren C. W.

    2016-06-01

    Collagen (I) impairs the targeting of nanoparticles to tumor cells by obstructing their diffusion inside dense tumor interstitial matrix. This potentially makes large nanoparticles (>50 nm) reside near the tumor vessels and thereby compromises their functionality. Here we propose a strategy to locally improve nanoparticle transport inside collagen (I) component of the tumor tissue. We first used heat generating gold nanorods to alter collagen (I) matrix by local temperature elevation. We then explored this impact on the transport of 50 nm and 120 nm inorganic nanoparticles inside collagen (I). We demonstrated an increase in average diffusivity of 50 nm and 120 nm in the denatured collagen (I) by ~14 and ~21 fold, respectively, compared to intact untreated collagen (I) matrix. This study shows how nanoparticle-mediated hyperthermia inside tumor tissue can improve the transport of large nanoparticles through collagen (I) matrix. The ability to increase nanoparticles diffusion inside tumor stroma allows their targeting or other functionalities to take effect, thereby significantly improving cancer therapeutic or diagnostic outcome.Collagen (I) impairs the targeting of nanoparticles to tumor cells by obstructing their diffusion inside dense tumor interstitial matrix. This potentially makes large nanoparticles (>50 nm) reside near the tumor vessels and thereby compromises their functionality. Here we propose a strategy to locally improve nanoparticle transport inside collagen (I) component of the tumor tissue. We first used heat generating gold nanorods to alter collagen (I) matrix by local temperature elevation. We then explored this impact on the transport of 50 nm and 120 nm inorganic nanoparticles inside collagen (I). We demonstrated an increase in average diffusivity of 50 nm and 120 nm in the denatured collagen (I) by ~14 and ~21 fold, respectively, compared to intact untreated collagen (I) matrix. This study shows how nanoparticle-mediated hyperthermia inside

  3. Analysis of the major epitope of the alpha2 chain of bovine type I collagen in children with bovine gelatin allergy.

    PubMed

    Hori, Hisae; Hattori, Shunji; Inouye, Sakae; Kimura, Akinori; Irie, Shinkichi; Miyazawa, Hiroshi; Sakaguchi, Masahiro

    2002-10-01

    Anaphylaxis to measles, mumps, and rubella vaccines has been reported. It has been found that most of these reactions to live vaccines are caused by type I allergy with the bovine gelatin present in the vaccines as an allergen. Gelatin mainly includes denatured type I collagen, which consists of alpha1 and alpha2 chains. We previously reported that allergic reactions to gelatin are caused by the type I collagen alpha2 (alpha2[I]) chain. To aid in the development of gelatin that has little or no allergenicity in human subjects, we investigated epitopes of bovine alpha2(I) chain with use of IgE in gelatin-sensitive children. Serum samples were collected from 15 patients who had systemic allergic reactions to vaccines and high levels of specific IgE to bovine gelatin. Eleven overlapping recombinant proteins that cover bovine alpha2(I) were prepared with a bacterial expression vector. We examined IgE reactivity to these recombinant proteins by means of ELISA. Fifteen peptides covering a major reactive recombinant protein were synthesized. The IgE-reacting epitope was identified by means of IgE-ELISA inhibition with these synthetic peptides and pooled serum from the patients. We found that of the 15 patients, 13 showed IgE reactivity to a recombinant protein (no. 3) spanning the central region of the collagenous domain ((418)Gly-(662)Pro). Furthermore, all 13 patients showed IgE reactivity to the 4-kd recombinant protein (no. 3a) spanning the region from (461)Pro to (500)Glu. In IgE-ELISA inhibition we found that a minimum IgE epitope of gelatin allergen was composed of the 10-amino-acid sequence (485)Ile-Pro-Gly-Glu-Phe-Gly-Leu-Pro-Gly-Pro(494). This sequence is not observed in the human type I collagen alpha1 and alpha2 chains, nor is it found in the bovine type I collagen alpha1 chain. We found that Ile-Pro-Gly-Glu-Phe-Gly-Leu-Pro-Gly-Pro is a major IgE epitope of the alpha2 chain of bovine type I collagen in patients with gelatin allergy. The degree of anaphylaxis

  4. Collagen Type I Selectively Activates Ectodomain Shedding of the Discoidin Domain Receptor 1: Involvement of Src Tyrosine Kinase

    PubMed Central

    Slack, Barbara E.; Siniaia, Marina S.; Blusztajn, Jan K.

    2008-01-01

    The discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that is highly expressed in breast carcinoma cells. Upon binding to collagen, DDR1 undergoes autophosphorylation followed by limited proteolysis to generate a tyrosine phosphorylated C-terminal fragment (CTF). Although it was postulated that this fragment is formed as a result of shedding of the N-terminal ectodomain, collagen-dependent release of the DDR1 extracellular domain has not been demonstrated. We now report that, in conjunction with CTF formation, collagen type I stimulates concentration-dependent, saturable shedding of the DDR1 ectodomain from two carcinoma cell lines, and from transfected cells. In contrast, collagen did not promote cleavage of other transmembrane proteins including the amyloid precursor protein (APP), ErbB2, and E-cadherin. Collagen-dependent tyrosine phosphorylation and proteolysis of DDR1 in carcinoma cells were reduced by a pharmacologic Src inhibitor. Moreover, expression of a dominant negative Src mutant protein in human embryonic kidney cells inhibited collagen-dependent phosphorylation and shedding of co-transfected DDR1. The hydroxamate-based metalloproteinase inhibitor TAPI-1 (tumor necrosis factor-α protease inhibitor-1), and tissue inhibitor of metalloproteinase (TIMP)-3, also blocked collagen-evoked DDR1 shedding, but did not reduce levels of the phosphorylated CTF. Neither shedding nor CTF formation were affected by the γ-secretase inhibitor, L-685,458. The results demonstrate that collagen-evoked ectodomain cleavage of DDR1 is mediated in part by Src-dependent activation or recruitment of a matrix- or disintegrin metalloproteinase, and that CTF formation can occur independently of ectodomain shedding. Delayed shedding of the DDR1 ectodomain may represent a mechanism that limits DDR1-dependent cell adhesion and migration on collagen matrices. PMID:16440311

  5. Evaluation effect of low level Helium-Neon laser and Iranian propolis extract on Collagen Type I gene expression by human gingival fibroblasts: an in vitro study.

    PubMed

    Eslami, Hosein; Motahari, Paria; Safari, Ebrahim; Seyyedi, Maryam

    2017-06-30

    production of collagen by fibroblast cells is a key component in wound healing. Several studies have shown that low level laser therapy (LLLT) and propolis extract stimulate collagen Type I production. The aim of this study is to evaluation the combined effect of LLL helium neon (632.8 nm) and Iranian propolis extract on collagen Type I gene expression by human gingival fibroblasts (HGF3-PI 53). Human gingival fibroblasts after culturing divided into six experimental groups: G1-control group, which received no irradiation and propolis extract, G2-irradiated at1.5 J/cm 2 , G3-irradiated at 0.15 J/cm 2 , G4-recived extract of propolis, G5- combined extract of propolis and 1.5 J/cm 2 laser irradiation and G6- combined extract of propolis and 0.15 J/cm 2 laser irradiation. The experiments were conducted in triplicate. After 24 hour, the total RNA was extracted and cDNA synthesis was performed. Type I collagen mRNA expression was determined with real time PCR. The obtained results illustrated a statistically significant difference between G3 (0.15 J/cm 2 ) and G1 (control group) in levels of collagen Type I messenger RNA (mRNA) expression (p<0.05). The irradiated cells showed a 1.4 times increase in mRNA expression of the collagen Type I gene. Expression of this gene decreases in other groups that this difference was statistically significant. LLLT in different dosage and propolis extract may result in decreased or increased collagen type I gene expression. However this effect should be investigated in clinical studies.

  6. Evaluation effect of low level Helium-Neon laser and Iranian propolis extract on Collagen Type I gene expression by human gingival fibroblasts: an in vitro study

    PubMed Central

    Eslami, Hosein; Motahari, Paria; Safari, Ebrahim; Seyyedi, Maryam

    2017-01-01

    Back ground and aim production of collagen by fibroblast cells is a key component in wound healing. Several studies have shown that low level laser therapy (LLLT) and propolis extract stimulate collagen Type I production. The aim of this study is to evaluation the combined effect of LLL helium neon (632.8 nm) and Iranian propolis extract on collagen Type I gene expression by human gingival fibroblasts (HGF3-PI 53). Methods and materials Human gingival fibroblasts after culturing divided into six experimental groups: G1-control group, which received no irradiation and propolis extract, G2-irradiated at1.5 J/cm2, G3-irradiated at 0.15 J/cm2, G4-recived extract of propolis, G5- combined extract of propolis and 1.5 J/cm2 laser irradiation and G6- combined extract of propolis and 0.15 J/cm2 laser irradiation. The experiments were conducted in triplicate. After 24 hour, the total RNA was extracted and cDNA synthesis was performed. Type I collagen mRNA expression was determined with real time PCR. Results The obtained results illustrated a statistically significant difference between G3 (0.15 J/cm2) and G1 (control group) in levels of collagen Type I messenger RNA (mRNA) expression (p<0.05). The irradiated cells showed a 1.4 times increase in mRNA expression of the collagen Type I gene. Expression of this gene decreases in other groups that this difference was statistically significant. Conclusion LLLT in different dosage and propolis extract may result in decreased or increased collagen type I gene expression. However this effect should be investigated in clinical studies. PMID:28785130

  7. Design and validation of an immunoaffinity LC-MS/MS assay for the quantification of a collagen type II neoepitope peptide in human urine: application as a biomarker of osteoarthritis.

    PubMed

    Nemirovskiy, Olga; Li, Wenlin Wendy; Szekely-Klepser, Gabriella

    2010-01-01

    Biomarkers play an increasingly important role for drug efficacy and safety evaluation in all stages of drug development. It is especially important to develop and validate sensitive and selective biomarkers for diseases where the onset of the disease is very slow and/or the disease progression is hard to follow, i.e., osteoarthritis (OA). The degradation of Type II collagen has been associated with the disease state of OA. Matrix metalloproteinases (MMPs) are enzymes that catalyze the degradation of collagen and therefore pursued as potential targets for the treatment of OA. Peptide biomarkers of MMP activity related to type II collagen degradation were identified and the presence of these peptides in MMP digests of human articular cartilage (HAC) explants and human urine were confirmed. An immunoaffinity LC/MS/MS assay for the quantification of the most abundant urinary type II collagen neoepitope (uTIINE) peptide, a 45-mer with 5 HO-proline residues was developed and clinically validated. The assay has subsequently been applied to analyze human urine samples from clinical studies. We have shown that the assay is able to differentiate between symptomatic OA and normal subjects, indicating that uTIINE can be used as potential biomarker for OA. This chapter discusses the assay procedure and provides information on the validation experiments used to evaluate the accuracy, precision, and selectivity data with attention to the specific challenges related to the quantification of endogenous protein/peptide biomarker analytes. The generalized approach can be used as a follow-up to studies whereby proteomics-based urinary biomarkers are identified and an assay needs to be developed. Considerations for the validation of such an assay are described.

  8. Consortium for Osteogenesis Imperfecta Mutations in the Helical Domain of Type I Collagen: Regions Rich in Lethal Mutations Align With Collagen Binding Sites for Integrins and Proteoglycans

    PubMed Central

    Marini, Joan C.; Forlino, Antonella; Cabral, Wayne A.; Barnes, Aileen M.; San Antonio, James D.; Milgrom, Sarah; Hyland, James C.; Körkkö, Jarmo; Prockop, Darwin J.; De Paepe, Anne; Coucke, Paul; Symoens, Sofie; Glorieux, Francis H.; Roughley, Peter J.; Lund, Alan M.; Kuurila-Svahn, Kaija; Hartikka, Heini; Cohn, Daniel H.; Krakow, Deborah; Mottes, Monica; Schwarze, Ulrike; Chen, Diana; Yang, Kathleen; Kuslich, Christine; Troendle, James; Dalgleish, Raymond; Byers, Peter H.

    2014-01-01

    Osteogenesis imperfecta (OI) is a generalized disorder of connective tissue characterized by fragile bones and easy susceptibility to fracture. Most cases of OI are caused by mutations in type I collagen. We have identified and assembled structural mutations in type I collagen genes (COL1A1 and COL1A2, encoding the proα1(I) and proα2(I) chains, respectively) that result in OI. Quantitative defects causing type I OI were not included. Of these 832 independent mutations, 682 result in substitution for glycine residues in the triple helical domain of the encoded protein and 150 alter splice sites. Distinct genotype–phenotype relationships emerge for each chain. One-third of the mutations that result in glycine substitutions in α1(I) are lethal, especially when the substituting residues are charged or have a branched side chain. Substitutions in the first 200 residues are nonlethal and have variable outcome thereafter, unrelated to folding or helix stability domains. Two exclusively lethal regions (helix positions 691–823 and 910–964) align with major ligand binding regions (MLBRs), suggesting crucial interactions of collagen monomers or fibrils with integrins, matrix metalloproteinases (MMPs), fibronectin, and cartilage oligomeric matrix protein (COMP). Mutations in COL1A2 are predominantly nonlethal (80%). Lethal substitutions are located in eight regularly spaced clusters along the chain, supporting a regional model. The lethal regions align with proteoglycan binding sites along the fibril, suggesting a role in fibril–matrix interactions. Recurrences at the same site in α2(I) are generally concordant for outcome, unlike α1(I). Splice site mutations comprise 20% of helical mutations identified in OI patients, and may lead to exon skipping, intron inclusion, or the activation of cryptic splice sites. Splice site mutations in COL1A1 are rarely lethal; they often lead to frameshifts and the mild type I phenotype. In α2(I), lethal exon skipping events are

  9. Chondroitin sulphates A, B and C, collagen types I-IV and fibronectin in venous sinus of the red pulp in human spleen.

    PubMed

    Rovenská, E; Michalka, P; Papincák, J; Durdík, S; Jakubovský, J

    2005-01-01

    The morphological relationship of chondroitin sulphates A, B, and C, collagen types I-IV and fibronectin in the wall of venous sinuses of the red pulp in human spleen has not been a focus of interest among morphologists. Regarding the hypothesis that the structure of the spleen lends it the function of a blood filter the substances described in our study might play a significant role in the functional morphology. Of 146 human spleen surgical specimens, groups of 12 specimens each were examined under a light microscope using the method of antibodies against fibronectin, against collagen types I-IV and against chondroitin sulphates A, B, and C. The sections of the red pulp of human spleen stained with hematoxylin and eosin provided limited information about the wall of the sinuses. Chondroitin sulphates A and B were observed on the surface of sinus-lining cells (SLC), and fibronectin was detected on the surface of the annular fibers. Collagen type 11 was observed almost in the same places as chondroitin sulphates A and B. Collagen type IV was present in annular fibers of the wall of the sinus and in the basement membrane, like fibronectin. Chondroitin sulphate was not present in the walls of sinuses. Binding of antibodies against chondroitin sulphate A and against chondroitin sulphate B indicates the presence of chondroitin sulfates on the surface of SLC, where they probably play a role in helping the human organism to recognize alien and self substances. The presence of chondroitin,sulphates A and B probably affects inhibition of binding of cells with collagen type I, but not with fibronectin.

  10. Motor Physical Therapy Affects Muscle Collagen Type I and Decreases Gait Speed in Dystrophin-Deficient Dogs

    PubMed Central

    Gaiad, Thaís P.; Araujo, Karla P. C.; Serrão, Júlio C.; Miglino, Maria A.; Ambrósio, Carlos Eduardo

    2014-01-01

    Golden Retriever Muscular Dystrophy (GRMD) is a dystrophin-deficient canine model genetically homologous to Duchenne Muscular Dystrophy (DMD) in humans. Muscular fibrosis secondary to cycles of degeneration/regeneration of dystrophic muscle tissue and muscular weakness leads to biomechanical adaptation that impairs the quality of gait. Physical therapy (PT) is one of the supportive therapies available for DMD, however, motor PT approaches have controversial recommendations and there is no consensus regarding the type and intensity of physical therapy. In this study we investigated the effect of physical therapy on gait biomechanics and muscular collagen deposition types I and III in dystrophin-deficient dogs. Two dystrophic dogs (treated dogs-TD) underwent a PT protocol of active walking exercise, 3×/week, 40 minutes/day, 12 weeks. Two dystrophic control dogs (CD) maintained their routine of activities of daily living. At t0 (pre) and t1 (post-physical therapy), collagen type I and III were assessed by immunohistochemistry and gait biomechanics were analyzed. Angular displacement of shoulder, elbow, carpal, hip, stifle and tarsal joint and vertical (Fy), mediolateral (Fz) and craniocaudal (Fx) ground reaction forces (GRF) were assessed. Wilcoxon test was used to verify the difference of biomechanical variables between t0 and t1, considering p<.05. Type I collagen of endomysium suffered the influence of PT, as well as gait speed that had decreased from t0 to t1 (p<.000). The PT protocol employed accelerates morphological alterations on dystrophic muscle and promotes a slower velocity of gait. Control dogs which maintained their routine of activities of daily living seem to have found a better balance between movement and preservation of motor function. PMID:24713872

  11. Sika Deer Antler Collagen Type I-Accelerated Osteogenesis in Bone Marrow Mesenchymal Stem Cells via the Smad Pathway

    PubMed Central

    Li, Na; Zhang, Min; Drummen, Gregor P. C.; Zhao, Yu; Tan, Yin Fen; Luo, Su; Qu, Xiao Bo

    2016-01-01

    Deer antler preparations have been used to strengthen bones for centuries. It is particularly rich in collagen type I. This study aimed to unravel part of the purported bioremedial effect of Sika deer antler collagen type I (SDA-Col I) on bone marrow mesenchymal stem cells. The results suggest that SDA-Col I might be used to promote and regulate osteoblast proliferation and differentiation. SDA-Col I might potentially provide the basis for novel therapeutic strategies in the treatment of bone injury and/or in scaffolds for bone replacement strategies. Finally, isolation of SDA-Col I from deer antler represents a renewable, green, and uncomplicated way to obtain a biomedically valuable therapeutic. PMID:27066099

  12. PEP-1-SIRT2-induced matrix metalloproteinase-1 and -13 modulates type II collagen expression via ERK signaling in rabbit articular chondrocytes.

    PubMed

    Eo, Seong-Hui; Choi, Soo Young; Kim, Song Ja

    2016-11-01

    Matrix metalloproteinases (MMPs) are critical for the degradation of the extracellular matrix (ECM), which includes cartilage-specific collagen types I, II and XI. We previously found that PEP-1-sirtuin (SIRT)2 could induce dedifferentiation of articular chondrocytes; however, the underlying mechanisms remains unclear. We addressed this in the present study by examining the association between PEP-1-SIRT2 and the expression of MMP-1 and MMP-13 and type II collagen in rabbit articular chondrocytes. We found that PEP-1-SIRT2 increased MMP-1 and -13 expression in a dose- and time-dependent manner, as determined by western blotting. A similar trend in MMP-1 and -13 levels was observed in cultures during expansion to four passages. Pharmacological inhibition of MMP-1 and -13 blocked the PEP-1-SIRT2-induced decrease in type II collagen level. Phosphorylation of extracellular regulated kinase (ERK) was increased by PEP-1-SIRT2; however, treatment with the mitogen-activated protein kinase inhibitor PD98059 suppressed PEP-1-SIRT2-induced MMP-1 and -13 expression and dedifferentiation while restoring type II collagen expression in passage 2 cells. These results suggest that PEP-1-SIRT2 promotes MMP-induced dedifferentiation via ERK signaling in articular chondrocytes. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. New lethal disease involving type I and III collagen defect resembling geroderma osteodysplastica, De Barsy syndrome, and Ehlers-Danlos syndrome IV.

    PubMed

    Jukkola, A; Kauppila, S; Risteli, L; Vuopala, K; Risteli, J; Leisti, J; Pajunen, L

    1998-06-01

    We describe the clinical findings and biochemical features of a male child suffering from a so far undescribed lethal connective tissue disorder characterised by extreme hypermobility of the joints, lax skin, cataracts, severe growth retardation, and insufficient production of type I and type III procollagens. His features are compared with Ehlers-Danlos type IV, De Barsy syndrome, and geroderma osteodysplastica, as these disorders show some symptoms and signs shared with our patient. The child died because of failure of the connective tissue structures joining the skull and the spine, leading to progressive spinal stenosis. The aortic valve was translucent and insufficient. The clinical symptoms and signs, together with histological findings, suggested a collagen defect. Studies on both skin fibroblast cultures and the patient's serum showed reduced synthesis of collagen types I and III at the protein and RNA levels. The sizes of the mRNAs and newly synthesised proteins were normal, excluding gross structural abnormalities. These findings are not in accordance with any other collagen defect characterised so far.

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

    PubMed

    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.

  15. Histochemical analysis of collagen fibers in giant cell fibroma and inflammatory fibrous hyperplasia.

    PubMed

    Schmidt, Mônica Jarema; Tschoeke, André; Noronha, Lúcia; Moraes, Rafaela Scariot de; Mesquita, Ricardo Alves; Grégio, Ana Maria Trindade; Alanis, Luciana Reis Azevedo; Ignácio, Sérgio Aparecido; Santos, Jean Nunes Dos; Lima, Antonio Adilson Soares de; Luiz, Teixeira Suelen; Michels, Arielli Carine; Aguiar, Maria Cássia Ferreira; Johann, Aline Cristina Batista Rodrigues

    2016-06-01

    The aim was to investigate collagen fibers in giant cell fibroma, inflammatory fibrous hyperplasia, and oral normal mucosa. Sixty-six cases were stained with picrosirius red. The slides were observed under polarization, followed by the measurement of the area and the percentage of the type I and type III collagens. The age and gender were obtained from the clinical records. No differences could be observed in both the area and percentage of the type I and type III collagens within the categories of lesions and normal mucosa. In the giant cells fibroma, a greater area and percentage of type I collagen could be identified in individuals of less than 41.5 years (p<0.05). The distribution of type I and type III collagen fibers in the studied lesions followed a similar pattern to that observed in the normal mucosa, indicating a normal collagen maturation process of type III to I. The study supports that multinucleated and stellate cells of the giant cell fibroma appear to be functional within collagen types III and I turnover. The greater amount of type I collagen identified in giant cell fibroma in individuals of less than 41.5 years reinforce the neoplastic nature of lesion. Copyright © 2016 Elsevier GmbH. All rights reserved.

  16. Amino acid substitutions of conserved residues in the carboxyl-terminal domain of the [alpha]I(X) chain of type X collagen occur in two unrelated families with metaphyseal chondrodysplasia type Schmid

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wallis, G.A.; Rash, B.; Sweetman, W.A.

    1994-02-01

    Type X collagen is a homotrimeric, short-chain, nonfibrillar extracellular-matrix component that is specifically and transiently synthesized by hypertrophic chondrocytes at the site of endochondral ossification. The precise function of type X collagen is not known, but its specific pattern of expression suggests that mutations within the encoding gene (COL10A1) that alter the structure or synthesis of the protein may cause heritable forms of chondrodysplasia. The authors used the PCR and the SSCP techniques to analyze the coding and upstream promoter regions of the COL10A1 gene in a number of individuals with forms of chondrodysplasia. Using this approach, they identified twomore » individuals with metaphyseal chondrodysplasia type Schmid (MCDS) with SSCP changes in the region of the gene encoding the carboxyl-terminal domain. Sequence analysis demonstrated that the individuals were heterozygous for two unique single-base-pair transitions that led to the substitution of the highly conserved amino acid residue tyrosine at position 598 by aspartic acid in one person and of leucine at position 614 by proline in the other. The substitution at residue 598 segregated with the phenotype in a family of eight (five affected and three unaffected) related persons. The substitutions at residue 614 occurred in a sporadically affected individual but not in her unaffected mother and brother. Additional members of this family were not available for further study. These results suggest that certain amino acid substitutions within the carboxyl-terminal domain of the chains of the type X collagen molecule cause MCDS. These amino acid substitutions are likely to alter either chain recognition or assembly of the type X collagen molecule, thereby depleting the amount of normal type X collagen deposited in the extracellular matrix, with consequent aberrations in bone growth and development. 36 refs., 5 figs.« less

  17. UPR transducer BBF2H7 allows export of type II collagen in a cargo- and developmental stage–specific manner

    PubMed Central

    Toyama, Takuya; Nakamura, Yuki; Tamada, Kentaro; Shimizu, Hitomi; Ninagawa, Satoshi; Okada, Tetsuya; Ishikawa-Fujiwara, Tomoko; Aoyama, Eriko; Takigawa, Masaharu

    2017-01-01

    The unfolded protein response (UPR) handles unfolded/misfolded proteins accumulated in the endoplasmic reticulum (ER). However, it is unclear how vertebrates correctly use the total of ten UPR transducers. We have found that ER stress occurs physiologically during early embryonic development in medaka fish and that the smooth alignment of notochord cells requires ATF6 as a UPR transducer, which induces ER chaperones for folding of type VIII (short-chain) collagen. After secretion of hedgehog for tissue patterning, notochord cells differentiate into sheath cells, which synthesize type II collagen. In this study, we show that this vacuolization step requires both ATF6 and BBF2H7 as UPR transducers and that BBF2H7 regulates a complete set of genes (Sec23/24/13/31, Tango1, Sedlin, and KLHL12) essential for the enlargement of COPII vesicles to accommodate long-chain collagen for export, leading to the formation of the perinotochordal basement membrane. Thus, the most appropriate UPR transducer is activated to cope with the differing physiological ER stresses of different content types depending on developmental stage. PMID:28500182

  18. Differential proteomics reveals S100-A11 as a key factor in aldosterone-induced collagen expression in human cardiac fibroblasts.

    PubMed

    Martínez-Martínez, Ernesto; Ibarrola, Jaime; Lachén-Montes, Mercedes; Fernández-Celis, Amaya; Jaisser, Frederic; Santamaría, Enrique; Fernández-Irigoyen, Joaquín; López-Andrés, Natalia

    2017-08-23

    Aldosterone (Aldo) could induce cardiac fibrosis, a hallmark of heart disease. Aldo direct effects on collagen production in cardiac fibroblasts remain controversial. Our aim is to characterize changes in the proteome of adult human cardiac fibroblasts treated with Aldo to identify new proteins altered that might be new therapeutic targets in cardiovascular diseases. Aldo increased collagens expressions in human cardiac fibroblasts. Complementary, using a quantitative proteomic approach, 30 proteins were found differentially expressed between control and Aldo-treated cardiac fibroblasts. Among these proteins, 7 were up-regulated and 23 were down-regulated by Aldo. From the up-regulated proteins, collagen type I, collagen type III, collagen type VI and S100-A11 were verified by Western blot. Moreover, protein interaction networks revealed a functional link between a third of Aldo-modulated proteome and specific survival routes. S100-A11 was identified as a possible link between Aldo and collagen. Interestingly, CRISPR/Cas9-mediated knock-down of S100-A11 blocked Aldo-induced collagen production in human cardiac fibroblasts. In adult human cardiac fibroblasts treated with Aldo, proteomic analyses revealed an increase in collagen production. S100-A11 was identified as a new regulator of Aldo-induced collagen production in human cardiac fibroblasts. These data could identify new candidate proteins for the treatment of cardiac fibrosis in cardiovascular diseases. S100-A11 is identified by a proteomic approach as a novel regulator of Aldosterone-induced collagen production in human cardiac fibroblasts. Our data could identify new candidate proteins of interest for the treatment of cardiac fibrosis in cardiovascular diseases. Copyright © 2017. Published by Elsevier B.V.

  19. Type XVIII collagen degradation products in acute lung injury

    PubMed Central

    Perkins, Gavin D; Nathani, Nazim; Richter, Alex G; Park, Daniel; Shyamsundar, Murali; Heljasvaara, Ritva; Pihlajaniemi, Taina; Manji, Mav; Tunnicliffe, W; McAuley, Danny; Gao, Fang; Thickett, David R

    2009-01-01

    Introduction In acute lung injury, repair of the damaged alveolar-capillary barrier is an essential part of recovery. Endostatin is a 20 to 28 kDa proteolytic fragment of the basement membrane collagen XVIII, which has been shown to inhibit angiogenesis via action on endothelial cells. We hypothesised that endostatin may have a role in inhibiting lung repair in patients with lung injury. The aims of the study were to determine if endostatin is elevated in the plasma/bronchoalveolar lavage fluid of patients with acute lung injury and ascertain whether the levels reflect the severity of injury and alveolar inflammation, and to assess if endostatin changes occur early after the injurious lung stimuli of one lung ventilation and lipopolysaccharide (LPS) challenge. Methods Endostatin was measured by ELISA and western blotting. Results Endostatin is elevated within the plasma and bronchoalveolar lavage fluid of patients with acute lung injury. Lavage endostatin reflected the degree of alveolar neutrophilia and the extent of the loss of protein selectivity of the alveolar-capillary barrier. Plasma levels of endostatin correlated with the severity of physiological derangement. Western blotting confirmed elevated type XVIII collagen precursor levels in the plasma and lavage and multiple endostatin-like fragments in the lavage of patients. One lung ventilation and LPS challenge rapidly induce increases in lung endostatin levels. Conclusions Endostatin may adversely affect both alveolar barrier endothelial and epithelial cells, so its presence within both the circulation and the lung may have a pathophysiological role in acute lung injury that warrants further evaluation. PMID:19358707

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

    NASA Astrophysics Data System (ADS)

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

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

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

    NASA Astrophysics Data System (ADS)

    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.

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

    USDA-ARS?s Scientific Manuscript database

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

  3. Cell-populated collagen lattice contraction model for the investigation of fibroblast collagen interactions.

    PubMed

    Ehrlich, H Paul; Moyer, Kurtis E

    2013-01-01

    The fibroblast-populated collagen lattice (FPCL) was intended to act as the dermal component for "skin-equivalent" or artificial skin developed for skin grafting burn patients. The "skin-equivalent" was clinically unsuccessful as a skin graft, but today it is successfully used as a dressing for the management of chronic wounds. The FPCL has, however, become an instrument for investigating cell-connective tissue interactions within a three-dimensional matrix. Through the capacity of cell compaction of collagen fibrils, the FPCL undergoes a reduction in volume referred to as lattice contraction. Lattice contraction proceeds by cell-generated forces that reduce the water mass between collagen fibers, generating a closer relationship between collagen fibers. The compaction of collagen fibers is responsible for the reduction in the FPCL volume. Cell-generated forces through the linkage of collagen fibers with fibroblast's cytoskeletal actin-rich microfilament structures are responsible for the completion of the collagen matrix compaction. The type of culture dish used to cast FPCL as well as the cell number will dictate the mechanism for compacting collagen matrices. Fibroblasts, at moderate density, cast as an FPCL within a petri dish and released from the surface of the dish soon after casting compact collagen fibers through cell tractional forces. Fibroblasts at moderate density cast as an FPCL within a tissue culture dish and not released for 4 days upon release show rapid lattice contraction through a mechanism of cell contraction forces. Fibroblasts at high density cast in an FPCL within a petri dish, released from the surface of the dish soon after casting, compact a collagen lattice very rapidly through forces related to cell elongation. The advantage of the FPCL contraction model is the study of cells in the three-dimensional environment, which is similar to the environment from which these cells were isolated. In this chapter methods are described for

  4. Study on the kinetic self-assembly of type I collagen from tilapia (Oreochromis niloticus) skin using the fluorescence probe thioflavin T.

    PubMed

    Yan, Mingyan; Wang, Xinping

    2018-05-27

    The kinetic self-assembly of type I collagen from tilapia (Oreochromis niloticus) skin was characterized by the fluorescence method based on thioflavin T (ThT). The fluorescence probe could bind to the active monomeric collagen with a higher ordered degree of molecule, which displayed the pH and ionic strength dependence, the binding constant higher at neutral pH and proportional to the NaCl concentration. Compared to the turbidity method, ThT was more suitable to characterize the nucleation phase of collagen self-assembly. The nucleus size was determined through the ThT fluorescence and linear-polymerization model. At various pH and ionic strength, the nucleus size was nearly identical, either one or two monomers, demonstrating that one or two active monomeric collagen formed into the nucleus and different pH and ionic strength didn't alter the self-assembly mechanism of collagen. This approach was beneficial to advance the understanding of the kinetic self-assembly of the fish-sourced collagen in vitro. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Electrochemically Preadsorbed Collagen Promotes Adult Human Mesenchymal Stem Cell Adhesion

    PubMed Central

    Benavidez, Tomás E.; Wechsler, Marissa E.; Farrer, Madeleine M.; Bizios, Rena

    2016-01-01

    The present article reports on the effect of electric potential on the adsorption of collagen type I (the most abundant component of the organic phase of bone) onto optically transparent carbon electrodes (OTCE) and its mediation on subsequent adhesion of adult, human, mesenchymal stem cells (hMSCs). For this purpose, adsorption of collagen type I was investigated as a function of the protein concentration (0.01, 0.1, and 0.25 mg/mL) and applied potential (open circuit potential [OCP; control], +400, +800, and +1500 mV). The resulting substrate surfaces were characterized using spectroscopic ellipsometry, atomic force microscopy, and cyclic voltammetry. Adsorption of collagen type I onto OTCE was affected by the potential applied to the sorbent surface and the concentration of protein. The higher the applied potential and protein concentration, the higher the adsorbed amount (Γcollagen). It was also observed that the application of potential values higher than +800 mV resulted in the oxidation of the adsorbed protein. Subsequent adhesion of hMSCs on the OTCEs (precoated with the collagen type I films) under standard cell culture conditions for 2 h was affected by the extent of collagen preadsorbed onto the OTCE substrates. Specifically, enhanced hMSCs adhesion was observed when the Γcollagen was the highest. When the collagen type I was oxidized (under applied potential equal to +1500 mV), however, hMSCs adhesion was decreased. These results provide the first correlation between the effects of electric potential on protein adsorption and subsequent modulation of anchorage-dependent cell adhesion. PMID:26549607

  6. Interleukin-1 alpha modulates collagen gene expression in cultured synovial cells.

    PubMed Central

    Mauviel, A; Teyton, L; Bhatnagar, R; Penfornis, H; Laurent, M; Hartmann, D; Bonaventure, J; Loyau, G; Saklatvala, J; Pujol, J P

    1988-01-01

    The effects of porcine interleukin-1 (IL-1) alpha on collagen production were studied in cultured human rheumatoid synovial cells. Addition of 0.05-5 ng of IL-1/ml into the cultures resulted in a dose-dependent decreased rate of collagen released into the medium over 24 h. To determine whether this inhibition was due to secondary action of prostaglandin E2 (PGE2) secreted in response to IL-1, cultures were incubated in presence of various inhibitors of arachidonate metabolism. Depending on the cell strains, these inhibitors were able to suppress or diminish the effect of IL-1, suggesting that PGE2 is involved in the mechanism. Depression of collagen production caused by IL-1 mainly affected type I collagen and therefore led to a change in the type I/type III collagen ratio in the extracellular medium. Steady-state levels of mRNA for types I and III procollagens were estimated by dot-blot hybridization and compared with the amounts of respective collagens produced in the same cultures. IL-1 generally increased procollagen type I mRNA, but to a variable extent, as did indomethacin (Indo). Depending on the cell strain, the combination of indo and IL-1 could elevate the mRNA level of type I procollagen compared with Indo alone. These results did not correlate with the production rate of collagen in the medium, which was diminished by exposure to IL-1. The level of mRNA for collagen type III was not greatly changed by incubation with IL-1, and a better correlation was generally observed with the amount of type III collagen found in the medium. These data suggest that an additional control mechanism at translational or post-translational level must exist, counterbalancing the stimulatory effect of IL-1 on collagen mRNA transcription. It is likely that IL-1 could modulate the production of collagen in synovial cells by an interplay of different mechanisms, some of them limiting the effect of primary elevation of the steady-state mRNA level. Images Fig. 3. Fig. 4. Fig. 5

  7. Loss of Type I Collagen Telopeptide Lysyl Hydroxylation Causes Musculoskeletal Abnormalities in a Zebrafish Model of Bruck Syndrome

    PubMed Central

    Gistelinck, Charlotte; Witten, Paul Eckhard; Huysseune, Ann; Symoens, Sofie; Malfait, Fransiska; Larionova, Daria; Simoens, Pascal; Dierick, Manuel; Van Hoorebeke, Luc; De Paepe, Anne; Kwon, Ronald Y; Weis, MaryAnn; Eyre, David R; Willaert, Andy; Coucke, Paul J

    2017-01-01

    Bruck syndrome (BS) is a disorder characterized by joint flexion contractures and skeletal dysplasia that shows strong clinical overlap with the brittle bone disease Osteogenesis Imperfecta (OI). BS is caused by bi-allelic mutations in either the FKBP10 or the PLOD2 gene. PLOD2 encodes the lysyl hydroxylase 2 (LH2) enzyme, which is responsible for the hydroxylation of lysine residues in fibrillar collagen telopeptides. This hydroxylation directs cross-linking of collagen fibrils in the extracellular matrix, which is necessary to provide stability and tensile integrity to the collagen fibrils. To further elucidate the function of LH2 in vertebrate skeletal development, we created a zebrafish model harboring a homozygous plod2 nonsense mutation resulting in reduced telopeptide hydroxylation and cross-linking of bone type I collagen. Adult plod2 mutants present with a shortened body axis and severe skeletal abnormalities with evidence of bone fragility and fractures. The vertebral column of plod2 mutants is short and scoliotic with compressed vertebrae that show excessive bone formation at the vertebral end plates, and increased tissue mineral density in the vertebral centra. The muscle fibers of mutant zebrafish have a reduced diameter near the horizontal myoseptum. The endomysium, a layer of connective tissue ensheathing the individual muscle fibers, is enlarged. Transmission electron microscopy of mutant vertebral bone shows type I collagen fibrils that are less organized with loss of the typical plywood-like structure. In conclusion, plod2 mutant zebrafish show molecular and tissue abnormalities in the musculoskeletal system that are concordant with clinical findings in BS patients. Therefore, the plod2 zebrafish mutant is a promising model for the elucidation of the underlying pathogenetic mechanisms leading to BS and the development of novel therapeutic avenues in this syndrome. PMID:27541483

  8. Loss of Type I Collagen Telopeptide Lysyl Hydroxylation Causes Musculoskeletal Abnormalities in a Zebrafish Model of Bruck Syndrome.

    PubMed

    Gistelinck, Charlotte; Witten, Paul Eckhard; Huysseune, Ann; Symoens, Sofie; Malfait, Fransiska; Larionova, Daria; Simoens, Pascal; Dierick, Manuel; Van Hoorebeke, Luc; De Paepe, Anne; Kwon, Ronald Y; Weis, MaryAnn; Eyre, David R; Willaert, Andy; Coucke, Paul J

    2016-11-01

    Bruck syndrome (BS) is a disorder characterized by joint flexion contractures and skeletal dysplasia that shows strong clinical overlap with the brittle bone disease osteogenesis imperfecta (OI). BS is caused by biallelic mutations in either the FKBP10 or the PLOD2 gene. PLOD2 encodes the lysyl hydroxylase 2 (LH2) enzyme, which is responsible for the hydroxylation of lysine residues in fibrillar collagen telopeptides. This hydroxylation directs crosslinking of collagen fibrils in the extracellular matrix, which is necessary to provide stability and tensile integrity to the collagen fibrils. To further elucidate the function of LH2 in vertebrate skeletal development, we created a zebrafish model harboring a homozygous plod2 nonsense mutation resulting in reduced telopeptide hydroxylation and crosslinking of bone type I collagen. Adult plod2 mutants present with a shortened body axis and severe skeletal abnormalities with evidence of bone fragility and fractures. The vertebral column of plod2 mutants is short and scoliotic with compressed vertebrae that show excessive bone formation at the vertebral end plates, and increased tissue mineral density in the vertebral centra. The muscle fibers of mutant zebrafish have a reduced diameter near the horizontal myoseptum. The endomysium, a layer of connective tissue ensheathing the individual muscle fibers, is enlarged. Transmission electron microscopy of mutant vertebral bone shows type I collagen fibrils that are less organized with loss of the typical plywood-like structure. In conclusion, plod2 mutant zebrafish show molecular and tissue abnormalities in the musculoskeletal system that are concordant with clinical findings in BS patients. Therefore, the plod2 zebrafish mutant is a promising model for the elucidation of the underlying pathogenetic mechanisms leading to BS and the development of novel therapeutic avenues in this syndrome. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for

  9. Natural Type II Collagen Hydrogel, Fibrin Sealant, and Adipose-Derived Stem Cells as a Promising Combination for Articular Cartilage Repair.

    PubMed

    Lazarini, Mariana; Bordeaux-Rego, Pedro; Giardini-Rosa, Renata; Duarte, Adriana S S; Baratti, Mariana Ozello; Zorzi, Alessandro Rozim; de Miranda, João Batista; Lenz Cesar, Carlos; Luzo, Ângela; Olalla Saad, Sara Teresinha

    2017-10-01

    Objective Articular cartilage is an avascular tissue with limited ability of self-regeneration and the current clinical treatments have restricted capacity to restore damages induced by trauma or diseases. Therefore, new techniques are being tested for cartilage repair, using scaffolds and/or stem cells. Although type II collagen hydrogel, fibrin sealant, and adipose-derived stem cells (ASCs) represent suitable alternatives for cartilage formation, their combination has not yet been investigated in vivo for focal articular cartilage defects. We performed a simple experimental procedure using the combination of these 3 compounds on cartilage lesions of rabbit knees. Design The hydrogel was developed in house and was first tested in vitro for chondrogenic differentiation. Next, implants were performed in chondral defects with or without ASCs and the degree of regeneration was macroscopically and microscopically evaluated. Results Production of proteoglycans and the increased expression of collagen type II (COL2α1), aggrecan (ACAN), and sex-determining region Y-box 9 (SOX9) confirmed the chondrogenic character of ASCs in the hydrogel in vitro. Importantly, the addition of ASC induced a higher overall repair of the chondral lesions and a better cellular organization and collagen fiber alignment compared with the same treatment without ASCs. This regenerating tissue also presented the expression of cartilage glycosaminoglycan and type II collagen. Conclusions Our results indicate that the combination of the 3 compounds is effective for articular cartilage repair and may be of future clinical interest.

  10. Collagen XIX Is Expressed by Interneurons and Contributes to the Formation of Hippocampal Synapses

    PubMed Central

    Su, Jianmin; Gorse, Karen; Ramirez, Francesco; Fox, Michael A.

    2010-01-01

    Extracellular matrix (ECM) molecules contribute to the formation and maintenance of synapses in the mammalian nervous system. We previously discovered a family of nonfibrillar collagens that organize synaptic differentiation at the neuromuscular junction (NMJ). Although many NMJ-organizing cues contribute to central nervous system (CNS) synaptogenesis, whether similar roles for collagens exist at central synapses remained unclear. In the present study we discovered that col19a1, the gene encoding nonfibrillar collagen XIX, is expressed by subsets of hippocampal neurons. Colocalization with the interneuron-specific enzyme glutamate decarboxylase 67 (Gad67), but not other cell-type-specific markers, suggests that hippocampal expression of col19a1 is restricted to interneurons. However, not all hippocampal interneurons express col19a1 mRNA; subsets of neuropeptide Y (NPY)-, somatostatin (Som)-, and calbindin (Calb)-immunoreactive interneurons express col19a1, but those containing parvalbumin (Parv) or calretinin (Calr) do not. To assess whether collagen XIX is required for the normal formation of hippocampal synapses, we examined synaptic morphology and composition in targeted mouse mutants lacking collagen XIX. We show here that subsets of synaptotagmin 2 (Syt2)-containing hippocampal nerve terminals appear malformed in the absence of collagen XIX. The presence of Syt2 in inhibitory hippocampal synapses, the altered distribution of Gad67 in collagen XIX-deficient subiculum, and abnormal levels of gephyrin in collagen XIX-deficient hippocampal extracts all suggest inhibitory synapses are affected by the loss of collagen XIX. Together, these data not only reveal that collagen XIX is expressed by central neurons, but show for the first time that a nonfibrillar collagen is necessary for the formation of hippocampal synapses. PMID:19937713

  11. Time-dependent effects of intermittent hydrostatic pressure on articular chondrocyte type II collagen and aggrecan mRNA expression.

    PubMed

    Smith, R L; Lin, J; Trindade, M C; Shida, J; Kajiyama, G; Vu, T; Hoffman, A R; van der Meulen, M C; Goodman, S B; Schurman, D J; Carter, D R

    2000-01-01

    The normal loading of joints during daily activities causes the articular cartilage to be exposed to high levels of intermittent hydrostatic pressure. This study quantified effects of intermittent hydrostatic pressure on expression of mRNA for important extracellular matrix constituents. Normal adult bovine articular chondrocytes were isolated and tested in primary culture, either as high-density monolayers or formed aggregates. Loaded cells were exposed to 10 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for periods of 2, 4, 8, 12, and 24 hrs. Other cells were intermittently loaded for a period of 4 hrs per day for 4 days. Semiquantitative reverse transcription polymerase chain reaction assays were used to assess mRNA signal levels for collagen types II and I and aggrecan. The results showed that type II collagen mRNA signal levels exhibited a biphasic pattern, with an initial increase of approximately five-fold at 4 and 8 hrs that subsequently decreased by 24 hrs. In contrast, aggrecan mRNA signal increased progressively up to three-fold throughout the loading period. Changing the loading profile to 4 hrs per day for 4 days increased the mRNA signal levels for type II collagen nine-fold and for aggrecan twenty-fold when compared to unloaded cultures. These data suggest that specific mechanical loading protocols may be required to optimally promote repair and regeneration of diseased joints.

  12. Type XVII collagen (BP180) can function as a cell-matrix adhesion molecule via binding to laminin 332

    PubMed Central

    Van den Bergh, F.; Eliason, S.L.; Giudice, G.J.

    2010-01-01

    Collagen XVII (COL17) is a transmembrane glycoprotein that is expressed on the basal surface of basal epidermal keratinocytes. Previous observations have led to the hypothesis that an interaction between COL17 and laminin 332, an extracellular matrix protein, contributes to the attachment of the basal keratinocyte to the basement membrane. In order to isolate and manipulate COL17 interactions with ECM components, we induced COL17 expression in two cells lines, SK-MEL1 and K562, that exhibit little or no capacity to attach to our test substrates, including laminin 332, types I and IV collagen, and fibronectin. Cells expressing high levels of COL17 preferentially adhered to a laminin 332 matrix, and, to a lesser extent, type IV collagen, while showing little or no binding to type I collagen or fibronectin. A quantitative analysis of cell adhesive forces revealed that, compared with COL17-negative cells, COL17-positive cells required over 7-fold greater force to achieve 50% detachment from a laminin 332 substrate. When a cell preparation (either K562 or SK-MEL1) with heterogeneous COL17 expression levels was allowed to attach to a laminin 332 matrix, the COL17-positive and COL17-negative cells differentially sorted to the bound and unbound cell fractions, respectively. COL17-dependent attachment to laminin 332 could be reduced or abolished by siRNA-mediated knockdown of COL17 expression or by adding to the assay wells specific antibodies against COL17 or laminin 332. These findings provide strong support for the hypothesis that cell surface COL17 can interact with laminin 332 and, together, participate in the adherence of a cell to the extracellular matrix. PMID:21034821

  13. Cartilage canals in the distal intermediate ridge of the tibia of fetuses and foals are surrounded by different types of collagen.

    PubMed

    Hellings, Ingunn Risnes; Dolvik, Nils Ivar; Ekman, Stina; Olstad, Kristin

    2017-10-01

    Some epiphyseal growth cartilage canals are surrounded by a ring of hypereosinophilic matrix consisting of collagen type I. Absence of the collagen type I ring may predispose canal vessels to failure and osteochondrosis, which can lead to fragments in joints (osteochondrosis dissecans). It is not known whether the ring develops in response to programming or biomechanical force. The distribution that may reveal the function of the ring has only been described in the distal femur of a limited number of foals. It is also not known which cells are responsible for producing the collagen ring. The aims of the current study were to examine fetuses and foals to infer whether the ring forms in response to biomechanical force or programming, to describe distribution and to investigate which cell type produces the ring. The material consisted of 46 fetuses and foals from 293 days of gestation to 142 days old, of both sexes and different breeds, divided into three groups, designated the naïve group up to and including the day of birth, the adapting group from 2 days up to and including 14 days old, and the loaded group from 15 days and older. The distal tibia was sawn into parasagittal slabs and the cranial half of the central slab from the intermediate ridge was examined by light microscopy and immunohistochemical staining for collagen type I. Presence, completeness and location of the collagen ring was compared, as was the quantity of perivascular mesenchymal cells. An eosinophilic ring present on HE-stained sections was seen in every single fetus and foal examined, which corresponded to collagen type I in immunostained sections. A higher proportion of cartilage canals were surrounded by an eosinophilic ring in the naïve and adapting groups at 73 and 76%, respectively, compared with the loaded group at 51%. When considering only patent canals, the proportion of canals with an eosinophilic ring was higher in the adapting and loaded than the naïve group of foals. The

  14. Marine Origin Collagens and Its Potential Applications

    PubMed Central

    Silva, Tiago H.; Moreira-Silva, Joana; Marques, Ana L. P.; Domingues, Alberta; Bayon, Yves; Reis, Rui L.

    2014-01-01

    Collagens are the most abundant high molecular weight proteins in both invertebrate and vertebrate organisms, including mammals, and possess mainly a structural role, existing different types according with their specific organization in distinct tissues. From this, they have been elected as one of the key biological materials in tissue regeneration approaches. Also, industry is constantly searching for new natural sources of collagen and upgraded methodologies for their production. The most common sources are from bovine and porcine origin, but other ways are making their route, such as recombinant production, but also extraction from marine organisms like fish. Different organisms have been proposed and explored for collagen extraction, allowing the sustainable production of different types of collagens, with properties depending on the kind of organism (and their natural environment) and extraction methodology. Such variety of collagen properties has been further investigated in different ways to render a wide range of applications. The present review aims to shed some light on the contribution of marine collagens for the scientific and technological development of this sector, stressing the opportunities and challenges that they are and most probably will be facing to assume a role as an alternative source for industrial exploitation. PMID:25490254

  15. Molecular dynamics simulations on networks of heparin and collagen.

    PubMed

    Kulke, Martin; Geist, Norman; Friedrichs, Wenke; Langel, Walter

    2017-06-01

    Synthetic scaffolds containing collagen (Type I) are of increasing interest for bone tissue engineering, especially for highly porous biomaterials in combination with glycosaminoglycans. In experiments the integration of heparin during the fibrillogenesis resulted in different types of collagen fibrils, but models for this aggregation on a molecular scale were only tentative. We conducted molecular dynamic simulations investigating the binding of heparin to collagen and the influence of the telopeptides during collagen aggregation. This aims at explaining experimental findings on a molecular level. Novel structures for N- and C-telopeptides were developed with the TIGER2 replica exchange algorithm and dihedral principle component analysis. We present an extended statistical analysis of the mainly electrostatic interaction between heparin and collagen and identify several binding sites. Finally, we propose a molecular mechanism for the influence of glycosaminoglycans on the morphology of collagen fibrils. Proteins 2017; 85:1119-1130. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  16. Modern collagen wound dressings: function and purpose.

    PubMed

    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.

  17. Analysis of Therapeutic Effectiveness of Selected Types of Collagen in Prevention and Treatment of Degenerative Joint Disease.

    PubMed

    Tomaszewski, Wiesław; Paradowska, Anna

    2017-01-26

    Loss of collagen is a natural development accompanying aging of the body. It may be additionally accelerated by various conditions, including osteoarthritis (OA). Within the last two decades numerous and diverse studies have been conducted worldwide with the aim of identifying substances containing collagen, producing therapeutic preparations of expected effectiveness in the prevention and therapy of OA that would be safe to use and developing methods of delivering the final product into the body. The authors reviewed and analysed the latest available literature by selecting papers presenting the findings of studies conducted in line with the principles of Evidence-Based Medicine (EBM). The studies have been ordered from in vitro trials (studies on animals in the laboratory setting, use of tissue engineering methods to assess the effect of cartilage transplants, use of different collagen types for development of scaffolds etc.) to in vivo clinical trials. It appears that the findings of the latest multidimensional studies presented below, which confirm the therapeutic effectiveness of new-generation injectable medical collagen preparations, will help these medical products gain their well-deserved position in the comprehensive treatment of osteoarthritis both with respect to their analgesic properties as well as their ability to enable functional recovery and stimulate regeneration of tissues at the molecular level.

  18. Matrix metalloproteinase-2 and its correlation with basal membrane components laminin-5 and collagen type IV in paediatric burn patients measured with Surface Plasmon Resonance Imaging (SPRI) biosensors.

    PubMed

    Weremijewicz, Artur; Matuszczak, Ewa; Sankiewicz, Anna; Tylicka, Marzena; Komarowska, Marta; Tokarzewicz, Anna; Debek, Wojciech; Gorodkiewicz, Ewa; Hermanowicz, Adam

    2018-06-01

    The purpose of this study was the determination of matrix metalloproteinase-2 and its correlation with basal membrane components laminin-5 and collagen type IV in the blood plasma of burn patients measured with Surface Plasmon Resonance Imaging (SPRI) biosensors. 31 children scalded by hot water who were managed at the Department of Paediatric Surgery between 2014-2015, after primarily presenting with burns in 4-20% TBSA were included into the study (age 9 months up to 14 years, mean age 2,5+1 years). There were 10 girls and 21 boys. Venous blood samples were drawn 2-6h, and 12-16h after the thermal injury, and on the subsequent days 3, 5 and 7. The matrix metalloproteinase-2, collagen type IV and laminin-5 concentrations were assessed using Surface Plasmon Resonance Imaging by the investigators blinded to the other data. The MMP-2, laminin-5 and collagen type IV concentrations in the blood plasma of patients with burns, were highest 12-16h after thermal injury, the difference was statistically significant. The MMP-2, laminin-5 and collagen type IV concentrations measured 3 days, 5 days and 7 days after the thermal injury, slowly decreased over time, and on the 7th day reached the normal range, when compared with the concentration measured in controls. Current work is the first follow-up study regarding MMP-2 in burns. MMP-2, laminin-5 and collagen type IV levels were elevated early after burn injury in the plasma of studied patients, and were highest 12-16h after the injury. MMP-2, laminin-5 and collagen type IV levels were not proportional to the severity of the burn. We believe in the possibility that the gradual decrease of MMP-2, collagen type IV and laminin-5 concentrations could be connected with the process of healing, but to prove it, more investigation is needed in this area. The SPR imaging biosensor is a good diagnostic tool for determination of MMP-2, laminin-5 and collagen type IV in blood plasma of patients with burns. Copyright © 2017 Elsevier Ltd

  19. Biological Differences between Hanwoo longissimus dorsi and semimembranosus Muscles in Collagen Synthesis of Fibroblasts.

    PubMed

    Subramaniyan, Sivakumar Allur; Hwang, Inho

    2017-01-01

    Variations in physical toughness between muscles and animals are a function of growth rate and extend of collagen type I and III. The current study was designed to investigate the ability of growth rate, collagen concentration, collagen synthesizing and degrading genes on two different fibroblast cells derived from Hanwoo m. longissimus dorsi (LD) and semimembranosus (SM) muscles. Fibroblast cell survival time was determined for understanding about the characteristics of proliferation rate between the two fibroblasts. We examined the collagen concentration and protein expression of collagen type I and III between the two fibroblasts. The mRNA expression of collagen synthesis and collagen degrading genes to elucidate the molecular mechanisms on toughness and tenderness through collagen production between the two fibroblast cells. From our results the growth rate, collagen content and protein expression of collagen type I and III were significantly higher in SM than LD muscle fibroblast. The mRNA expressions of collagen synthesized genes were increased whereas the collagen degrading genes were decreased in SM than LD muscle. Results from confocal microscopical investigation showed increased fluorescence of collagen type I and III appearing stronger in SM than LD muscle fibroblast. These results implied that the locomotion muscle had higher fibroblast growth rate, leads to produce more collagen, and cause tougher than positional muscle. This in vitro study mirrored that background toughness of various muscles in live animal is likely associated with fibroblast growth pattern, collagen synthesis and its gene expression.

  20. Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers

    PubMed Central

    Juskaite, Victoria; Corcoran, David S; Leitinger, Birgit

    2017-01-01

    The collagen-binding receptor tyrosine kinase DDR1 (discoidin domain receptor 1) is a drug target for a wide range of human diseases, but the molecular mechanism of DDR1 activation is poorly defined. Here we co-expressed different types of signalling-incompetent DDR1 mutants (‘receiver’) with functional DDR1 (‘donor’) and demonstrate phosphorylation of receiver DDR1 by donor DDR1 in response to collagen. Making use of enforced covalent DDR1 dimerisation, which does not affect receptor function, we show that receiver dimers are phosphorylated in trans by the donor; this process requires the kinase activity of the donor but not that of the receiver. The receiver ectodomain is not required, but phosphorylation in trans is abolished by mutation of the transmembrane domain. Finally, we show that mutant DDR1 that cannot bind collagen is recruited into DDR1 signalling clusters. Our results support an activation mechanism whereby collagen induces lateral association of DDR1 dimers and phosphorylation between dimers. DOI: http://dx.doi.org/10.7554/eLife.25716.001 PMID:28590245

  1. The importance of size-exclusion characteristics of type I collagen in bonding to dentin matrices

    PubMed Central

    M, Takahashi; M, Nakajima; J, Tagami; DLS, Scheffel; RM, Carvalho; A, Mazzoni; M, Carrilho; A, Tezvergil-Mutluay; L, Breschi; L, Tjäderhane; SS, Jang; FR, Tay; KA, Agee; DH, Pashley

    2013-01-01

    The mineral phase of dentin is located primarily within collagen fibrils. During development, bone or dentin collagen fibrils are formed first and then water within the fibril is replaced with apatite crystallites. Mineralized collagen contains very little water. During dentin bonding, acid-etching of mineralized dentin solubilizes the mineral crystallites and replaces them with water. During the infiltration phase of dentin bonding, adhesive comonomers are supposed to replace all of the collagen water with adhesive monomers that are then polymerized into copolymers. The authors of a recently published review suggested that dental monomers were too large to enter and displace water from collagen fibrils. If that were true, the endogenous proteases bound to dentin collagen could be responsible for unimpeded collagen degradation that is responsible for the poor durability of resin-dentin bonds. The current work studied the size-exclusion characteristics of dentin collagen, using a gel-filtration-like column chromatography technique, using dentin powder instead of Sephadex. The elution volumes of test molecules, including adhesive monomers, revealed that adhesive monomers smaller than about 1000 Da can freely diffuse into collagen water, while molecules of 10,000 Da begin to be excluded, and bovine serum albumin (66,000 Da) was fully excluded. These results validate the concept that dental monomers can permeate between collagen molecules during infiltration by etch-and-rinse adhesives. PMID:23928333

  2. Influence of functionalized nanoparticles on conformational stability of type I collagen for possible biomedical applications.

    PubMed

    Kandamchira, Aswathy; Selvam, Sangeetha; Marimuthu, Nidhin; Janardhanan, Sreeram Kalarical; Fathima, Nishter Nishad

    2013-12-01

    Collagen-nanoparticle interactions are vital for many biomedical applications including drug delivery and tissue engineering applications. Iron oxide nanoparticles synthesized using starch template according to our earlier reported procedures were functionalized by treating them with Gum Arabic (GA), a biocompatible polysaccharide, so as to enhance the interaction between nanoparticle surfaces and collagen. Viscosity, circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) techniques have been used to study the collagen-nanoparticle interactions. The relative viscosity for collagen-nanoparticle conjugate was found to increase with increase in concentration of the nanoparticle within the concentration range investigated, which is due to the aggregation of protein onto the surface of nanoparticle. The CD spectra for the collagen-nanoparticle at different concentration ratios do not have much variation in the Rpn values (ratio of positive peak intensity over negative peak intensity) after functionalization with GA. The variation of molar ellipticity values for collagen-nanoparticle is due to the glycoprotein present in GA. The collagen triple helical structure is maintained after interaction with nanoparticles. The FTIR spectra of native collagen, Coll-Fs (nanoparticle without functionalization) and Coll-FsG (nanoparticle functionalized with GA) show clearly the amide I, II, III bands, with respect to collagen. The ability of polysaccharide stabilized/functionalized nanoparticles to maintain the collagen properties would help in its biomedical applications. © 2013.

  3. Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index

    PubMed Central

    Branly, Thomas; Contentin, Romain; Desancé, Mélanie; Jacquel, Thibaud; Bertoni, Lélia; Jacquet, Sandrine; Mallein-Gerin, Frédéric; Denoix, Jean-Marie; Audigié, Fabrice; Demoor, Magali

    2018-01-01

    Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-β3 alone showed promising result but the previously tested association of BMP-2 and TGF-β1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the

  4. Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index.

    PubMed

    Branly, Thomas; Contentin, Romain; Desancé, Mélanie; Jacquel, Thibaud; Bertoni, Lélia; Jacquet, Sandrine; Mallein-Gerin, Frédéric; Denoix, Jean-Marie; Audigié, Fabrice; Demoor, Magali; Galéra, Philippe

    2018-02-01

    Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-β3 alone showed promising result but the previously tested association of BMP-2 and TGF-β1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1 : Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the

  5. Metal stabilization of collagen and de novo designed mimetic peptides

    PubMed Central

    Parmar, Avanish S.; Xu, Fei; Pike, Douglas H.; Belure, Sandeep V.; Hasan, Nida F.; Drzewiecki, Kathryn E.; Shreiber, David I.; Nanda, Vikas

    2017-01-01

    We explore the design of metal binding sites to modulate triple-helix stability of collagen and collagen-mimetic peptides. Globular proteins commonly utilize metals to connect tertiary structural elements that are well separated in sequence, constraining structure and enhancing stability. It is more challenging to engineer structural metals into fibrous protein scaffolds, which lack the extensive tertiary contacts seen in globular proteins. In the collagen triple helix, the structural adjacency of the carboxy-termini of the three chains makes this region an attractive target for introducing metal binding sites. We engineered His3 sites based on structural modeling constraints into a series of designed homotrimeric and heterotrimeric peptides, assessing the capacity of metal binding to improve stability and in the case of heterotrimers, affect specificity of assembly. Notable enhancements in stability for both homo and heteromeric systems were observed upon addition of zinc(II) and several other metal ions only when all three histidine ligands were present. Metal binding affinities were consistent with the expected Irving-Williams series for imidazole. Unlike other metals tested, copper(II) also bound to peptides lacking histidine ligands. Acetylation of the peptide N-termini prevented copper binding, indicating proline backbone amide metal-coordination at this site. Copper similarly stabilized animal extracted Type I collagen in a metal specific fashion, highlighting the potential importance of metal homeostasis within the extracellular matrix. PMID:26225466

  6. Metal Stabilization of Collagen and de Novo Designed Mimetic Peptides.

    PubMed

    Parmar, Avanish S; Xu, Fei; Pike, Douglas H; Belure, Sandeep V; Hasan, Nida F; Drzewiecki, Kathryn E; Shreiber, David I; Nanda, Vikas

    2015-08-18

    We explore the design of metal binding sites to modulate triple-helix stability of collagen and collagen-mimetic peptides. Globular proteins commonly utilize metals to connect tertiary structural elements that are well separated in sequence, constraining structure and enhancing stability. It is more challenging to engineer structural metals into fibrous protein scaffolds, which lack the extensive tertiary contacts seen in globular proteins. In the collagen triple helix, the structural adjacency of the carboxy-termini of the three chains makes this region an attractive target for introducing metal binding sites. We engineered His3 sites based on structural modeling constraints into a series of designed homotrimeric and heterotrimeric peptides, assessing the capacity of metal binding to improve stability and in the case of heterotrimers, affect specificity of assembly. Notable enhancements in stability for both homo- and heteromeric systems were observed upon addition of zinc(II) and several other metal ions only when all three histidine ligands were present. Metal binding affinities were consistent with the expected Irving-Williams series for imidazole. Unlike other metals tested, copper(II) also bound to peptides lacking histidine ligands. Acetylation of the peptide N-termini prevented copper binding, indicating proline backbone amide metal-coordination at this site. Copper similarly stabilized animal extracted Type I collagen in a metal-specific fashion, highlighting the potential importance of metal homeostasis within the extracellular matrix.

  7. The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts.

    PubMed

    Yhee, Ji Young; Yoon, Hong Yeol; Kim, Hyunjoon; Jeon, Sangmin; Hergert, Polla; Im, Jintaek; Panyam, Jayanth; Kim, Kwangmeyung; Nho, Richard Seonghun

    2017-01-01

    Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (>33.6-78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosis-dependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts.

  8. The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts

    PubMed Central

    Yhee, Ji Young; Yoon, Hong Yeol; Kim, Hyunjoon; Jeon, Sangmin; Hergert, Polla; Im, Jintaek; Panyam, Jayanth; Kim, Kwangmeyung; Nho, Richard Seonghun

    2017-01-01

    Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (>33.6–78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosis-dependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts. PMID

  9. Collagen hydrolysate based collagen/hydroxyapatite composite materials

    NASA Astrophysics Data System (ADS)

    Ficai, Anton; Albu, Madalina Georgiana; Birsan, Mihaela; Sonmez, Maria; Ficai, Denisa; Trandafir, Viorica; Andronescu, Ecaterina

    2013-04-01

    The aim of this study was to study the influence of collagen hydrolysate (HAS) on the formation of ternary collagen-hydrolysate/hydroxyapatite composite materials (COLL-HAS/HA). During the precipitation process of HA, a large amount of brushite is resulted at pH = 7 but, practically pure HA is obtained at pH ⩾ 8. The FTIR data reveal the duplication of the most important collagen absorption bands due to the presence of the collagen hydrolysate. The presence of collagen hydrolysate is beneficial for the management of bone and joint disorders such as osteoarthritis and osteoporosis.

  10. Revealing Early Steps of α2β1 Integrin-mediated Adhesion to Collagen Type I by Using Single-Cell Force Spectroscopy

    PubMed Central

    Taubenberger, Anna; Cisneros, David A.; Friedrichs, Jens; Puech, Pierre-Henri; Muller, Daniel J.

    2007-01-01

    We have characterized early steps of α2β1 integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of α2β1 as a collagen type I receptor, α2β1-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas α2β1-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin–collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin–collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of α2β1-mediated adhesion as weak initial, single-integrin–mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility. PMID:17314408

  11. A Membrane-Type-1 Matrix Metalloproteinase (MT1-MMP) – Discoidin Domain Receptor 1 Axis Regulates Collagen-Induced Apoptosis in Breast Cancer Cells

    PubMed Central

    Assent, Delphine; Bourgot, Isabelle; Hennuy, Benoît; Geurts, Pierre; Noël, Agnès; Foidart, Jean-Michel; Maquoi, Erik

    2015-01-01

    During tumour dissemination, invading breast carcinoma cells become confronted with a reactive stroma, a type I collagen-rich environment endowed with anti-proliferative and pro-apoptotic properties. To develop metastatic capabilities, tumour cells must acquire the capacity to cope with this novel microenvironment. How cells interact with and respond to their microenvironment during cancer dissemination remains poorly understood. To address the impact of type I collagen on the fate of tumour cells, human breast carcinoma MCF-7 cells were cultured within three-dimensional type I collagen gels (3D COL1). Using this experimental model, we have previously demonstrated that membrane type-1 matrix metalloproteinase (MT1-MMP), a proteinase overexpressed in many aggressive tumours, promotes tumour progression by circumventing the collagen-induced up-regulation of BIK, a pro-apoptotic tumour suppressor, and hence apoptosis. Here we performed a transcriptomic analysis to decipher the molecular mechanisms regulating 3D COL1-induced apoptosis in human breast cancer cells. Control and MT1-MMP expressing MCF-7 cells were cultured on two-dimensional plastic plates or within 3D COL1 and a global transcriptional time-course analysis was performed. Shifting the cells from plastic plates to 3D COL1 activated a complex reprogramming of genes implicated in various biological processes. Bioinformatic analysis revealed a 3D COL1-mediated alteration of key cellular functions including apoptosis, cell proliferation, RNA processing and cytoskeleton remodelling. By using a panel of pharmacological inhibitors, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase specifically activated by collagen, as the initiator of 3D COL1-induced apoptosis. Our data support the concept that MT1-MMP contributes to the inactivation of the DDR1-BIK signalling axis through the cleavage of collagen fibres and/or the alteration of DDR1 receptor signalling unit, without triggering a

  12. A theoretical approach of the relationships between collagen content, collagen cross-links and meat tenderness.

    PubMed

    Lepetit, J

    2007-05-01

    This work concerns the relationship between meat tenderness and the rubber-like properties, i.e. pressure and elastic modulus, that endomysium and perimysium connective tissues develop when meat has been heated to a temperature above which collagen contracts. For rest length meats with similar intramuscular connective tissue morphology, and which are at the same ageing state and pH, the elastic modulus of the collagenous fraction of connective tissues is approximately proportional to the total number of collagen cross-links present per volume of meat. Calculations from various published experiments concerned with the effect on tenderness of muscle type, animal age, type, and sex from different species show that this modulus follows most of the variations of meat toughness. Moreover, the proportionality between the increase in this elastic modulus and the increase in meat toughness approaches unity in situations where toughness mainly depends on connective tissues. This work demonstrates the decisive role of rubber-like properties of connective tissues in meat tenderness variations.

  13. Expression of collagen in ovular membranes of pregnant smokers and non-smokers: a pilot study.

    PubMed

    Negrini, Romulo; Araujo Júnior, Edward; Piato, Sebastião; Chade, Milca Cezar; Rios, Adriana Ribeiro Santos; Silva, Maria Antonieta Galvão; Aldrighi, José Mendes

    2015-09-01

    Our study compared the amount of total collagen and type I collagen in ovular membranes of pregnant smokers and non-smokers. The study group consisted of 14 pregnant smokers at 24-36 weeks of gestation; 39 pregnant non-smokers between 24-36 weeks of gestation comprised the control group. The expressions of total collagen and type I collagen were analyzed using two histological sections of the fetal membranes. The assessment of total collagen was performed using the Picro-Cirius red stain, and type I collagen expression was determined by means of immunohistochemistry The Mann-Whitney test was applied to verify possible differences between the groups. The average area covered by total collagen was lower in smokers (20630.45 microm2) as compared to non-smokers (24058.61 microm2), although the difference was not statistically significant (p = 0.454). Comparison involving collagen type I deemed similar results (20001.33 microm2 vs. 25328.29 microm2, p = 0.158). The amount of total collagen and type I collagen was lower in ovular membranes of pregnant smokers as compared to non-smokers, although the difference was not statistically significant.

  14. Evaluation of the efficacy of 100% Type-I collagen membrane of bovine origin in the treatment of human gingival recession: A clinical study

    PubMed Central

    Soni, Nitin; Sikri, Poonam; Kapoor, Daljit; Soni, Bhavita Wadhwa; Jain, Rachna

    2014-01-01

    Background: Various treatment modalities have been devised for gingival recession, which is one of the most common signs of periodontal disease. The present study evaluates the efficacy of bioresorbable 100% type I collagen membrane of bovine origin in the treatment of human gingival recession. Materials and Methods: Twenty cases of Miller's class I or class II localized gingival recession defects on the facial surface were treated with 100% type I collagen membrane of bovine origin in conjunction with coronally positioned flap. Pre-operative and post-operative assessments were performed with respect to probing pocket depth, clinical attachment level and clinical recession at 12, 24 and 36 weeks. The data thus collected were analyzed statistically. Results: Statistically significant improvement based on Student's t test was found in all the three clinical parameters. Conclusion: Bioresorbable 100% type I collagen membrane of bovine origin has given inspiring results in the treatment of human gingival recession defects, thereby justifying the use of this material wherever indicated. PMID:25565742

  15. Pirfenidone inhibits proliferation, arrests the cell cycle, and downregulates heat shock protein-47 and collagen type I in rat hepatic stellate cells in vitro.

    PubMed

    Xiang, Xian-Hong; Jiang, Tian-Peng; Zhang, Shuai; Song, Jie; Li, Xing; Yang, Jian-Yong; Zhou, Shi

    2015-07-01

    Pirfenidone (esbiret) is an established anti-fibrotic and anti-inflammatory drug used to treat idiopathic pulmonary fibrosis. In the present study, the dose-dependent effects of pirfenidone on the cell cycle, proliferation and expression of heat shock protein (HSP)-47 and collagen type I in a cultured rat hepatic stellate cell line (HSC-T6) were investigated. Following pirfenidone treatment, cell proliferation was determined using the cell counting kit-8 assay and the cell cycle was measured using flow cytometry. HSP-47 expression was estimated using western blot analysis and collagen type I mRNA was assessed using reverse transcription quantitative polymerase chain reaction. Pirfenidone induced significant dose-dependent inhibition of proliferation in HSC-T6 cells. Cell viability was unaffected by treatment with pirfenidone (0, 10 or 100 µM) for 24 and 72 h. However, after 24 h, HSC-T6 cells exhibited dose-dependent decreases in HSP-47 protein and collagen I mRNA levels. In conclusion, pirfenidone inhibited HSC-T6 cell proliferation, arrested the cell cycle and reduced the expression of HSP-47 and collagen type I, indicating that pirfenidone may be a promising drug in the treatment of liver fibrosis.

  16. Saponin Isolation as Main Ingredients of Insecticide and Collagen Type I From Crown of Thorn-Starfish (Acanthaster planci)

    NASA Astrophysics Data System (ADS)

    Wijanarko, Anondho; Januardi Ginting, Mikael; Sahlan, Muhamad; Krisanta Endah Savitri, Imelda; Florensia, Yunita; Sudiarta, Maria Regina; Pastika, Satria; Rafiki, Fakhri; Hermansyah, Heri

    2017-10-01

    The outbreaks of crown of thorns starfish (Acanthaster planci) resulted in the severe destruction of coral reefs in a large number of Indonesia’s marine ecosystem, especially in the western part. At the moment, control efforts are proven to be ineffective because of its high cost and labor intensive. Recent research found that A. planci contain saponins that act as cytotoxic compound and can be used as an environment-friendly insecticide to eradicate Kalotermitidae pest. Saponins extracted by maceration using ethanol 96.0% with a total yield of saponins 9.04% and 4.66% for two test. Purification of saponin was achieved by utilization of activated carbon with a mass of carbon:volume sample 1:2 (w/v) and stirred for 20 minutes. Sapogenin can be isolated by hydrolyzing using hydrochloric acid, and thus 168.4 mg sapogenin is obtained. In addition to saponins, A. planci also contains collagen Type I. Collagen isolation by multistage extraction began with extracting the collagen with alkaline solvent, with water, NaOH 0.1 M, and Ca(OH)2 0.2 M as the solvent variations. The second step is acid-enzymatic extraction by pepsin digestion in 0.5 M acetic acid. Collagen extract will be further purified by salting out and dialysis method to obtain pure collagen yield called Pepsin Solubilized Collagens (PSC). Characterization of PSC consists of quantitative and qualitative analysis such as Lowry method, gel electrophoresis, UV spectroscopy, amino acid composition analysis, and Scanning Electron Microscopy (SEM). The result shows Ca(OH)2 0.2 M as the best extraction solvent with 2.26% yield of PSC.

  17. Rheological characterization of the nucleus pulposus and dense collagen scaffolds intended for functional replacement.

    PubMed

    Bron, J L; Koenderink, G H; Everts, V; Smit, T H

    2009-05-01

    Lumbar discectomy is an effective therapy for neurological decompression in patients suffering from sciatica due to a herniated nucleus pulposus (NP). However, high numbers of patients suffering from persisting postoperative low back pain have resulted in many strategies targeting the regeneration of the NP. For successful regeneration, the stiffness of scaffolds is increasingly recognized as a potent mechanical cue for the differentiation and biosynthetic response of (stem) cells. The aim of the current study is to characterize the viscoelastic properties of the NP and to develop dense collagen scaffolds with similar properties. The scaffolds consisted of highly dense (0.5%-12%) type I collagen matrices, prepared by plastic compression. The complex modulus of the NP was 22 kPa (at 10 rad s(-1)), which should agree with a scaffold with a collagen concentration of 23%. The loss tangent, indicative of energy dissipation, is higher for the NP (0.28) than for the scaffolds (0.12) and was not dependent of the collagen density. Gamma sterilization of the scaffolds increased the shear moduli but also resulted in more brittle behavior and a reduced swelling capacity. In conclusion, by tuning the collagen density, we can approach the stiffness of the NP. Therefore, dense collagen is a promising candidate for tissue engineering of the NP that deserves further study, such as the addition of other proteins.

  18. Endocytosis of collagen by hepatic stellate cells regulates extracellular matrix dynamics

    PubMed Central

    Bi, Yan; Mukhopadhyay, Dhriti; Drinane, Mary; Ji, Baoan; Li, Xing; Cao, Sheng

    2014-01-01

    Hepatic stellate cells (HSCs) generate matrix, which in turn may also regulate HSCs function during liver fibrosis. We hypothesized that HSCs may endocytose matrix proteins to sense and respond to changes in microenvironment. Primary human HSCs, LX2, or mouse embryonic fibroblasts (MEFs) [wild-type; c-abl−/−; or Yes, Src, and Fyn knockout mice (YSF−/−)] were incubated with fluorescent-labeled collagen or gelatin. Fluorescence-activated cell sorting analysis and confocal microscopy were used for measuring cellular internalization of matrix proteins. Targeted PCR array and quantitative real-time PCR were used to evaluate gene expression changes. HSCs and LX2 cells endocytose collagens in a concentration- and time-dependent manner. Endocytosed collagen colocalized with Dextran 10K, a marker of macropinocytosis, and 5-ethylisopropyl amiloride, an inhibitor of macropinocytosis, reduced collagen internalization by 46%. Cytochalasin D and ML7 blocked collagen internalization by 47% and 45%, respectively, indicating that actin and myosin are critical for collagen endocytosis. Wortmannin and AKT inhibitor blocked collagen internalization by 70% and 89%, respectively, indicating that matrix macropinocytosis requires phosphoinositide-3-kinase (PI3K)/AKT signaling. Overexpression of dominant-negative dynamin-2 K44A blocked matrix internalization by 77%, indicating a role for dynamin-2 in matrix macropinocytosis. Whereas c-abl−/− MEF showed impaired matrix endocytosis, YSF−/− MEF surprisingly showed increased matrix endocytosis. It was also associated with complex gene regulations that related with matrix dynamics, including increased matrix metalloproteinase 9 (MMP-9) mRNA levels and zymographic activity. HSCs endocytose matrix proteins through macropinocytosis that requires a signaling network composed of PI3K/AKT, dynamin-2, and c-abl. Interaction with extracellular matrix regulates matrix dynamics through modulating multiple gene expressions including MMP-9

  19. Endocytosis of collagen by hepatic stellate cells regulates extracellular matrix dynamics.

    PubMed

    Bi, Yan; Mukhopadhyay, Dhriti; Drinane, Mary; Ji, Baoan; Li, Xing; Cao, Sheng; Shah, Vijay H

    2014-10-01

    Hepatic stellate cells (HSCs) generate matrix, which in turn may also regulate HSCs function during liver fibrosis. We hypothesized that HSCs may endocytose matrix proteins to sense and respond to changes in microenvironment. Primary human HSCs, LX2, or mouse embryonic fibroblasts (MEFs) [wild-type; c-abl(-/-); or Yes, Src, and Fyn knockout mice (YSF(-/-))] were incubated with fluorescent-labeled collagen or gelatin. Fluorescence-activated cell sorting analysis and confocal microscopy were used for measuring cellular internalization of matrix proteins. Targeted PCR array and quantitative real-time PCR were used to evaluate gene expression changes. HSCs and LX2 cells endocytose collagens in a concentration- and time-dependent manner. Endocytosed collagen colocalized with Dextran 10K, a marker of macropinocytosis, and 5-ethylisopropyl amiloride, an inhibitor of macropinocytosis, reduced collagen internalization by 46%. Cytochalasin D and ML7 blocked collagen internalization by 47% and 45%, respectively, indicating that actin and myosin are critical for collagen endocytosis. Wortmannin and AKT inhibitor blocked collagen internalization by 70% and 89%, respectively, indicating that matrix macropinocytosis requires phosphoinositide-3-kinase (PI3K)/AKT signaling. Overexpression of dominant-negative dynamin-2 K44A blocked matrix internalization by 77%, indicating a role for dynamin-2 in matrix macropinocytosis. Whereas c-abl(-/-) MEF showed impaired matrix endocytosis, YSF(-/-) MEF surprisingly showed increased matrix endocytosis. It was also associated with complex gene regulations that related with matrix dynamics, including increased matrix metalloproteinase 9 (MMP-9) mRNA levels and zymographic activity. HSCs endocytose matrix proteins through macropinocytosis that requires a signaling network composed of PI3K/AKT, dynamin-2, and c-abl. Interaction with extracellular matrix regulates matrix dynamics through modulating multiple gene expressions including MMP-9

  20. A collagen-based scaffold delivering exogenous microrna-29B to modulate extracellular matrix remodeling.

    PubMed

    Monaghan, Michael; Browne, Shane; Schenke-Layland, Katja; Pandit, Abhay

    2014-04-01

    Directing appropriate extracellular matrix remodeling is a key aim of regenerative medicine strategies. Thus, antifibrotic interfering RNA (RNAi) therapy with exogenous microRNA (miR)-29B was proposed as a method to modulate extracellular matrix remodeling following cutaneous injury. It was hypothesized that delivery of miR-29B from a collagen scaffold will efficiently modulate the extracellular matrix remodeling response and reduce maladaptive remodeling such as aggressive deposition of collagen type I after injury. The release of RNA from the scaffold was assessed and its ability to silence collagen type I and collagen type III expression was evaluated in vitro. When primary fibroblasts were cultured with scaffolds doped with miR-29B, reduced levels of collagen type I and collagen type III mRNA expression were observed for up to 2 weeks of culture. When the scaffolds were applied to full thickness wounds in vivo, reduced wound contraction, improved collagen type III/I ratios and a significantly higher matrix metalloproteinase (MMP)-8: tissue inhibitor of metalloproteinase (TIMP)-1 ratio were detected when the scaffolds were functionalized with miR-29B. Furthermore, these effects were significantly influenced by the dose of miR-29B in the collagen scaffold (0.5 versus 5 μg). This study shows a potential of combining exogenous miRs with collagen scaffolds to improve extracellular matrix remodeling following injury.

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

    PubMed

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

    2012-10-01

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

  2. Collagen-Gold Nanoparticle Conjugates for Versatile Biosensing

    PubMed Central

    Unser, Sarah; Holcomb, Samuel; Cary, ReJeana; Sagle, Laura

    2017-01-01

    Integration of noble metal nanoparticles with proteins offers promising potential to create a wide variety of biosensors that possess both improved selectivity and versatility. The multitude of functionalities that proteins offer coupled with the unique optical properties of noble metal nanoparticles can allow for the realization of simple, colorimetric sensors for a significantly larger range of targets. Herein, we integrate the structural protein collagen with 10 nm gold nanoparticles to develop a protein-nanoparticle conjugate which possess the functionality of the protein with the desired colorimetric properties of the nanoparticles. Applying the many interactions that collagen undergoes in the extracellular matrix, we are able to selectively detect both glucose and heparin with the same collagen-nanoparticle conjugate. Glucose is directly detected through the cross-linking of the collagen fibrils, which brings the attached nanoparticles into closer proximity, leading to a red-shift in the LSPR frequency. Conversely, heparin is detected through a competition assay in which heparin-gold nanoparticles are added to solution and compete with heparin in the solution for the binding sites on the collagen fibrils. The collagen-nanoparticle conjugates are shown to detect both glucose and heparin in the physiological range. Lastly, glucose is selectively detected in 50% mouse serum with the collagen-nanoparticle devices possessing a linear range of 3–25 mM, which is also within the physiologically relevant range. PMID:28212282

  3. Lack of Collagen VI Promotes Wound-Induced Hair Growth.

    PubMed

    Chen, Peiwen; Cescon, Matilde; Bonaldo, Paolo

    2015-10-01

    Collagen VI is an extracellular matrix molecule that is abundantly expressed in the skin. However, the role of collagen VI in hair follicle growth is unknown. Here, we show that collagen VI is strongly deposited in hair follicles, and is markedly upregulated by skin wounding. Lack of collagen VI in Col6a1(-/-) mice delays hair cycling and growth under physiological conditions, but promotes wound-induced hair regrowth without affecting skin regeneration. Conversely, addition of purified collagen VI rescues the abnormal wound-induced hair regrowth in Col6a1(-/-) mice. Mechanistic studies revealed that the increased wound-induced hair regrowth of Col6a1(-/-) mice is triggered by activation of the Wnt/β-catenin signaling pathway, and is abolished by inhibition of this pathway. These findings highlight the essential relationships between extracellular matrix (ECM) and hair follicle regeneration, and suggest that collagen VI could be a potential therapeutic target for hair loss and other skin-related diseases.

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

    PubMed

    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.

  5. A novel deletion/insertion mutation in the mRNA transcribed from one {alpha}1(I) collagen allele in a family with dominant type III OI and germline mosaicism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, O.; Masters, C.; Lewis, M.B.

    1994-09-01

    In an 8-year-old girl and her father, both of whom have severe type III OI, we have previously used RNA/RNA hybrid analysis to demonstrate a mismatch in the region of {alpha}1(I) mRNA coding for aa 558-861. We used SSCP to further localize the abnormality to a subregion coding for aa 579-679. This region was subcloned and sequenced. Each patient`s cDNA has a deletion of the sequences coding for the last residue of exon 34, and all of exons 35 and 36 (aa 604-639), followed by an insertion of 156 nt from the 3{prime}-end of intron 36. PCR amplification of leukocytemore » DNA from the patients and the clinically normal paternal grandmother yielded two fragments: a 1007 bp fragment predicted from normal genomic sequences and a 445 bp fragment. Subcloning and sequencing of the shorter genomic PCR product confirmed the presence of a 565 bp genomic deletion from the end of exon 34 to the middle of intron 36. The abnormal protein is apparently synthesized and incorporated into helix. The inserted nucleotides are in frame with the collagenous sequence and contain no stop codons. They encode a 52 aa non-collagenous region. The fibroblast procollagen of the patients has both normal and electrophoretically delayed pro{alpha}(I) bands. The electrophoretically delayed procollagen is very sensitive to pepsin or trypsin digestion, as predicted by its non-collagenous sequence, and cannot be visualized as collagen. This unique OI collagen mutation is an excellent candidate for molecular targeting to {open_quotes}turn off{close_quotes} a dominant mutant allele.« less

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

    PubMed

    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.

  7. Loss of Matrix Metalloproteinase-2 Amplifies Murine Toxin-Induced Liver Fibrosis by Upregulating Collagen I Expression

    PubMed Central

    Radbill, Brian D.; Gupta, Ritu; Ramirez, Maria Celeste M.; DiFeo, Analisa; Martignetti, John A.; Alvarez, Carlos E.; Friedman, Scott L.; Narla, Goutham; Vrabie, Raluca; Bowles, Robert; Saiman, Yedidya

    2010-01-01

    Background and Aims Matrix metalloproteinase-2 (MMP-2), a type IV collagenase secreted by activated hepatic stellate cells (HSCs), is upregulated in chronic liver disease and is considered a profibrotic mediator due to its proliferative effect on cultured HSCs and ability to degrade normal liver matrix. Although associative studies and cell culture findings suggest that MMP-2 promotes hepatic fibrogenesis, no in vivo model has definitively established a pathologic role for MMP-2 in the development and progression of liver fibrosis. We therefore examined the impact of MMP-2 deficiency on liver fibrosis development during chronic CCl4 liver injury and explored the effect of MMP-2 deficiency and overexpression on collagen I expression. Methods Following chronic CCl4 administration, liver fibrosis was analyzed using Sirius Red staining with quantitative morphometry and real-time polymerase chain reaction (PCR) in MMP-2−/− mice and age-matched MMP-2+/+ controls. These studies were complemented by analyses of cultured human stellate cells. Results MMP-2−/− mice demonstrated an almost twofold increase in fibrosis which was not secondary to significant differences in hepatocellular injury, HSC activation or type I collagenase activity; however, type I collagen messenger RNA (mRNA) expression was increased threefold in the MMP-2−/− group by real-time PCR. Furthermore, targeted reduction of MMP-2 in cultured HSCs using RNA interference significantly increased collagen I mRNA and protein, while overexpression of MMP-2 resulted in decreased collagen I mRNA. Conclusions These findings suggest that increased MMP-2 during the progression of liver fibrosis may be an important mechanism for inhibiting type I collagen synthesis by activated HSCs, thereby providing a protective rather than pathologic role. PMID:20563750

  8. How electrostatic networks modulate specificity and stability of collagen.

    PubMed

    Zheng, Hongning; Lu, Cheng; Lan, Jun; Fan, Shilong; Nanda, Vikas; Xu, Fei

    2018-06-12

    One-quarter of the 28 types of natural collagen exist as heterotrimers. The oligomerization state of collagen affects the structure and mechanics of the extracellular matrix, providing essential cues to modulate biological and pathological processes. A lack of high-resolution structural information limits our mechanistic understanding of collagen heterospecific self-assembly. Here, the 1.77-Å resolution structure of a synthetic heterotrimer demonstrates the balance of intermolecular electrostatics and hydrogen bonding that affects collagen stability and heterospecificity of assembly. Atomistic simulations and mutagenesis based on the solved structure are used to explore the contributions of specific interactions to energetics. A predictive model of collagen stability and specificity is developed for engineering novel collagen structures.

  9. Investigating the relationship between changes in collagen fiber orientation during skin aging and collagen/water interactions by polarized-FTIR microimaging.

    PubMed

    Eklouh-Molinier, Christophe; Happillon, Teddy; Bouland, Nicole; Fichel, Caroline; Diébold, Marie-Danièle; Angiboust, Jean-François; Manfait, Michel; Brassart-Pasco, Sylvie; Piot, Olivier

    2015-09-21

    Upon chronological aging, human skin undergoes structural and molecular modifications, especially at the level of type I collagen. This macromolecule is one of the main dermal structural proteins and presents several age-related alterations. It exhibits a triple helical structure and assembles itself to form fibrils and fibers. In addition, water plays an important role in stabilizing the collagen triple helix by forming hydrogen-bonds between collagen residues. However, the influence of water on changes of dermal collagen fiber orientation with age has not been yet understood. Polarized-Fourier Transform Infrared (P-FTIR) imaging is an interesting biophotonic approach to determine in situ the orientation of type I collagen fibers, as we have recently shown by comparing skin samples of different ages. In this work, P-FTIR spectral imaging was performed on skin samples from two age groups (35- and 38-year-old on the one hand, 60- and 66-year-old on the other hand), and our analyses were focused on the effect of H2O/D2O substitution. Spectral data were processed with fuzzy C-means (FCM) clustering in order to distinguish different orientations of collagen fibers. We demonstrated that the orientation was altered with aging, and that D2O treatment, affecting primarily highly bound water molecules, is more marked for the youngest skin samples. Collagen-bound water-related spectral markers were also highlighted. Our results suggest a weakening of water/collagen interactions with age. This non-destructive and label-free methodology allows us to understand better the importance of bound water in collagen fiber orientation alterations occurring with skin aging. Obtaining such structural information could find benefits in dermatology as well as in cosmetics.

  10. Skin Collagen Glycation, Glycoxidation, and Crosslinking Are Lower in Subjects With Long-Term Intensive Versus Conventional Therapy of Type 1 Diabetes

    PubMed Central

    Monnier, Vincent M.; Bautista, Oliver; Kenny, David; Sell, David R.; Fogarty, John; Dahms, William; Cleary, Patricia A.; Lachin, John; Genuth, Saul

    2010-01-01

    The relationships between long-term intensive control of glycemia and indicators of skin collagen glycation (furosine), glycoxidation (pentosidine and N∊-[carboxymethyl]-lysine [CML]), and crosslinking (acid and pepsin solubility) were examined in 216 patients with type 1 diabetes from the primary prevention and secondary intervention cohorts of the Diabetes Control and Complications Trial. By comparison with conventional treatment, 5 years of intensive treatment was associated with 30–32% lower furosine, 9% lower pentosidine, 9–13% lower CML, 24% higher acid-soluble collagen, and 50% higher pepsin-soluble collagen. All of these differences were statistically significant in the subjects of the primary prevention cohort (P < 0 .006–0.001) and also of the secondary intervention cohort (P < 0.015–0.001) with the exception of CML and acid-soluble collagen. Age- and duration-adjusted collagen variables were significantly associated with the HbA1c value nearest the biopsy and with cumulative prior HbA1c values. Multiple logistic regression analyses with six nonredundant collagen parameters as independent variables and various expressions of retinopathy, nephropathy, and neuropathy outcomes as dependent variables showed that the complications were significantly associated with the full set of collagen variables. Surprisingly, the percentage of total variance (R2) in complications explained by the collagen variables ranged from 19 to 36% with the intensive treatment and from 14 to 51% with conventional treatment. These associations generally remained significant even after adjustment for HbA1c, and, most unexpectedly, in conventionally treated subjects, glycated collagen was the parameter most consistently associated with diabetic complications. Continued monitoring of these subjects may determine whether glycation products in the skin, and especially the early Amadori product (furosine), have the potential to be predictors of the future risk of developing

  11. Upregulation of distinct collagen transcripts in post-surgery scar tissue: a study of conjunctival fibrosis.

    PubMed

    Seet, Li-Fong; Toh, Li Zhen; Chu, Stephanie W L; Finger, Sharon N; Chua, Jocelyn L L; Wong, Tina T

    2017-06-01

    Excessive accumulation of collagen is often used to assess the development of fibrosis. This study aims to identify collagen genes that define fibrosis in the conjunctiva following glaucoma filtration surgery (GFS). Using the mouse model of GFS, we have identified collagen transcripts that were upregulated in the fibrotic phase of wound healing via RNA-seq. The collagen transcripts that were increased the most were encoded by Col8a1 , Col11a1 and Col8a2 Further analysis of the Col8a1 , Col11a1 and Col8a2 transcripts revealed their increase by 67-, 54- and 18-fold, respectively, in the fibrotic phase, compared with 12-fold for Col1a1 , the most commonly evaluated collagen gene for fibrosis. However, only type I collagen was significantly upregulated at the protein level in the fibrotic phase. Type VIII and type I collagens colocalized in fibrous structures and in ACTA2-positive pericytes, and appeared to compensate for each other in expression levels. Type XI collagen showed low colocalization with both type VIII and type I collagens but can be found in association with macrophages. Furthermore, we show that both mouse and human conjunctival fibroblasts expressed elevated levels of the most highly expressed collagen genes in response to TGFβ2 treatment. Importantly, conjunctival tissues from individuals whose GF surgeries have failed due to scarring showed 3.60- and 2.78-fold increases in type VIII and I collagen transcripts, respectively, compared with those from individuals with no prior surgeries. These data demonstrate that distinct collagen transcripts are expressed at high levels in the conjunctiva after surgery and their unique expression profiles may imply differential influences on the fibrotic outcome. © 2017. Published by The Company of Biologists Ltd.

  12. Biochemical and biophysical characterization of collagens of marine sponge, Ircinia fusca (Porifera: Demospongiae: Irciniidae).

    PubMed

    Pallela, Ramjee; Bojja, Sreedhar; Janapala, Venkateswara Rao

    2011-07-01

    Collagens were isolated and partially characterized from the marine demosponge, Ircinia fusca from Gulf of Mannar (GoM), India, with an aim to develop potentially applicable collagens from unused and under-used resources. The yield of insoluble, salt soluble and acid soluble forms of collagens was 31.71 ± 1.59, 20.69 ± 1.03, and 17.38 ± 0.87 mg/g dry weight, respectively. Trichrome staining, Scanning & Transmission Electron microscopic (SEM & TEM) studies confirmed the presence of collagen in the isolated, terminally globular irciniid filaments. The partially purified (gel filtration chromatography), non-fibrillar collagens appeared as basement type collagenous sheets under light microscopy whereas the purified fibrillar collagens appeared as fibrils with a repeated band periodicity of 67 nm under Atomic Force Microscope (AFM). The non-fibrillar and fibrillar collagens were seen to have affinity for anti-collagen type IV and type I antibodies raised against human collagens, respectively. The macromolecules, i.e., total protein, carbohydrate and lipid contents within the tissues were also quantified. The present information on the three characteristic irciniid collagens (filamentous, fibrillar and non-fibrillar) could assist the future attempts to unravel the therapeutically important, safer collagens from marine sponges for their use in pharmaceutical and cosmeceutical industries. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Lumican Deficiency Results In Cardiomyocyte Hypertrophy With Altered Collagen Assembly

    PubMed Central

    Dupuis, Loren E.; Berger, Matthew G.; Feldman, Samuel; Doucette, Lorna; Fowlkes, Vennece; Chakravarti, Shukti; Thibaudeau, Sarah; Alcala, Nicolas E.; Bradshaw, Amy D.; Kern, Christine B.

    2015-01-01

    The ability of the heart to adapt to increased stress is dependent on modification of its extracellular matrix (ECM) architecture that is established during postnatal development as cardiomyocytes differentiate, a process that is poorly understood. We hypothesized that the small leucine-rich proteoglycan (SLRP) lumican (LUM), which binds collagen and facilitates collagen assembly in other tissues, may play a critical role in establishing the postnatal murine myocardial ECM. Although previous studies suggest LUM deficient mice (lum−/−) exhibit skin anomalies consistent with Ehlers-Danlos syndrome, lum−/− hearts have not been evaluated. These studies show LUM was immunolocalized to non-cardiomyocytes of the cardiac ventricles and its expression increased throughout development. Lumican deficiency resulted in significant (50%) perinatal death and further examination of the lum−/− neonatal hearts revealed an increase in myocardial tissue without a significant increase in cell proliferation. However cardiomyocytes from surviving postnatal day 0 (P0), 1 month (1 mo) and adult (4 mo) lum−/− hearts were significantly larger than their wild type (WT) littermates. Immunohistochemistry revealed that the increased cardiomyocyte size in the lum−/− hearts correlated with alteration of the cardiomyocyte pericellular ECM components collagenα1(I) and the class I SLRP decorin (DCN). Western blot analysis demonstrated that the ratio of glycosaminoglycan (GAG) decorated DCN to core DCN was reduced in P0 and 1 mo lum−/− hearts. There was also a reduction in the β and γ forms of collagenα1(I) in lum−/− hearts. While the total insoluble collagen content was significantly reduced, the fibril size was increased in lum−/− hearts, indicating LUM may play a role in collagen fiber stability and lateral fibril assembly. These results suggest that LUM controls cardiomyocyte growth by regulating the pericellular ECM and also indicates that LUM may coordinate

  14. Live imaging of collagen deposition during skin development and repair in a collagen I - GFP fusion transgenic zebrafish line.

    PubMed

    Morris, Josephine L; Cross, Stephen J; Lu, Yinhui; Kadler, Karl E; Lu, Yongbo; Dallas, Sarah L; Martin, Paul

    2018-06-06

    Fibrillar collagen is a major component of many tissues but has been difficult to image in vivo using transgenic approaches because of problems associated with establishing cells and organisms that generate GFP-fusion collagens that can polymerise into functional fibrils. Here we have developed and characterised GFP and mCherry collagen-I fusion zebrafish lines with basal epidermal-specific expression. We use these lines to reveal the dynamic nature of collagen-I fibril deposition beneath the developing embryonic epidermis, as well as the repair of this collagen meshwork following wounding. Transmission electron microscope studies show that these transgenic lines faithfully reproduce the collagen ultrastructure present in wild type larval skin. During skin development we show that collagen I is deposited by basal epidermal cells initially in fine filaments that are largely randomly orientated but are subsequently aligned into a cross-hatch, orthogonal sub-epithelial network by embryonic day 4. Following skin wounding, we see that sub-epidermal collagen is re-established in the denuded domain, initially as randomly orientated wisps that subsequently become bonded to the undamaged collagen and aligned in a way that recapitulates developmental deposition of sub-epidermal collagen. Crossing our GFP-collagen line against one with tdTomato marking basal epidermal cell membranes reveals how much more rapidly wound re-epithelialisation occurs compared to the re-deposition of collagen beneath the healed epidermis. By use of other tissue specific drivers it will be possible to establish zebrafish lines to enable live imaging of collagen deposition and its remodelling in various other organs in health and disease. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Sorption of sodium hydroxide by type I collagen and bovine corneas.

    PubMed

    Whikehart, D R; Edwards, W C; Pfister, R R

    1991-01-01

    There are no quantitative studies on the uptake of alkali into corneal tissues. To study this phenomenon, both type I collagen and bovine corneas were incubated in sodium hydroxide (NaOH) under varying conditions for periods up to 27.5 h. The sorption (absorption or adsorption) of the alkali to protein and tissue was measured as the quantity of NaOH no longer available for titration to neutrality with hydrochloric acid. Sorption was found to be dependent on the concentration of NaOH (0.01-1 N) but independent of the incubation temperature (4-35 degrees C). In whole cornea, sorption of 1 N NaOH began immediately and increased with time up to 6 h. After 6 h, sorption decreased, together with the observed degradation and solubilization of the tissue. Stripping of the corneal endothelium alone or of the endothelium and epithelium increased sorption in a similar manner when compared to whole corneas for periods up to 4 h. These observations are compatible with ionic and nonionic bonding of hydroxide ions to collagen (including that of the cornea) and the subsequent release of hydroxide ions during hydrolysis of the protein itself. Indirect evidence also suggests the inclusion of quantities of unbound hydroxide ions in hydrated gels of glycosaminoglycans. It is proposed that in a chemical burn of the cornea, alkali is both stored in the tissue (by sorption) and reacted with it (by hydrolysis), without any net consumption of alkali taking place.

  16. Regional alterations of type I collagen in rat tibia induced by skeletal unloading

    NASA Technical Reports Server (NTRS)

    Shiiba, Masashi; Arnaud, Sara B.; Tanzawa, Hideki; Kitamura, Eiji; Yamauchi, Mitsuo

    2002-01-01

    Skeletal unloading induces loss of mineral density in weight-bearing bones that leads to inferior bone mechanical strength. This appears to be caused by a failure of bone formation; however, its mechanisms still are not well understood. The objective of this study was to characterize collagen, the predominant matrix protein in bone, in various regions of tibia of rats that were subjected to skeletal unloading by 4 weeks tail suspension. Sixteen male Sprague-Dawley rats (4 months old) were divided into tail suspension and ambulatory controls (eight rats each). After the tail suspension, tibias from each animal were collected and divided into five regions and collagen was analyzed. The collagen cross-linking and the extent of lysine (Lys) hydroxylation in unloaded bones were significantly altered in proximal epiphysis, diaphysis, and, in particular, proximal metaphysis but not in distal regions. The pool of immature/nonmineralized collagen measured by its extractability with a chaotropic solvent was significantly increased in proximal metaphysis. These results suggest that skeletal unloading induced an accumulation of post-translationally altered nonmineralized collagen and that these changes are bone region specific. These alterations might be caused by impaired osteoblastic function/differentiation resulting in a mineralization defect.

  17. The effect of different collagen modifications for titanium and titanium nitrite surfaces on functions of gingival fibroblasts.

    PubMed

    Ritz, U; Nusselt, T; Sewing, A; Ziebart, T; Kaufmann, K; Baranowski, A; Rommens, P M; Hofmann, Alexander

    2017-01-01

    Targeted modifications of the bulk implant surfaces using bioactive agents provide a promising tool for improvement of the long-term bony and soft tissue integration of dental implants. In this study, we assessed the cellular responses of primary human gingival fibroblasts (HGF) to different surface modifications of titanium (Ti) and titanium nitride (TiN) alloys with type I collagen or cyclic-RGDfK-peptide in order to define a modification improving long-term implants in dental medicine. Employing Ti and TiN implants, we compared the performance of simple dip coating and anodic immobilization of type I collagen that provided collagen layers of two different thicknesses. HGF were seeded on the different coated implants, and adhesion, proliferation, and gene expression were analyzed. Although there were no strong differences in initial cell adhesion between the groups at 2 and 4 hours, we found that all surface modifications induced higher proliferation rates as compared to the unmodified controls. Consistently, gene expression levels of cell adhesion markers (focal adhesion kinase (FAK), integrin beta1, and vinculin), cell differentiation markers (FGFR1, TGFb-R1), extracellular protein markers (type I collagen, vimentin), and cytoskeletal protein marker aktinin-1 were consistently higher in all surface modification groups at two different time points of investigation as compared to the unmodified controls. Our results indicate that simple dip coating of Ti and TiN with collagen is sufficient to induce in vitro cellular responses that are comparable to those of more reliable coating methods like anodic adsorption, chemical cross-linking, or RGD coating. TiN alloys do not possess any positive or adverse effects on HGF. Our results demonstrate a simple, yet effective, method for collagen coating on titanium implants to improve the long term integration and stability of dental implants.

  18. Cyclophilin B Deficiency Causes Abnormal Dentin Collagen Matrix.

    PubMed

    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.

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

    PubMed

    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.

  20. Polymerized-type I collagen downregulates inflammation and improves clinical outcomes in patients with symptomatic knee osteoarthritis following arthroscopic lavage: a randomized, double-blind, and placebo-controlled clinical trial.

    PubMed

    Furuzawa-Carballeda, Janette; Lima, Guadalupe; Llorente, Luis; Nuñez-Álvarez, Carlos; Ruiz-Ordaz, Blanca H; Echevarría-Zuno, Santiago; Hernández-Cuevas, Virgilio

    2012-01-01

    Polymerized-type I collagen (polymerized collagen) is a downmodulator of inflammation and cartilage regenerator biodrug. To evaluate the effect of intraarticular injections of polymerized collagen after arthroscopic lavage on inflammation and clinical improvement in patients with knee osteoarthritis (OA). Patients (n = 19) were treated with 6 intraarticular injections of 2 mL of polymerized collagen (n = 10) or 2 mL of placebo (n = 9) during 3 months. Followup was 3 months. The primary endpoints included Lequesne index, pain on a visual analogue scale (VAS), WOMAC, analgesic usage, the number of Tregs and proinflammatory/anti-inflammatory cytokine-expressing peripheral cells. Secondary outcomes were Likert score and drug evaluation. Clinical and immunological improvement was determined if the decrease in pain exceeds 20 mm on a VAS, 20% of clinical outcomes, and inflammatory parameters from baseline. Urinary levels of C-terminal crosslinking telopeptide of collagen type II (CTXII) and erythrocyte sedimentation rate (ESR) were determined. Polymerized collagen was safe and well tolerated. Patients had a statistically significant improvement (P < 0.05) from baseline versus polymerized collagen and versus placebo at 6 months on Lequesne index, VAS, ESR, Tregs IL-1β, and IL-10 peripheral-expressing cells. Urinary levels of CTXII were decreased 44% in polymerized collagen versus placebo. No differences were found on incidence of adverse events between groups. Polymerized collagen is safe and effective on downregulation of inflammation in patients with knee OA.

  1. Polymerized-Type I Collagen Downregulates Inflammation and Improves Clinical Outcomes in Patients with Symptomatic Knee Osteoarthritis Following Arthroscopic Lavage: A Randomized, Double-Blind, and Placebo-Controlled Clinical Trial

    PubMed Central

    Furuzawa-Carballeda, Janette; Lima, Guadalupe; Llorente, Luis; Nuñez-Álvarez, Carlos; Ruiz-Ordaz, Blanca H.; Echevarría-Zuno, Santiago; Hernández-Cuevas, Virgilio

    2012-01-01

    Objectives. Polymerized-type I collagen (polymerized collagen) is a downmodulator of inflammation and cartilage regenerator biodrug. Aim. To evaluate the effect of intraarticular injections of polymerized collagen after arthroscopic lavage on inflammation and clinical improvement in patients with knee osteoarthritis (OA). Methods. Patients (n = 19) were treated with 6 intraarticular injections of 2 mL of polymerized collagen (n = 10) or 2 mL of placebo (n = 9) during 3 months. Followup was 3 months. The primary endpoints included Lequesne index, pain on a visual analogue scale (VAS), WOMAC, analgesic usage, the number of Tregs and proinflammatory/anti-inflammatory cytokine-expressing peripheral cells. Secondary outcomes were Likert score and drug evaluation. Clinical and immunological improvement was determined if the decrease in pain exceeds 20 mm on a VAS, 20% of clinical outcomes, and inflammatory parameters from baseline. Urinary levels of C-terminal crosslinking telopeptide of collagen type II (CTXII) and erythrocyte sedimentation rate (ESR) were determined. Results. Polymerized collagen was safe and well tolerated. Patients had a statistically significant improvement (P < 0.05) from baseline versus polymerized collagen and versus placebo at 6 months on Lequesne index, VAS, ESR, Tregs IL-1β, and IL-10 peripheral-expressing cells. Urinary levels of CTXII were decreased 44% in polymerized collagen versus placebo. No differences were found on incidence of adverse events between groups. Conclusion. Polymerized collagen is safe and effective on downregulation of inflammation in patients with knee OA. PMID:22545014

  2. Monitoring fibrous scaffold guidance of three-dimensional collagen organisation using minimally-invasive second harmonic generation.

    PubMed

    Delaine-Smith, Robin M; Green, Nicola H; Matcher, Stephen J; MacNeil, Sheila; Reilly, Gwendolen C

    2014-01-01

    The biological and mechanical function of connective tissues is largely determined by controlled cellular alignment and therefore it seems appropriate that tissue-engineered constructs should be architecturally similar to the in vivo tissue targeted for repair or replacement. Collagen organisation dictates the tensile properties of most tissues and so monitoring the deposition of cell-secreted collagen as the construct develops is essential for understanding tissue formation. In this study, electrospun fibres with a random or high degree of orientation, mimicking two types of tissue architecture found in the body, were used to culture human fibroblasts for controlling cell alignment. The minimally-invasive technique of second harmonic generation was used with the aim of monitoring and profiling the deposition and organisation of collagen at different construct depths over time while construct mechanical properties were also determined over the culture period. It was seen that scaffold fibre organisation affected cell migration and orientation up to 21 days which in turn had an effect on collagen organisation. Collagen in random fibrous constructs was deposited in alternating configurations at different depths however a high degree of organisation was observed throughout aligned fibrous constructs orientated in the scaffold fibre direction. Three-dimensional second harmonic generation images showed that deposited collagen was more uniformly distributed in random constructs but aligned constructs were more organised and had higher intensities. The tensile properties of all constructs increased with increasing collagen deposition and were ultimately dictated by collagen organisation. This study highlights the importance of scaffold architecture for controlling the development of well-organised tissue engineered constructs and the usefulness of second harmonic generation imaging for monitoring collagen maturation in a minimally invasive manner.

  3. Deposition of collagen type I onto skeletal endothelium reveals a new role for blood vessels in regulating bone morphology

    PubMed Central

    Ben Shoham, Adi; Rot, Chagai; Stern, Tomer; Krief, Sharon; Akiva, Anat; Dadosh, Tali; Sabany, Helena; Lu, Yinhui; Kadler, Karl E.

    2016-01-01

    Recently, blood vessels have been implicated in the morphogenesis of various organs. The vasculature is also known to be essential for endochondral bone development, yet the underlying mechanism has remained elusive. We show that a unique composition of blood vessels facilitates the role of the endothelium in bone mineralization and morphogenesis. Immunostaining and electron microscopy showed that the endothelium in developing bones lacks basement membrane, which normally isolates the blood vessel from its surroundings. Further analysis revealed the presence of collagen type I on the endothelial wall of these vessels. Because collagen type I is the main component of the osteoid, we hypothesized that the bone vasculature guides the formation of the collagenous template and consequently of the mature bone. Indeed, some of the bone vessels were found to undergo mineralization. Moreover, the vascular pattern at each embryonic stage prefigured the mineral distribution pattern observed one day later. Finally, perturbation of vascular patterning by overexpressing Vegf in osteoblasts resulted in abnormal bone morphology, supporting a role for blood vessels in bone morphogenesis. These data reveal the unique composition of the endothelium in developing bones and indicate that vascular patterning plays a role in determining bone shape by forming a template for deposition of bone matrix. PMID:27621060

  4. Effects of flexibility of the α2 chain of type I collagen on collagenase cleavage.

    PubMed

    Mekkat, Arya; Poppleton, Erik; An, Bo; Visse, Robert; Nagase, Hideaki; Kaplan, David L; Brodsky, Barbara; Lin, Yu-Shan

    2018-05-12

    Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage. Copyright © 2018. Published by Elsevier Inc.

  5. Interaction of von Willebrand factor domains with collagen investigated by single molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Posch, Sandra; Obser, Tobias; König, Gesa; Schneppenheim, Reinhard; Tampé, Robert; Hinterdorfer, Peter

    2018-03-01

    von Willebrand factor (VWF) is a huge multimeric protein that plays a key role in primary hemostasis. Sites for collagen binding, an initial event of hemostasis, are located in the VWF-domains A1 and A3. In this study, we investigated single molecule interactions between collagen surfaces and wild type VWF A1A2A3 domain constructs, as well as clinically relevant VWF A3 domain point mutations, such as p.Ser1731Thr, p.Gln1734His, and p.His1786Arg. For this, we utilized atomic force microscopy based single molecular force spectroscopy. The p.Ser1731Thr mutant had no impact on the VWF-collagen type III and VI interactions, while the p.Gln1734His and p.His1786Arg mutants showed a slight increase in bond stability to collagen type III. This effect probably arises from additional hydrogen bonds that come along with the introduction of these mutations. Using the same mutants, but collagen type VI as a binding partner, resulted in a significant increase in bond stability. VWF domain A1 was reported to be essential for the interaction with collagen type VI and thus our findings strengthen the hypothesis that the VWF A1 domain can compensate for mutations in the VWF A3 domain. Additionally, our data suggest that the mutations could even stabilize the interaction between VWF and collagen without shear. VWF-collagen interactions seem to be an important system in which defective interactions between one VWF domain and one type of collagen can be compensated by alternative binding events.

  6. Advanced glycation end-products and methionine sulphoxide in skin collagen of patients with type 1 diabetes.

    PubMed

    Yu, Y; Thorpe, S R; Jenkins, A J; Shaw, J N; Sochaski, M A; McGee, D; Aston, C E; Orchard, T J; Silvers, N; Peng, Y G; McKnight, J A; Baynes, J W; Lyons, T J

    2006-10-01

    We determined whether oxidative damage in collagen is increased in (1) patients with diabetes; (2) patients with diabetic complications; and (3) subjects from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) study, with comparison of subjects from the former standard vs intensive treatment groups 4 years after DCCT completion. We quantified the early glycation product fructose-lysine, the two AGEs N (epsilon)-(carboxymethyl)lysine (CML) and pentosidine, and the oxidised amino acid methionine sulphoxide (MetSO) in skin collagen from 96 patients with type 1 diabetes (taken from three groups: DCCT/EDIC patients and clinic patients from South Carolina and Scotland) and from 78 healthy subjects. Fructose-lysine was increased in diabetic patients (p<0.0001), both with or without complications (p<0.0001). Controlling for HbA(1c), rates of accumulation of AGEs were higher in diabetic patients than control subjects, regardless of whether the former had complications (CML and pentosidine given as log(e)[pentosidine]) or not (CML only) (all p<0.0001). MetSO (log(e)[MetSO]) also accumulated more rapidly in diabetic patients with complications than in controls (p<0.0001), but rates were similar in patients without complications and controls. For all three products, rates of accumulation with age were significantly higher in diabetic patients with complications than in those without (all p<0.0001). At 4 years after the end of the DCCT, no differences were found between the previous DCCT management groups for fructose-lysine, AGEs or MetSO. The findings suggest that in type 1 diabetic patients enhanced oxidative damage to collagen is associated with the presence of vascular complications.

  7. Collagen-PVA aligned nanofiber on collagen sponge as bi-layered scaffold for surface cartilage repair.

    PubMed

    Lin, Hsin-Yi; Tsai, Wen-Chi; Chang, Shih-Hsing

    2017-05-01

    Researchers have made bi-layered scaffolds but mostly for osteochondral repairs. The anatomic structure of human cartilage has different zones and that each has varying matrix morphology and mechanical properties is often overlooked. Two bi-layered collagen-based composites were made to replicate the superficial and transitional zones of an articular cartilage. Aligned and random collagen-PVA nanofibers were electrospun onto a freeze-dried collagen sponge to make the aligned and random composites, respectively. The morphology, swelling ratio, degradation and tensile properties of the two composites were examined. Primary porcine chondrocytes were cultured on the composites for three weeks and their proliferation and secretion of glycosaminoglycan (GAG) and type II collagen were measured. The influences of the cell culture on the tensile properties of the composites were studied. The nanofiber layer remained adhered to the sponge after three weeks of cell culture. Both composites lost 30-35% of their total weight in a saline buffer after three weeks. The tensile strength and Young's modulus of both composites increased after three weeks of chondrocyte culture (p < 0.05). The aligned composite with extracellular matrix deposition had a Young's modulus (0.35 MPa) similar to that of articular cartilage reported in literature (0.36-0.8 MPa). The chondrocytes on both aligned and random composites proliferated and secreted similar amounts of GAG and type II collagen. They were seen embedded in lacunae after three weeks. The aligned composite may be more suitable for articular cartilage repair because of the higher tensile strength from the aligned nanofibers on the surface that can better resist wear.

  8. Autoantibody profile in the experimental model of scleroderma induced by type V human collagen

    PubMed Central

    Callado, Maria R M; Viana, Vilma S T; Vendramini, Margarete B G; Leon, Elaine P; Bueno, Cleonice; Velosa, Ana P P; Teodoro, Walcy R; Yoshinari, Natalino H

    2007-01-01

    The aim of this study is to evaluate the humoral autoimmune response in the experimental model of systemic sclerosis (SSc) induced by human type V collagen (huCol V). New Zealand rabbits were immunized with huCol V in Freund's complete adjuvant (FCA) and boosted twice with 15 days intervals with huCol V in Freund's incomplete adjuvant. Control groups included animals injected only with FCA or bovine serum albumin. Bleeding was done at days 0, 30, 75 and 120. Tissue specimens were obtained for histopathological investigation. Serological analysis included detection of antibodies against huCol V and anti-topoisomerase I (Anti-Scl70) by enzyme-linked immunosorbent assay, antinuclear antibodies (ANA) by indirect immunofluorescence, and rheumatoid factor (RF) by a latex agglutination test. Target antigens were characterized by immunoblot. Histological analysis revealed extracellular matrix remodeling with fibrosis and vasculitis. Anti-Scl70 and ANA were detected as early as 30 days in all huCol V animals. The universal ANA staining pattern was Golgi-like. This serum reactivity was not abolished by previous absorption with huCol V. Characterization of the target antigen by immunoblot revealed two major protein fractions of 175 000 and 220 000 MW. Similarly to ANA, there was a gradual increase of reactivity throughout the immunization and also it was not abolished by preincubation of serum samples with huCol V. RF testing was negative in hyperimmune sera. Conclusion: The production of autoantibodies, including anti-Scl70, a serological marker for SSc associated with histopathological alterations, validates huCol V induced-experimental model and brings out its potential for understanding the pathophysiology of SSc. PMID:17442023

  9. Abnormal Collagen Metabolism in Cultured Skin Fibroblasts from Patients with Duchenne Muscular Dystrophy

    NASA Astrophysics Data System (ADS)

    Rodemann, H. Peter; Bayreuther, Klaus

    1984-08-01

    Total collagen synthesis is decreased by about 29% (P < 0.01) in skin fibroblasts established in vitro from male patients with Duchenne muscular dystrophy (DMD) as compared with that in normal male skin fibroblasts in vitro. The reduction in collagen synthesis is associated with an approximately 2-fold increase in collagen degradation in DMD fibroblasts. Correlated to these alterations in the metabolism of collagen, DMD fibroblasts express a significantly higher hydroxyproline/proline ratio (DMD: 1.36-1.45; P < 0.01) than do normal fibroblasts (controls: 0.86-0.89). The increased hydroxylation of proline residues of collagen (composed of type I and type III) could be the cause for the enhanced degradation of collagen in DMD fibroblasts.

  10. Comparative Proteomic Analysis of Normal and Collagen IX Null Mouse Cartilage Reveals Altered Extracellular Matrix Composition and Novel Components of the Collagen IX Interactome*

    PubMed Central

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

    2013-01-01

    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, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGFβ-induced protein βig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the

  11. Type VII collagen regulates expression of OATP1B3, promotes front-to-rear polarity and increases structural organisation in 3D spheroid cultures of RDEB tumour keratinocytes

    PubMed Central

    Dayal, Jasbani H. S.; Cole, Clare L.; Pourreyron, Celine; Watt, Stephen A.; Lim, Yok Zuan; Salas-Alanis, Julio C.; Murrell, Dedee F.; McGrath, John A.; Stieger, Bruno; Jahoda, Colin; Leigh, Irene M.; South, Andrew P.

    2014-01-01

    ABSTRACT Type VII collagen is the main component of anchoring fibrils, structures that are integral to basement membrane homeostasis in skin. Mutations in the gene encoding type VII collagen COL7A1 cause recessive dystrophic epidermolysis bullosa (RDEB) an inherited skin blistering condition complicated by frequent aggressive cutaneous squamous cell carcinoma (cSCC). OATP1B3, which is encoded by the gene SLCO1B3, is a member of the OATP (organic anion transporting polypeptide) superfamily responsible for transporting a wide range of endogenous and xenobiotic compounds. OATP1B3 expression is limited to the liver in healthy tissues, but is frequently detected in multiple cancer types and is reported to be associated with differing clinical outcome. The mechanism and functional significance of tumour-specific expression of OATP1B3 has yet to be determined. Here, we identify SLCO1B3 expression in tumour keratinocytes isolated from RDEB and UV-induced cSCC and demonstrate that SLCO1B3 expression and promoter activity are modulated by type VII collagen. We show that reduction of SLCO1B3 expression upon expression of full-length type VII collagen in RDEB cSCC coincides with acquisition of front-to-rear polarity and increased organisation of 3D spheroid cultures. In addition, we show that type VII collagen positively regulates the abundance of markers implicated in cellular polarity, namely ELMO2, PAR3, E-cadherin, B-catenin, ITGA6 and Ln332. PMID:24357722

  12. Release of antibiotics from collagen dressing.

    PubMed

    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.

  13. The role of the non-collagenous matrix in tendon function.

    PubMed

    Thorpe, Chavaunne T; Birch, Helen L; Clegg, Peter D; Screen, Hazel R C

    2013-08-01

    Tendon consists of highly ordered type I collagen molecules that are grouped together to form subunits of increasing diameter. At each hierarchical level, the type I collagen is interspersed with a predominantly non-collagenous matrix (NCM) (Connect. Tissue Res., 6, 1978, 11). Whilst many studies have investigated the structure, organization and function of the collagenous matrix within tendon, relatively few have studied the non-collagenous components. However, there is a growing body of research suggesting the NCM plays an important role within tendon; adaptations to this matrix may confer the specific properties required by tendons with different functions. Furthermore, age-related alterations to non-collagenous proteins have been identified, which may affect tendon resistance to injury. This review focuses on the NCM within the tensional region of developing and mature tendon, discussing the current knowledge and identifying areas that require further study to fully understand structure-function relationships within tendon. This information will aid in the development of appropriate techniques for tendon injury prevention and treatment. © 2013 The Authors. International Journal of Experimental Pathology © 2013 International Journal of Experimental Pathology.

  14. Photo-induced processes in collagen-hypericin system revealed by fluorescence spectroscopy and multiphoton microscopy

    PubMed Central

    Hovhannisyan, V.; Guo, H. W.; Hovhannisyan, A.; Ghukasyan, V.; Buryakina, T.; Chen, Y. F.; Dong, C. Y.

    2014-01-01

    Collagen is the main structural protein and the key determinant of mechanical and functional properties of tissues and organs. Proper balance between synthesis and degradation of collagen molecules is critical for maintaining normal physiological functions. In addition, collagen influences tumor development and drug delivery, which makes it a potential cancer therapy target. Using second harmonic generation, two-photon excited fluorescence microscopy, and spectrofluorimetry, we show that the natural pigment hypericin induces photosensitized destruction of collagen-based tissues. We demonstrate that hypericin–mediated processes in collagen fibers are irreversible and may be used for the treatment of cancer and collagen-related disorders. PMID:24877000

  15. Photo-induced processes in collagen-hypericin system revealed by fluorescence spectroscopy and multiphoton microscopy.

    PubMed

    Hovhannisyan, V; Guo, H W; Hovhannisyan, A; Ghukasyan, V; Buryakina, T; Chen, Y F; Dong, C Y

    2014-05-01

    Collagen is the main structural protein and the key determinant of mechanical and functional properties of tissues and organs. Proper balance between synthesis and degradation of collagen molecules is critical for maintaining normal physiological functions. In addition, collagen influences tumor development and drug delivery, which makes it a potential cancer therapy target. Using second harmonic generation, two-photon excited fluorescence microscopy, and spectrofluorimetry, we show that the natural pigment hypericin induces photosensitized destruction of collagen-based tissues. We demonstrate that hypericin-mediated processes in collagen fibers are irreversible and may be used for the treatment of cancer and collagen-related disorders.

  16. Particulate wear debris activates protein tyrosine kinases and nuclear factor kappaB, which down-regulates type I collagen synthesis in human osteoblasts.

    PubMed

    Vermes, C; Roebuck, K A; Chandrasekaran, R; Dobai, J G; Jacobs, J J; Glant, T T

    2000-09-01

    Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a variety of cell types within the periprosthetic space including osteoblasts, which cells with an altered function may contribute to periprosthetic osteolysis. Exposure of osteoblast-like osteosarcoma cells or bone marrow-derived primary osteoblasts to either metallic or polymeric particles of phagocytosable sizes resulted in a marked decrease in the steady-state messenger RNA (mRNA) levels of procollagen alpha1[I] and procollagen alpha1[III]. In contrast, no significant effect was observed for the osteoblast-specific genes, such as osteonectin and osteocalcin (OC). In kinetic studies, particles once phagocytosed, maintained a significant suppressive effect on collagen gene expression and type I collagen synthesis for up to five passages. Large particles of a size that cannot be phagocytosed also down-regulated collagen gene expression suggesting that an initial contact between cells and particles can generate gene responsive signals independently of the phagocytosis process. Concerning such signaling, titanium particles rapidly increased protein tyrosine phosphorylation and nuclear transcription factor kappaB (NF-kappaB) binding activity before the phagocytosis of particles. Protein tyrosine kinase (PTK) inhibitors such as genistein and the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the suppressive effect of titanium on collagen gene expression suggesting particles suppress collagen gene expression through the NF-kappaB signaling pathway. These results provide a mechanism by which particulate wear debris can antagonize the transcription of the procollagen alpha1[I] gene in osteoblasts, which may contribute to reduced bone formation and progressive periprosthetic osteolysis.

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

    PubMed

    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

  18. Osteogenesis imperfecta type I: Molecular heterogeneity for COL1A1 null alleles of type I collagen

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Willing, M.C.; Deschenes, S.P.; Pitts, S.H.

    Osteogenesis imperfecta (OI) type I is the mildest form of inherited brittle-bone disease. Dermal fibroblasts from most affected individuals produce about half the usual amount of type I procollagen, as a result of a COL1A1 {open_quotes}null{close_quotes} allele. Using PCR amplification of genomic DNA from affected individuals, followed by denaturing gradient gel electrophoresis (DGGE) and SSCP, we identified seven different COL1A1 gene mutations in eight unrelated families with OI type I. Three families have single nucleotide substitutions that alter 5{prime} donor splice sites; two of these unrelated families have the same mutation. One family has a point mutation, in an exon,more » that creates a premature termination codon, and four have small deletions or insertions, within exons, that create translational frameshifts and new termination codons downstream of the mutation sites. Each mutation leads to both marked reduction in steady-state levels of mRNA from the mutant allele and a quantitative decrease in type I procollagen production. Our data demonstrate that different molecular mechanisms that have the same effect on type I collagen production result in the same clinical phenotype. 58 refs., 4 figs., 1 tab.« less

  19. Effect of collagen turnover on the accumulation of advanced glycation end products.

    PubMed

    Verzijl, N; DeGroot, J; Thorpe, S R; Bank, R A; Shaw, J N; Lyons, T J; Bijlsma, J W; Lafeber, F P; Baynes, J W; TeKoppele, J M

    2000-12-15

    Collagen molecules in articular cartilage have an exceptionally long lifetime, which makes them susceptible to the accumulation of advanced glycation end products (AGEs). In fact, in comparison to other collagen-rich tissues, articular cartilage contains relatively high amounts of the AGE pentosidine. To test the hypothesis that this higher AGE accumulation is primarily the result of the slow turnover of cartilage collagen, AGE levels in cartilage and skin collagen were compared with the degree of racemization of aspartic acid (% d-Asp, a measure of the residence time of a protein). AGE (N(epsilon)-(carboxymethyl)lysine, N(epsilon)-(carboxyethyl)lysine, and pentosidine) and % d-Asp concentrations increased linearly with age in both cartilage and skin collagen (p < 0.0001). The rate of increase in AGEs was greater in cartilage collagen than in skin collagen (p < 0.0001). % d-Asp was also higher in cartilage collagen than in skin collagen (p < 0.0001), indicating that cartilage collagen has a longer residence time in the tissue, and thus a slower turnover, than skin collagen. In both types of collagen, AGE concentrations increased linearly with % d-Asp (p < 0.0005). Interestingly, the slopes of the curves of AGEs versus % d-Asp, i.e. the rates of accumulation of AGEs corrected for turnover, were identical for cartilage and skin collagen. The present study thus provides the first experimental evidence that protein turnover is a major determinant in AGE accumulation in different collagen types. From the age-related increases in % d-Asp the half-life of cartilage collagen was calculated to be 117 years and that of skin collagen 15 years, thereby providing the first reasonable estimates of the half-lives of these collagens.

  20. Immunohistochemical and scanning electron microscopic comparison of the collagen network constructions between pig, goat and chicken livers.

    PubMed

    Nishimura, Shotaro; Sagara, Ayano; Oshima, Ichiro; Ono, Yoshitaka; Iwamoto, Hisao; Okano, Kaoru; Miyachi, Hideyuki; Tabata, Shoji

    2009-08-01

    The distribution and three-dimensional architecture of collagen fibers were compared between pig, goat and chicken livers. Immunohistochemical staining revealed that collagen type I was identified in the interlobular connective tissue region and intralobular areas in pigs and goats. Type III collagen was also identified in the interlobular connective tissue region and intralobular sinusoidal walls. In the chicken liver, only the circumference region of the vessels was immunostained with collagen type I and III antibodies and the interlobular connective tissue wall could not be distinguished clearly. In the intralobular region, collagen type I antibody immunoreacted around the hepatic cells but collagen type III antibody immunoreacted weakly. In the NaOH macerated specimen, well-developed collagen bundles formed the prominent interlobular walls in pigs. In contrast, the wall in the goat liver comprised a thin layer of the bundles. In the chicken liver, there were no notable collagen septa between lobules. The intralobular collagen construction was quite different between the animals, indicating a fragile collagen fibril networks in pigs, a robust framework in goats and dense fabric-like septa in chickens. These results indicate that the distinct collagen frameworks may contribute to the histological strength of the livers in each of the animal species.

  1. Dendritic cells: In vitro culture in two- and three-dimensional collagen systems and expression of collagen receptors in tumors and atherosclerotic microenvironments.

    PubMed

    Sprague, Leslee; Muccioli, Maria; Pate, Michelle; Singh, Manindra; Xiong, Chengkai; Ostermann, Alexander; Niese, Brandon; Li, Yihan; Li, Yandi; Courreges, Maria Cecilia; Benencia, Fabian

    2014-04-15

    Dendritic cells (DCs) are immune cells found in the peripheral tissues where they sample the organism for infections or malignancies. There they take up antigens and migrate towards immunological organs to contact and activate T lymphocytes that specifically recognize the antigen presented by these antigen presenting cells. In the steady state there are several types of resident DCs present in various different organs. For example, in the mouse, splenic DC populations characterized by the co-expression of CD11c and CD8 surface markers are specialized in cross-presentation to CD8 T cells, while CD11c/SIRP-1α DCs seem to be dedicated to activating CD4 T cells. On the other hand, DCs have also been associated with the development of various diseases such as cancer, atherosclerosis, or inflammatory conditions. In such disease, DCs can participate by inducing angiogenesis or immunosuppression (tumors), promoting autoimmune responses, or exacerbating inflammation (atherosclerosis). This change in DC biology can be prompted by signals in the microenvironment. We have previously shown that the interaction of DCs with various extracellular matrix components modifies the immune properties and angiogenic potential of these cells. Building on those studies, herewith we analyzed the angiogenic profile of murine myeloid DCs upon interaction with 2D and 3D type-I collagen environments. As determined by PCR array technology and quantitative PCR analysis we observed that interaction with these collagen environments induced the expression of particular angiogenic molecules. In addition, DCs cultured on collagen environments specifically upregulated the expression of CXCL-1 and -2 chemokines. We were also able to establish DC cultures on type-IV collagen environments, a collagen type expressed in pathological conditions such as atherosclerosis. When we examined DC populations in atherosclerotic veins of Apolipoprotein E deficient mice we observed that they expressed adhesion

  2. Toward angiogenesis of implanted bio-artificial liver using scaffolds with type I collagen and adipose tissue-derived stem cells

    PubMed Central

    Lee, Jae Geun; Bak, Seon Young; Nahm, Ji Hae; Lee, Sang Woo; Min, Seon Ok

    2015-01-01

    Backgrounds/Aims Stem cell therapies for liver disease are being studied by many researchers worldwide, but scientific evidence to demonstrate the endocrinologic effects of implanted cells is insufficient, and it is unknown whether implanted cells can function as liver cells. Achieving angiogenesis, arguably the most important characteristic of the liver, is known to be quite difficult, and no practical attempts have been made to achieve this outcome. We carried out this study to observe the possibility of angiogenesis of implanted bio-artificial liver using scaffolds. Methods This study used adipose tissue-derived stem cells that were collected from adult patients with liver diseases with conditions similar to the liver parenchyma. Specifically, microfilaments were used to create an artificial membrane and maintain the structure of an artificial organ. After scratching the stomach surface of severe combined immunocompromised (SCID) mice (n=4), artificial scaffolds with adipose tissue-derived stem cells and type I collagen were implanted. Expression levels of angiogenesis markers including vascular endothelial growth factor (VEGF), CD34, and CD105 were immunohistochemically assessed after 30 days. Results Grossly, the artificial scaffolds showed adhesion to the stomach and surrounding organs; however, there was no evidence of angiogenesis within the scaffolds; and VEGF, CD34, and CD105 expressions were not detected after 30 days. Conclusions Although implantation of cells into artificial scaffolds did not facilitate angiogenesis, the artificial scaffolds made with type I collagen helped maintain implanted cells, and surrounding tissue reactions were rare. Our findings indicate that type I collagen artificial scaffolds can be considered as a possible implantable biomaterial. PMID:26155277

  3. Toward angiogenesis of implanted bio-artificial liver using scaffolds with type I collagen and adipose tissue-derived stem cells.

    PubMed

    Lee, Jae Geun; Bak, Seon Young; Nahm, Ji Hae; Lee, Sang Woo; Min, Seon Ok; Kim, Kyung Sik

    2015-05-01

    Stem cell therapies for liver disease are being studied by many researchers worldwide, but scientific evidence to demonstrate the endocrinologic effects of implanted cells is insufficient, and it is unknown whether implanted cells can function as liver cells. Achieving angiogenesis, arguably the most important characteristic of the liver, is known to be quite difficult, and no practical attempts have been made to achieve this outcome. We carried out this study to observe the possibility of angiogenesis of implanted bio-artificial liver using scaffolds. This study used adipose tissue-derived stem cells that were collected from adult patients with liver diseases with conditions similar to the liver parenchyma. Specifically, microfilaments were used to create an artificial membrane and maintain the structure of an artificial organ. After scratching the stomach surface of severe combined immunocompromised (SCID) mice (n=4), artificial scaffolds with adipose tissue-derived stem cells and type I collagen were implanted. Expression levels of angiogenesis markers including vascular endothelial growth factor (VEGF), CD34, and CD105 were immunohistochemically assessed after 30 days. Grossly, the artificial scaffolds showed adhesion to the stomach and surrounding organs; however, there was no evidence of angiogenesis within the scaffolds; and VEGF, CD34, and CD105 expressions were not detected after 30 days. Although implantation of cells into artificial scaffolds did not facilitate angiogenesis, the artificial scaffolds made with type I collagen helped maintain implanted cells, and surrounding tissue reactions were rare. Our findings indicate that type I collagen artificial scaffolds can be considered as a possible implantable biomaterial.

  4. Tissue-engineered cartilaginous constructs for the treatment of caprine cartilage defects, including distribution of laminin and type IV collagen.

    PubMed

    Jeng, Lily; Hsu, Hu-Ping; Spector, Myron

    2013-10-01

    The purpose of this study was the immunohistochemical evaluation of (1) cartilage tissue-engineered constructs; and (2) the tissue filling cartilage defects in a goat model into which the constructs were implanted, particularly for the presence of the basement membrane molecules, laminin and type IV collagen. Basement membrane molecules are localized to the pericellular matrix in normal adult articular cartilage, but have not been examined in tissue-engineered constructs cultured in vitro or in tissue filling cartilage defects into which the constructs were implanted. Cartilaginous constructs were engineered in vitro using caprine chondrocyte-seeded type II collagen scaffolds. Autologous constructs were implanted into 4-mm-diameter defects created to the tidemark in the trochlear groove in the knee joints of skeletally mature goats. Eight weeks after implantation, the animals were sacrificed. Constructs underwent immunohistochemical and histomorphometric evaluation. Widespread staining for the two basement membrane molecules was observed throughout the extracellular matrix of in vitro and in vivo samples in a distribution unlike that previously reported for cartilage. At sacrifice, 70% of the defect site was filled with reparative tissue, which consisted largely of fibrous tissue and some fibrocartilage, with over 70% of the reparative tissue bonded to the adjacent host tissue. A novel finding of this study was the observation of laminin and type IV collagen in in vitro engineered cartilaginous constructs and in vivo cartilage repair samples from defects into which the constructs were implanted, as well as in normal caprine articular cartilage. Future work is needed to elucidate the role of basement membrane molecules during cartilage repair and regeneration.

  5. Tissue-Engineered Cartilaginous Constructs for the Treatment of Caprine Cartilage Defects, Including Distribution of Laminin and Type IV Collagen

    PubMed Central

    Jeng, Lily; Hsu, Hu-Ping

    2013-01-01

    The purpose of this study was the immunohistochemical evaluation of (1) cartilage tissue-engineered constructs; and (2) the tissue filling cartilage defects in a goat model into which the constructs were implanted, particularly for the presence of the basement membrane molecules, laminin and type IV collagen. Basement membrane molecules are localized to the pericellular matrix in normal adult articular cartilage, but have not been examined in tissue-engineered constructs cultured in vitro or in tissue filling cartilage defects into which the constructs were implanted. Cartilaginous constructs were engineered in vitro using caprine chondrocyte-seeded type II collagen scaffolds. Autologous constructs were implanted into 4-mm-diameter defects created to the tidemark in the trochlear groove in the knee joints of skeletally mature goats. Eight weeks after implantation, the animals were sacrificed. Constructs underwent immunohistochemical and histomorphometric evaluation. Widespread staining for the two basement membrane molecules was observed throughout the extracellular matrix of in vitro and in vivo samples in a distribution unlike that previously reported for cartilage. At sacrifice, 70% of the defect site was filled with reparative tissue, which consisted largely of fibrous tissue and some fibrocartilage, with over 70% of the reparative tissue bonded to the adjacent host tissue. A novel finding of this study was the observation of laminin and type IV collagen in in vitro engineered cartilaginous constructs and in vivo cartilage repair samples from defects into which the constructs were implanted, as well as in normal caprine articular cartilage. Future work is needed to elucidate the role of basement membrane molecules during cartilage repair and regeneration. PMID:23672504

  6. Activated hepatic stellate cells are dependent on self-collagen, cleaved by membrane type 1 matrix metalloproteinase for their growth.

    PubMed

    Birukawa, Naoko Kubo; Murase, Kazuyuki; Sato, Yasushi; Kosaka, Akemi; Yoneda, Akihiro; Nishita, Hiroki; Fujita, Ryosuke; Nishimura, Miyuki; Ninomiya, Takafumi; Kajiwara, Keiko; Miyazaki, Miyono; Nakashima, Yusuke; Ota, Sigenori; Murakami, Yuya; Tanaka, Yasunobu; Minomi, Kenjiro; Tamura, Yasuaki; Niitsu, Yoshiro

    2014-07-18

    Stellate cells are distributed throughout organs, where, upon chronic damage, they become activated and proliferate to secrete collagen, which results in organ fibrosis. An intriguing property of hepatic stellate cells (HSCs) is that they undergo apoptosis when collagen is resolved by stopping tissue damage or by treatment, even though the mechanisms are unknown. Here we disclose the fact that HSCs, normal diploid cells, acquired dependence on collagen for their growth during the transition from quiescent to active states. The intramolecular RGD motifs of collagen were exposed by cleavage with their own membrane type 1 matrix metalloproteinase (MT1-MMP). The following evidence supports this conclusion. When rat activated HSCs (aHSCs) were transduced with siRNA against the collagen-specific chaperone gp46 to inhibit collagen secretion, the cells underwent autophagy followed by apoptosis. Concomitantly, the growth of aHSCs was suppressed, whereas that of quiescent HSCs was not. These in vitro results are compatible with the in vivo observation that apoptosis of aHSCs was induced in cirrhotic livers of rats treated with siRNAgp46. siRNA against MT1-MMP and addition of tissue inhibitor of metalloproteinase 2 (TIMP-2), which mainly inhibits MT1-MMP, also significantly suppressed the growth of aHSCs in vitro. The RGD inhibitors echistatin and GRGDS peptide and siRNA against the RGD receptor αVβ1 resulted in the inhibition of aHSCs growth. Transduction of siRNAs against gp46, αVβ1, and MT1-MMP to aHSCs inhibited the survival signal of PI3K/AKT/IκB. These results could provide novel antifibrosis strategies. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Development and testing of a human collagen graft material.

    PubMed

    Quteish, D; Singh, G; Dolby, A E

    1990-06-01

    Human Type I collagen was extracted from placenta using pepsin and salt fractionation. The collagen was characterized by SDS-PAG electrophoresis dispersed in acidic medium, freeze-dried, and cross-linked in an 0.25% glutaraldehyde solution pH 4.5 for 2 days. After washing for 7 days and freeze drying the resultant collagen sponge was tested with regard to mechanical, physical, enzymatic degradation properties and biological responses. The modulus of elasticity was found to be 289 +/- 10 g/mm2 and the sponge was insoluble in water, buffered saline, or tissue culture medium over a period of 6 weeks with swelling occurring at less than 5% of volume. The sponge had a high fluid binding capacity, amounting to 56 +/- 5 mL tissue culture medium per gram of dry weight. Bacterial collagenase produced slow degradation of the sponge with complete disappearance by 24 h only when high concentrations (200 units enzyme per mg of the collagen sponge) were used. Cytotoxicity studies using human gingival and periodontal ligament fibroblasts revealed less than 5% apparent cytotoxicity or proliferation. Subcutaneous implantation was followed by resorption and vascularization over a period of 6-8 weeks. It was concluded that the collagen sponge prepared from human Type I collagen has potential as a graft material in oral surgical procedures.

  8. Localization of type IV collagen a 1 to a 6 chains in basement membrane during mouse molar germ development.

    PubMed

    Nagai, N; Nakano, K; Sado, Y; Naito, I; Gunduz, M; Tsujigiwa, H; Nagatsuka, H; Ninomiya, Y; Siar, C H

    2001-10-01

    The dental basement membrane (BM) putatively mediates epithelial-mesenchymal interactions during tooth morphogenesis and cytodifferentiation. Type IV collagen alpha chains, a major network-forming protein of the dental BM, was studied and results disclosed distinct expression patterns at different stages of mouse molar germ development. At the dental placode and bud stage, the BM of the oral epithelium expressed alpha 1, alpha 2, alpha 5 and alpha 6 chains while the gubernaculum dentis, in addition to the above four chains, also expressed a 4 chain. An asymmetrical expression for alpha 4, alpha 5 and alpha 6 chains was observed at the bud stage. At the early bell stage, the BM associated with the inner enamel epithelium (IEE) of molar germ expressed alpha 1, alpha 2 and alpha 4 chains while the BM of the outer enamel epithelium (OEE) expressed only alpha 1 and a 2 chains. With the onset of dentinogenesis, the collagen a chain profile of the IEE BM gradually disappeared. Howeverfrom the early to late bell stage, the gubernaculum dentis consistently expressed alpha 1, alpha 2, alpha 5 and a 6 chains resembling fetal oral mucosa. These findings suggest that stage- and position-specific distribution of type IV collagen alpha subunits occur during molar germ development and that these changes are essential for molar morphogenesis and cytodifferentiation.

  9. Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma

    PubMed Central

    Chen, Yulong; Terajima, Masahiko; Yang, Yanan; Sun, Li; Ahn, Young-Ho; Pankova, Daniela; Puperi, Daniel S.; Watanabe, Takeshi; Kim, Min P.; Blackmon, Shanda H.; Rodriguez, Jaime; Liu, Hui; Behrens, Carmen; Wistuba, Ignacio I.; Minelli, Rosalba; Scott, Kenneth L.; Sanchez-Adams, Johannah; Guilak, Farshid; Pati, Debananda; Thilaganathan, Nishan; Burns, Alan R.; Creighton, Chad J.; Martinez, Elisabeth D.; Zal, Tomasz; Grande-Allen, K. Jane; Yamauchi, Mitsuo; Kurie, Jonathan M.

    2015-01-01

    Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde–derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde–derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma. PMID:25664850

  10. Substitution of aspartate for glycine 103 of the type II collagen triple helical domain: Identification of the minimal mutation which can produce Kniest dysplasia

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wilkin, D.J.; Rimoin, D.L.; Cohn, D.H.

    1994-09-01

    Kniest dysplasia is an autosomal dominant chondrodysplasia which results from mutations in the gene for type II collagen, COL2A1. Characteristics of the disorder include a short trunk and extremities, mid-face hypoplasia, cleft palate, myopia, retinal detachment, and hearing loss. Recently, deletions of all or part of exon 12 have been identified in individuals with Kniest dysplasia, suggesting that mutations within this region of the protein may primarily result in the Kniest dysplasia phenotype. We used SSCP to analyze an amplified genomic DNA fragment containing exon 12 from 7 individuals with Kniest dysplasia. An abnormality was identified in one patient. DNAmore » sequence analysis demonstrated that the patient was heterozygous for a G to A transition that implied substitution of glycine{sup 103} of the triple helix by aspartate. The mutation was not observed in DNA from either of the proband`s parents. Protein microsequencing demonstrated expression of the abnormal allele in the proband`s cartilage, indicating that the Kniest phenotype results from the presence of abnormal type II collagen molecules in the extracellular matrix. These data demonstrate the minimal mutation which can produce Kniest dysplasia and further support the hypothesis that alteration of a domain which includes the region encoded by exon 12 in the type II collagen protein leads to this disorder. Experiments designed to identify specific effects that mutations in this region have on intermolecular interactions among abnormal type II collagen molecules and other components of the cartilage extracellular matrix may clarify the underlying pathophysiology of Kniest dysplasia.« less

  11. Effect of Cell Sheet Manipulation Techniques on the Expression of Collagen Type II and Stress Fiber Formation in Human Chondrocyte Sheets.

    PubMed

    Wongin, Sopita; Waikakul, Saranatra; Chotiyarnwong, Pojchong; Siriwatwechakul, Wanwipa; Viravaidya-Pasuwat, Kwanchanok

    2018-03-01

    Cell sheet technology is applied to human articular chondrocytes to construct a tissue-like structure as an alternative treatment for cartilage defect. The effect of a gelatin manipulator, as a cell sheet transfer system, on the quality of the chondrocyte sheets was investigated. The changes of important chondrogenic markers and stress fibers, resulting from the cell sheet manipulation, were also studied. The chondrocyte cell sheets were constructed with patient-derived chondrocytes using a temperature-responsive polymer and a gelatin manipulator as a transfer carrier. The properties of the cell sheets, including sizes, expression levels of collagen type II and I, and the localization of the stress fibers, were assessed and compared with those of the cell sheets harvested without the gelatin manipulator. Using the gelatin manipulator, the original size of the chondrocyte cell sheets was retained with abundant stress fibers, but with a decrease in the expression of collagen type II. Without the gelatin manipulator, although the cell shrinkage occurred, the cell sheet with suppressed stress fiber formation showed significantly higher levels of collagen type II. These results support our observations that stress fiber formation in chondrocyte cell sheets affected the production of chondrogenic markers. These densely packed tissue-like structures possessed a good chondrogenic activity, indicating their potential for use in autologous chondrocyte implantation to treat cartilage defects.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sweeney, Shawn M.; Orgel, Joseph P.; Fertala, Andrzej

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

  13. Suppression of type I collagen in human scleral fibroblasts treated with extremely low-frequency electromagnetic fields

    PubMed Central

    Wang, Jie; Cui, Jiefeng

    2013-01-01

    Purpose To investigate the expression differences of type I collagen (COL1A1) and its underlying mechanisms in human fetal scleral fibroblasts (HFSFs) that were treated with conditioned medium from retinal pigment epithelial (RPE) cells under extremely low-frequency electromagnetic fields (ELF-EMFs). Methods The ELF-EMFs used in this study were established by slidac and artificial coils. Growth of the treated HFSFs was evaluated by a cell-counting kit-8 assay. The expression of COL1A1 and matrix metalloproteinases-2 (MMP-2) in the treated HFSFs was detected by reverse transcription PCR (RT-PCR) and western blot, and the expression of transforming growth factor-β2 (TGF-β2) and basic fibroblast growth factor-2 (FGF-2) in RPE cells exposed to EMFs was detected by RT-PCR. The expression of COL1A1 and MMP-2 in HFSFs was further confirmed by immunofluorescence staining. Activation of extracellular signal-regulated kinase 1/2 (ERK1/2 also called p44/p42 mitogen-activated protein kinases [MAPK]) and p38 in HFSFs was measured by western blot. Results We found that exposure to ELF-EMFs resulted in a decreased proliferation rate of HFSFs and that addition of RPE supernatant medium could enhance this effect. Compared with that of the control cells, a significant decrease in collagen synthesis was detected in HFSFs under ELF-EMFs. However, the expression of MMP-2 was upregulated, which could be further enhanced via an RPE supernatant additive. The activities of ERK1/2 and p38 were significantly increased in HFSFs exposed to ELF-EMFs, and this effect could be enhanced by RPE supernatant medium additive. Conclusions Our results suggested that ELF-EMFs can inhibit the expression of type I collagen in HFSFs and contribute to the remodeling of the sclera. PMID:23592926

  14. Photodynamic Therapy Associated with a Blue Dye Papain-Based Gel and Evaluation of Its Degradation of Type I Collagen Fibers.

    PubMed

    Botta, Sergio Brossi; Ana, Patricia Aparecida; Gonçalves, Marcela Leticia Leal; Fernandes, Kristianne Porta Santos; Mesquita-Ferrari, Raquel Agnelli; de Araújo Prates, Renato; Brugnera, Aldo; Bussadori, Sandra Kalil

    2018-02-01

    The aim of this in vitro study was to evaluate the degradation of type I collagen fibers after treatment with a papain-based gel associated with a blue dye (PapaMBlue™) for use in antimicrobial photodynamic therapy. For such, 60 bioabsorbable membrane sponge discs were used. Group 1 was the negative control group. In groups 2, 3, and 4, the papain-based gel PapaMBlue gel was applied all over the samples for 4 min and irradiated using red laser (660 ± 10 nm) with 15, 30, and 40 J/cm 2 , respectively. In group 5, the papain-based gel was applied all over the samples for 4 min. In group 6, the photosensitizing dye was applied all over the samples for 4 min. The compositional analysis of the samples was performed using ATR-FTIR (attenuated total reflectance-Fourier transformed infrared spectroscopy). The data were statistically analyzed using ANOVA and Tukey's test (p < 0.05). Neither classic Papacarie™ nor the modified product with a photosensitizing agent (PapaMBlue) promoted collagen degradation. The irradiation of methylene blue added to papain gel with red light did not alter the chemical structure of type I collagen.

  15. Strategies for Directing the Structure and Function of 3D Collagen Biomaterials across Length Scales

    PubMed Central

    Walters, Brandan D.; Stegemann, Jan P.

    2013-01-01

    Collagen type I is a widely used natural biomaterial that has found utility in a variety of biological and medical applications. Its well characterized structure and role as an extracellular matrix protein make it a highly relevant material for controlling cell function and mimicking tissue properties. Collagen type I is abundant in a number of tissues, and can be isolated as a purified protein. This review focuses on hydrogel biomaterials made by reconstituting collagen type I from a solubilized form, with an emphasis on in vitro studies in which collagen structure can be controlled. The hierarchical structure of collagen from the nanoscale to the macroscale is described, with an emphasis on how structure is related to function across scales. Methods of reconstituting collagen into hydrogel materials are presented, including molding of macroscopic constructs, creation of microscale modules, and electrospinning of nanoscale fibers. The modification of collagen biomaterials to achieve desired structures and functions is also addressed, with particular emphasis on mechanical control of collagen structure, creation of collagen composite materials, and crosslinking of collagenous matrices. Biomaterials scientists have made remarkable progress in rationally designing collagen-based biomaterials and in applying them to both the study of biology and for therapeutic benefit. This broad review illustrates recent examples of techniques used to control collagen structure, and to thereby direct its biological and mechanical functions. PMID:24012608

  16. Molecular and ultrastructural studies of a fibrillar collagen from octocoral (Cnidaria).

    PubMed

    Orgel, Joseph P R O; Sella, Ido; Madhurapantula, Rama S; Antipova, Olga; Mandelberg, Yael; Kashman, Yoel; Benayahu, Dafna; Benayahu, Yehuda

    2017-09-15

    We report here the biochemical, molecular and ultrastructural features of a unique organization of fibrillar collagen extracted from the octocoral Sarcophyton ehrenbergi Collagen, the most abundant protein in the animal kingdom, is often defined as a structural component of extracellular matrices in metazoans. In the present study, collagen fibers were extracted from the mesenteries of S. ehrenbergi polyps. These fibers are organized as filaments and further compacted as coiled fibers. The fibers are uniquely long, reaching an unprecedented length of tens of centimeters. The diameter of these fibers is 9±0.37 μm. The amino acid content of these fibers was identified using chromatography and revealed close similarity in content to mammalian type I and II collagens. The ultrastructural organization of the fibers was characterized by means of high-resolution microscopy and X-ray diffraction. The fibers are composed of fibrils and fibril bundles in the range of 15 to 35 nm. These data indicate a fibrillar collagen possessing structural aspects of both types I and II collagen, a highly interesting and newly described form of fibrillar collagen organization. © 2017. Published by The Company of Biologists Ltd.

  17. Control of Collagen Production in Mouse Chondrocytes by Using a Combination of Bone Morphogenetic Protein-2 and Small Interfering RNA Targeting Col1a1 for Hydrogel-Based Tissue-Engineered Cartilage

    PubMed Central

    Perrier-Groult, Emeline; Pasdeloup, Marielle; Malbouyres, Marilyne; Galéra, Philippe

    2013-01-01

    Because articular cartilage does not self-repair, tissue-engineering strategies should be considered to regenerate this tissue. Autologous chondrocyte implantation is already used for treatment of focal damage of articular cartilage. Unfortunately, this technique includes a step of cell amplification, which results in dedifferentiation of chondrocytes, with expression of type I collagen, a protein characteristic of fibrotic tissues. Therefore, the risk of producing a fibrocartilage exists. The aim of this study was to propose a new strategy for authorizing the recovery of the differentiated status of the chondrocytes after their amplification on plastic. Because the bone morphogenetic protein (BMP)-2 and the transforming growth factor (TGF)-β1 are cytokines both proposed as stimulants for cartilage repair, we undertook a detailed comparative analysis of their biological effects on chondrocytes. As a cellular model, we used mouse chondrocytes after their expansion on plastic and we tested the capability of BMP-2 or TGF-β1 to drive their redifferentiation, with special attention given to the nature of the proteins synthesized by the cells. To prevent any fibrotic character of the newly synthesized extracellular matrix, we silenced type I collagen by transfecting small interfering RNA (siRNA) into the chondrocytes, before their exposure to BMP-2 or TGF-β1. Our results showed that addition of siRNA targeting the mRNA encoded by the Col1a1 gene (Col1a1 siRNA) and BMP-2 represents the most efficient combination to control the production of cartilage-characteristic collagen proteins. To go one step further toward scaffold-based cartilage engineering, Col1a1 siRNA-transfected chondrocytes were encapsulated in agarose hydrogel and cultured in vitro for 1 week. The analysis of the chondrocyte–agarose constructs by using real-time polymerase chain reaction, Western-blotting, immunohistochemistry, and electron microscopy techniques demonstrated that the BMP-2/Col1a1 si

  18. Quantification of collagen distributions in rat hyaline and fibro cartilages based on second harmonic generation imaging

    NASA Astrophysics Data System (ADS)

    Zhu, Xiaoqin; Liao, Chenxi; Wang, Zhenyu; Zhuo, Shuangmu; Liu, Wenge; Chen, Jianxin

    2016-10-01

    Hyaline cartilage is a semitransparent tissue composed of proteoglycan and thicker type II collagen fibers, while fibro cartilage large bundles of type I collagen besides other territorial matrix and chondrocytes. It is reported that the meniscus (fibro cartilage) has a greater capacity to regenerate and close a wound compared to articular cartilage (hyaline cartilage). And fibro cartilage often replaces the type II collagen-rich hyaline following trauma, leading to scar tissue that is composed of rigid type I collagen. The visualization and quantification of the collagen fibrillar meshwork is important for understanding the role of fibril reorganization during the healing process and how different types of cartilage contribute to wound closure. In this study, second harmonic generation (SHG) microscope was applied to image the articular and meniscus cartilage, and textural analysis were developed to quantify the collagen distribution. High-resolution images were achieved based on the SHG signal from collagen within fresh specimens, and detailed observations of tissue morphology and microstructural distribution were obtained without shrinkage or distortion. Textural analysis of SHG images was performed to confirm that collagen in fibrocartilage showed significantly coarser compared to collagen in hyaline cartilage (p < 0.01). Our results show that each type of cartilage has different structural features, which may significantly contribute to pathology when damaged. Our findings demonstrate that SHG microscopy holds potential as a clinically relevant diagnostic tool for imaging degenerative tissues or assessing wound repair following cartilage injury.

  19. Effects of in vivo static compressive loading on aggrecan and type II and X collagens in the rat growth plate extracellular matrix.

    PubMed

    Cancel, Mathilde; Grimard, Guy; Thuillard-Crisinel, Delphine; Moldovan, Florina; Villemure, Isabelle

    2009-02-01

    Mechanical loads are essential to normal bone growth, but excessive loads can lead to progressive deformities. In addition, growth plate extracellular matrix remodelling is essential to regulate the normal longitudinal bone growth process and to ensure physiological bone mineralization. In order to investigate the effects of static compression on growth plate extracellular matrix using an in vivo animal model, a loading device was used to precisely apply a compressive stress of 0.2 MPa for two weeks on the seventh caudal vertebra (Cd7) of rats during the pubertal growth spurt. Control, sham and loaded groups were studied. Growth modulation was quantified based on calcein labelling, and three matrix components (type II and X collagens, and aggrecan) were assessed using immunohistochemistry/safranin-O staining. As well, extracellular matrix components and enzymes (MMP-3 and -13, ADAMTS-4 and -5) were studied by qRT-PCR. Loading reduced Cd7 growth by 29% (p<0.05) and 15% (p=0.07) when compared to controls and shams respectively. No significant change could be observed in the mRNA expression of collagens and the proteolytic enzyme MMP-13. However, MMP-3 was significantly increased in the loaded group as compared to the control group (p<0.05). No change was observed in aggrecan and ADAMTS-4 and -5 expression. Low immunostaining for type II and X collagens was observed in 83% of the loaded rats as compared to the control rats. This in vivo study shows that, during pubertal growth spurt, two-week static compression reduced caudal vertebrae growth rates; this mechanical growth modulation occurred with decreased type II and X collagen proteins in the growth plate.

  20. Effects of increased collagen-matrix density on the mechanical properties and in vivo absorbability of hydroxyapatite-collagen composites as artificial bone materials.

    PubMed

    Yunoki, Shunji; Sugiura, Hiroaki; Ikoma, Toshiyuki; Kondo, Eiji; Yasuda, Kazunori; Tanaka, Junzo

    2011-02-01

    The aim of this study was to evaluate the effects of increased collagen-matrix density on the mechanical properties and in vivo absorbability of porous hydroxyapatite (HAp)-collagen composites as artificial bone materials. Seven types of porous HAp-collagen composites were prepared from HAp nanocrystals and dense collagen fibrils. Their densities and HAp/collagen weight ratios ranged from 122 to 331 mg cm⁻³ and from 20/80 to 80/20, respectively. The flexural modulus and strength increased with an increase in density, reaching 2.46 ± 0.48 and 0.651 ± 0.103 MPa, respectively. The porous composites with a higher collagen-matrix density exhibited much higher mechanical properties at the same densities, suggesting that increasing the collagen-matrix density is an effective way of improving the mechanical properties. It was also suggested that other structural factors in addition to collagen-matrix density are required to achieve bone-like mechanical properties. The in vivo absorbability of the composites was investigated in bone defects of rabbit femurs, demonstrating that the absorption rate decreased with increases in the composite density. An exhaustive increase in density is probably limited by decreases in absorbability as artificial bones.

  1. Effects of various salts on structural polymorphism of reconstituted type I collagen fibrils.

    PubMed

    Li, Yuping; Douglas, Elliot P

    2013-12-01

    Even though the behavior of collagen monomers self-assembling into fibrils is commonly understood in terms of hydrophobic and electrostatic interactions, the mechanisms that drive their ordered, longitudinal alignment to form a characteristic periodicity are still unclear. By introducing various salts into the collagen fibrillogenesis system, the intermolecular interactions of fibril formation were studied. We found that the pH and ion species play a critical role in forming native fibrils. Turbidity and electron microscopy revealed that collagen self-assembled into fibrils with a native banding pattern in the presence of multivalent ions. The isoelectric point of collagen in 12mM of NaCl is pH 8.9; it shifted to pH 9.4 and pH 7.0 after adding 10mM CaCl2 and Na2SO4, respectively. Native fibrils were reconstituted at pH 7.4 in salts with divalent anions and at pH 9.0 in salts with divalent cations. Circular dichroism spectroscopy showed a loss of helicity in the conditions where fibrillogenesis was unable to be achieved. The multivalent ions not only change the surface charge of collagen, but also facilitate the formation of fibrils with the native D-periodic banding pattern. It is likely that the binding multivalent ions induce the like-charge attraction and facilitate monomers' longitudinal registration to form fibrils with the native banding. Published by Elsevier B.V.

  2. Increase in type II collagen turnover after iron depletion in patients with hereditary haemochromatosis.

    PubMed

    Richette, Pascal; Eymard, Claire; Deberg, Michelle; Vidaud, Dominique; de Kerguenec, Caroline; Valla, Dominique; Vicaut, Eric; Bardin, Thomas; Henrotin, Yves

    2010-04-01

    To determine the effects of iron depletion on serum levels of joint biomarkers and on joint symptoms in patients with hereditary haemochromatosis (HH). Levels of biomarkers were measured in 18 patients with HH at the time of diagnosis and after iron depletion. The markers were type II collagen degradation (Coll2-1) and its nitrated form (Coll2-1NO(2)), type II procollagen synthesis (CPII), MPO, COMP and HA. For each patient, demographic data were collected and the global joint pain (visual analogue scale) was assessed before and after iron depletion by phlebotomy. A total of 18 patients [10 males; mean (s.d.) age 48 (11) years] were homozygous for the C282Y mutation. No patient had liver dysfunction. Ferritin level before iron removal was 627.5 (range 133-3276) microg/l, and duration of the iron depletion phase was 295 (70-670) days. Serum levels of both Coll2-1 and CPII were significantly increased from diagnosis after iron depletion: 80.1 (55.6-113.5) vs 96.0 (48.8-136.3) nM (P = 0.004) and 731.4 (374.2-1012.3) vs 812.8 (535.8-1165.6) ng/ml (P = 0.03), respectively. Levels of other biomarkers were not modified by iron depletion. Ferritin level, which at baseline was correlated with body iron store (r = 0.63; P = 0.008), was significantly correlated with HA level measured before iron depletion (r = 0.60; P = 0.01). Global joint pain was not correlated with ferritin concentration and did not significantly decrease after iron depletion: 43 (19-73) vs 36 (16-67) mm (P = 0.07). In patients with HH, cartilage homoeostasis is modified by iron excess and an increase in type II collagen turnover occurs after excess iron removal.

  3. A quantitative comparison of morphological and histological characteristics of collagen in the rabbit medial collateral ligament.

    PubMed

    Wan, Chao; Hao, Zhixiu; Wen, Shizhu

    2013-12-01

    Collagen fiber is one of the critical factors in determining mechanical properties of ligaments and both the morphological and histological characteristics of collagen have been widely studied. However, there was still no consensus about whether the morphological characteristics of collagen correlated with its histological characteristics in physiological ligaments. Rabbit medial collateral ligaments (MCLs) were measured under a transmission electron microscope and a polarized light microscope plus picrosirius red-staining to obtain the distributions of collagen fibril diameters and types at different anatomical sites of rabbit MCLs, respectively. The correlation between the fibril diameter and type was determined by a correlation analysis. The collagen fibril diameters at the different anatomical sites had different distributions (unimodal or bimodal) and mean fibril diameters were found to increase significantly from the anterior part to the posterior part (P=0.0482) as well as from the proximal to the distal sections (P=0.0208). Type I collagen in the core portion of MCLs was significantly less than at the other four peripheral areas (P<0.005) but no significant variation was found in each respective portion (P>0.05). The low coefficient in the correlation analysis (r=0.3759) demonstrated collagen fibril diameters had no correlation with collagen types. This may provide a new view of collagen types in studying the mechanical behavior of ligaments. Copyright © 2013 Elsevier GmbH. All rights reserved.

  4. FKBP65-dependent peptidyl-prolyl isomerase activity potentiates the lysyl hydroxylase 2-driven collagen cross-link switch.

    PubMed

    Chen, Yulong; Terajima, Masahiko; Banerjee, Priyam; Guo, Houfu; Liu, Xin; Yu, Jiang; Yamauchi, Mitsuo; Kurie, Jonathan M

    2017-04-05

    Bruck Syndrome is a connective tissue disease associated with inactivating mutations in lysyl hydroxylase 2 (LH2/PLOD2) or FK506 binding protein 65 (FKBP65/FKBP10). However, the functional relationship between LH2 and FKBP65 remains unclear. Here, we postulated that peptidyl prolyl isomerase (PPIase) activity of FKBP65 positively modulates LH2 enzymatic activity and is critical for the formation of hydroxylysine-aldehyde derived intermolecular collagen cross-links (HLCCs). To test this hypothesis, we analyzed collagen cross-links in Fkbp10-null and -wild-type murine embryonic fibroblasts. Although LH2 protein levels did not change, FKBP65 deficiency significantly diminished HLCCs and increased the non-hydroxylated lysine-aldehyde-derived collagen cross-links (LCCs), a pattern consistent with loss of LH2 enzymatic activity. The HLCC-to-LCC ratio was rescued in FKBP65-deficient murine embryonic fibroblasts by reconstitution with wild-type but not mutant FKBP65 that lacks intact PPIase domains. Findings from co-immunoprecipitation, protein-fragment complementation, and co-immunofluorescence assays showed that LH2 and FKBP65 are part of a common protein complex. We conclude that FKBP65 regulates LH2-mediated collagen cross-linking. Because LH2 promotes fibrosis and cancer metastasis, our findings suggest that pharmacologic strategies to target FKBP65 and LH2 may have complementary therapeutic activities.

  5. FKBP65-dependent peptidyl-prolyl isomerase activity potentiates the lysyl hydroxylase 2-driven collagen cross-link switch

    PubMed Central

    Chen, Yulong; Terajima, Masahiko; Banerjee, Priyam; Guo, Houfu; Liu, Xin; Yu, Jiang; Yamauchi, Mitsuo; Kurie, Jonathan M.

    2017-01-01

    Bruck Syndrome is a connective tissue disease associated with inactivating mutations in lysyl hydroxylase 2 (LH2/PLOD2) or FK506 binding protein 65 (FKBP65/FKBP10). However, the functional relationship between LH2 and FKBP65 remains unclear. Here, we postulated that peptidyl prolyl isomerase (PPIase) activity of FKBP65 positively modulates LH2 enzymatic activity and is critical for the formation of hydroxylysine-aldehyde derived intermolecular collagen cross-links (HLCCs). To test this hypothesis, we analyzed collagen cross-links in Fkbp10-null and –wild-type murine embryonic fibroblasts. Although LH2 protein levels did not change, FKBP65 deficiency significantly diminished HLCCs and increased the non-hydroxylated lysine-aldehyde–derived collagen cross-links (LCCs), a pattern consistent with loss of LH2 enzymatic activity. The HLCC-to-LCC ratio was rescued in FKBP65-deficient murine embryonic fibroblasts by reconstitution with wild-type but not mutant FKBP65 that lacks intact PPIase domains. Findings from co-immunoprecipitation, protein-fragment complementation, and co-immunofluorescence assays showed that LH2 and FKBP65 are part of a common protein complex. We conclude that FKBP65 regulates LH2-mediated collagen cross-linking. Because LH2 promotes fibrosis and cancer metastasis, our findings suggest that pharmacologic strategies to target FKBP65 and LH2 may have complementary therapeutic activities. PMID:28378777

  6. Hydroxyl Radical Modification of Collagen Type II Increases Its Arthritogenicity and Immunogenicity

    PubMed Central

    Shahab, Uzma; Ahmad, Saheem; Moinuddin; Dixit, Kiran; Habib, Safia; Alam, Khursheed; Ali, Asif

    2012-01-01

    Background The oxidation of proteins by endogenously generated free radicals causes structural modifications in the molecules that lead to generation of neo-antigenic epitopes that have implications in various autoimmune disorders, including rheumatoid arthritis (RA). Collagen induced arthritis (CIA) in rodents (rats and mice) is an accepted experimental model for RA. Methodology/Principal Findings Hydroxyl radicals were generated by the Fenton reaction. Collagen type II (CII) was modified by •OH radical (CII-OH) and analysed by ultraviolet-visible (UV-VIS), fluorescence and circular dichroism (CD) spectroscopy. The immunogenicity of native and modified CII was checked in female Lewis rats and specificity of the induced antibodies was ascertained by enzyme linked immunosorbent assay (ELISA). The extent of CIA was evaluated by visual inspection. We also estimated the oxidative and inflammatory markers in the sera of immunized rats. A slight change in the triple helical structure of CII as well as fragmentation was observed after hydroxyl radical modification. The modified CII was found to be highly arthritogenic and immunogenic as compared to the native form. The CII-OH immunized rats exhibited increased oxidative stress and inflammation as compared to the CII immunized rats in the control group. Conclusions/Significance Neo-antigenic epitopes were generated on •OH modified CII which rendered it highly immunogenic and arthritogenic as compared to the unmodified form. Since the rodent CIA model shares many features with human RA, these results illuminate the role of free radicals in human RA. PMID:22319617

  7. Attachment, proliferation and collagen type I mRNA expression of human gingival fibroblasts on different biodegradable membranes.

    PubMed

    Hakki, Sema S; Korkusuz, Petek; Purali, Nuhan; Bozkurt, Buket; Kus, Mahmut; Duran, Ismet

    2013-01-01

    The purpose of this study was to investigate adhesion, proliferation and type I collagen (COL I) mRNA expression of gingival fibroblasts on different membranes used in periodontal applications. Collagen (C), acellular dermal matrix (ADM) and polylactic acid; polyglycolic acid; lactide/glycolide copolymer (PLGA) biodegradable membranes were combined with gingival fibroblasts in culture and incubated for 48 h. Cell adhesion was examined with scanning electron and confocal microscopy. MTT assay was used to measure proliferation. COL I mRNA expression was assessed using quantitative-polymerase chain reaction (QPCR). The PLGA group exhibited the lowest cell survival on day 5 and 10, and lowest cell proliferation on days 5, 10 and 14. While cell proliferation was similar in C and ADM groups, the C membrane showed a slightly greater increase in viable cells to day 10. Confocal and scanning electron microscopy confirmed the results of proliferation and MTT assays. The highest COL I mRNA expression was noted in the PLGA membrane group when compared to the C (p < 0.01) and ADM (p < 0.05) membrane groups. These data revealed that adherence and proliferation of primary gingival fibroblasts on collagen-based C and ADM membranes is better than that seen with PLGA membranes, and thus may be preferable in the treatment of gingival recession defects.

  8. Influence of collagen-fibril-based coatings containing decorin and biglycan on osteoblast behavior.

    PubMed

    Douglas, Timothy; Hempel, Ute; Mietrach, Carolin; Viola, Manuela; Vigetti, Davide; Heinemann, Sascha; Bierbaum, Susanne; Scharnweber, Dieter; Worch, Hartmut

    2008-03-01

    Collagen is used as a scaffold material for tissue engineering as well as a coating material for implants with a view to enhancing osseointegration by mimicry of the bone extracellular matrix in vivo. The biomimicry strategy can be taken further by incorporating the small leucine-rich proteoglycans (SLRPs) decorin and biglycan, which are expressed in bone. Both bind to fibrils during fibrillogenesis in vitro. In this study, the ability of collagen types I, II, and III to bind decorin and biglycan was compared. Collagen type II bound significantly more SLRPs in fibrils than collagen I and III, with more biglycan than decorin bound by all three collagen types. Therefore, type II fibrils with bound decorin or biglycan or neither were used to coat titanium surfaces. Bioavailability of SLRPs was confirmed by direct ELISA after SLRP biotinilation. The in vitro behavior of osteoblasts from rat calvaria (rOs) and human knee (hOs) cultured on different surfaces was compared. Proliferation and collagen synthesis were determined. Also, the influence of SLRPs on the formation of focal adhesions by rO was investigated. Biglycan enhanced the formation of focal adhesions after 2 and 24 h. Decorin and biglycan affected rO and hO proliferation and collagen synthesis differently. Biglycan stimulated hO proliferation significantly but had no effect on rO proliferation, and also inhibited rO collagen synthesis significantly while not affecting hO collagen synthesis. Decorin promoted hO proliferation slightly but did not influence rO proliferation. The results could be relevant when designing implant coatings or tissue engineering scaffolds. (c) 2007 Wiley Periodicals, Inc.

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

    PubMed Central

    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

  10. Drosophila type IV collagen mutation associates with immune system activation and intestinal dysfunction.

    PubMed

    Kiss, Márton; Kiss, András A; Radics, Monika; Popovics, Nikoletta; Hermesz, Edit; Csiszár, Katalin; Mink, Mátyás

    2016-01-01

    The basal lamina (BM) contains numerous components with a predominance of type IV collagens. Clinical manifestations associated with mutations of the human COL4A1 gene include perinatal cerebral hemorrhage and porencephaly, hereditary angiopathy, nephropathy, aneurysms and muscle cramps (HANAC), ocular dysgenesis, myopathy, Walker–Warburg syndrome and systemic tissue degeneration. In Drosophila, the phenotype associated with dominant temperature sensitive mutations of col4a1 include severe myopathy resulting from massive degradation of striated muscle fibers, and in the gut, degeneration of circular visceral muscle cells and epithelial cells following detachment from the BM. In order to determine the consequences of altered BMfunctions due to aberrant COL4A1 protein, we have carried out a series of tests using Drosophila DTS-L3 mutants from our allelic series of col4a1 mutations with confirmed degeneration of various cell types and lowest survival rate among the col4a1 mutant lines at restrictive temperature. Results demonstrated epithelial cell degeneration in the gut, shortened gut, enlarged midgut with multiple diverticulae, intestinal dysfunction and shortened life span. Midgut immunohistochemistry analyses confirmed altered expression and distribution of BM components integrin PSI and PSII alpha subunits, laminin gamma 1, and COL4A1 both in larvae and adults. Global gene expression analysis revealed activation of the effector AMP genes of the primary innate immune system including Metchnikowin, Diptericin, Diptericin B, and edin that preceded morphological changes. Attacin::GFP midgut expression pattern further supported these changes. An increase in ROS production and changes in gut bacterial flora were also noted and may have further enhanced an immune response. The phenotypic features of Drosophila col4a1 mutants confirmed an essential role for type IV collagen in maintaining epithelial integrity, gut morphology and intestinal function and suggest that

  11. Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs.

    PubMed

    Jabłońska-Trypuć, Agata; Matejczyk, Marzena; Rosochacki, Stanisław

    2016-01-01

    The main group of enzymes responsible for the collagen and other protein degradation in extracellular matrix (ECM) are matrix metalloproteinases (MMPs). Collagen is the main structural component of connective tissue and its degradation is a very important process in the development, morphogenesis, tissue remodeling, and repair. Typical structure of MMPs consists of several distinct domains. MMP family can be divided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other non-classified MMPs. MMPs and their inhibitors have multiple biological functions in all stages of cancer development: from initiation to outgrowth of clinically relevant metastases and likewise in apoptosis and angiogenesis. MMPs and their inhibitors are extensively examined as potential anticancer drugs. MMP inhibitors can be divided into two main groups: synthetic and natural inhibitors. Selected synthetic inhibitors are in clinical trials on humans, e.g. synthetic peptides, non-peptidic molecules, chemically modified tetracyclines, and bisphosphonates. Natural MMP inhibitors are mainly isoflavonoids and shark cartilage.

  12. Collagen-Like Proteins in Pathogenic E. coli Strains

    PubMed Central

    Ghosh, Neelanjana; McKillop, Thomas J.; Jowitt, Thomas A.; Howard, Marjorie; Davies, Heather; Holmes, David F.; Roberts, Ian S.; Bella, Jordi

    2012-01-01

    The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages. PMID:22701585

  13. Disruption of fibronectin matrix affects type IV collagen, fibrillin and laminin deposition into extracellular matrix of human trabecular meshwork (HTM) cells.

    PubMed

    Filla, Mark S; Dimeo, Kaylee D; Tong, Tiegang; Peters, Donna M

    2017-12-01

    Fibronectin fibrils are a major component of the extracellular matrix (ECM) of the trabecular meshwork (TM). They are a key mediator of the formation of the ECM which controls aqueous humor outflow and contributes to the pathogenesis of glaucoma. The purpose of this work was to determine if a fibronectin-binding peptide called FUD, derived from the Streptococcus pyogenes Functional Upstream Domain of the F1 adhesin protein, could be used to control fibronectin fibrillogenesis and hence ECM formation under conditions where its expression was induced by treatment with the glucocorticoid dexamethasone. FUD was very effective at preventing fibronectin fibrillogenesis in the presence or absence of steroid treatment as well as the removal of existing fibronectin fibrils. Disruption of fibronectin fibrillogenesis by FUD also disrupted the incorporation of type IV collagen, laminin and fibrillin into the ECM. The effect of FUD on these other protein matrices, however, was found to be dependent upon the maturity of the ECM when FUD was added. FUD effectively disrupted the incorporation of these other proteins into matrices when added to newly confluent cells that were forming a nascent ECM. In contrast, FUD had no effect on these other protein matrices if the cell cultures already possessed a pre-formed, mature ECM. Our studies indicate that FUD can be used to control fibronectin fibrillogenesis and that these fibrils play a role in regulating the assembly of other ECM protein into matrices involving type IV collagen, laminin, and fibrillin within the TM. This suggests that under in vivo conditions, FUD would selectively disrupt fibronectin fibrils and de novo assembly of other proteins into the ECM. Finally, our studies suggest that targeting fibronectin fibril assembly may be a viable treatment for POAG as well as other glaucomas involving excessive or abnormal matrix deposition of the ECM. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

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

    2008-01-01

    Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The “cell interaction domain” is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The “matrix interaction domain” may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging. PMID:18487200

  17. Collagen type IV alpha 1 (COL4A1) and collagen type XIII alpha 1 (COL13A1) produced in cancer cells promote tumor budding at the invasion front in human urothelial carcinoma of the bladder

    PubMed Central

    Miyake, Makito; Hori, Shunta; Morizawa, Yosuke; Tatsumi, Yoshihiro; Toritsuka, Michihiro; Ohnishi, Sayuri; Shimada, Keiji; Furuya, Hideki; Khadka, Vedbar S.; Deng, Youping; Ohnishi, Kenta; Iida, Kota; Gotoh, Daisuke; Nakai, Yasushi; Inoue, Takeshi; Anai, Satoshi; Torimoto, Kazumasa; Aoki, Katsuya; Tanaka, Nobumichi; Konishi, Noboru; Fujimoto, Kiyohide

    2017-01-01

    Current knowledge of the molecular mechanism driving tumor budding is limited. Here, we focused on elucidating the detailed mechanism underlying tumor budding in urothelial cancer of the bladder. Invasive urothelial cancer was pathologically classified into three groups as follows: nodular, trabecular, and infiltrative (tumor budding). Pathohistological analysis of the orthotopic tumor model revealed that human urothelial cancer cell lines MGH-U3, UM-UC-14, and UM-UC-3 displayed typical nodular, trabecular, and infiltrative patterns, respectively. Based on the results of comprehensive gene expression analysis using microarray (25 K Human Oligo chip), we identified two collagens, COL4A1 and COL13A1, which may contribute to the formation of the infiltrative pattern. Visualization of protein interaction networks revealed that proteins associated with connective tissue disorders, epithelial-mesenchymal transition, growth hormone, and estrogen were pivotal factors in tumor cells. To evaluate the invasion pattern of tumor cells in vitro, 3-D collective cell invasion assay using Matrigel was performed. Invadopodial formation was evaluated using Gelatin Invadopodia Assay. Knockdown of collagens with siRNA led to dramatic changes in invasion patterns and a decrease in invasion capability through decreased invadopodia. The in vivo orthotopic experimental model of bladder tumors showed that intravesical treatment with siRNA targeting COL4A1 and COL13A1 inhibited the formation of the infiltrative pattern. COL4A1 and COL13A1 production by cancer cells plays a pivotal role in tumor invasion through the induction of tumor budding. Blocking of these collagens may be an attractive therapeutic approach for treatment of human urothelial cancer of the bladder. PMID:28415608

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

    PubMed

    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

  19. Isolation, characterization and biological evaluation of jellyfish collagen for use in biomedical applications.

    PubMed

    Addad, Sourour; Exposito, Jean-Yves; Faye, Clément; Ricard-Blum, Sylvie; Lethias, Claire

    2011-01-01

    Fibrillar collagens are the more abundant extracellular proteins. They form a metazoan-specific family, and are highly conserved from sponge to human. Their structural and physiological properties have been successfully used in the food, cosmetic, and pharmaceutical industries. On the other hand, the increase of jellyfish has led us to consider this marine animal as a natural product for food and medicine. Here, we have tested different Mediterranean jellyfish species in order to investigate the economic potential of their collagens. We have studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using Rhizostoma pulmo oral arms and the pepsin extraction method (2-10 mg collagen/g of wet tissue). Although a significant yield was obtained with Cotylorhiza tuberculata (0.45 mg/g), R. pulmo was used for further experiments, this jellyfish being considered as harmless to humans and being an abundant source of material. Then, we compared the biological properties of R. pulmo collagen with mammalian fibrillar collagens in cell cytotoxicity assays and cell adhesion. There was no statistical difference in cytotoxicity (p > 0.05) between R. pulmo collagen and rat type I collagen. However, since heparin inhibits cell adhesion to jellyfish-native collagen by 55%, the main difference is that heparan sulfate proteoglycans could be preferentially involved in fibroblast and osteoblast adhesion to jellyfish collagens. Our data confirm the broad harmlessness of jellyfish collagens, and their biological effect on human cells that are similar to that of mammalian type I collagen. Given the bioavailability of jellyfish collagen and its biological properties, this marine material is thus a good candidate for replacing bovine or human collagens in selected biomedical applications.

  20. Electrophoretic mobility patterns of collagen following laser welding

    NASA Astrophysics Data System (ADS)

    Bass, Lawrence S.; Moazami, Nader; Pocsidio, Joanne O.; Oz, Mehmet C.; LoGerfo, Paul; Treat, Michael R.

    1991-06-01

    Clinical application of laser vascular anastomosis in inhibited by a lack of understanding of its mechanism. Whether tissue fusion results from covalent or non-covalent bonding of collagen and other structural proteins is unknown. We compared electrophoretic mobility of collagen in laser treated and untreated specimens of rat tail tendon (>90% type I collagen) and rabbit aorta. Welding was performed, using tissue shrinkage as the clinical endpoint, using the 808 nm diode laser (power density 14 watts/cm2) and topical indocyanine green dye (max absorption 805 nm). Collagen was extracted with 8 M urea (denaturing), 0.5 M acetic acid (non-denaturing) and acetic acid/pepsin (cleaves non- helical protein). Mobility patterns on gel electrophoresis (SDS-PAGE) after urea or acetic acid extraction were identical in the lasered and control tendon and vessel (confirmed by optical densitometry), revealing no evidence of formation of novel covalent bonds. Alpha and beta band intensity was diminished in pepsin incubated lasered specimens compared with controls (optical density ratio 0.00 +/- 9 tendon, 0.65 +/- 0.12 aorta), indicating the presence of denatured collagen. With the laser parameters used, collagen is denatured without formation of covalent bonds, suggesting that non-covalent interaction between denatured collagen molecules may be responsible for the weld. Based on this mechanism, welding parameters can be chosen which produce collagen denaturation without cell death.

  1. Differences in collagen distribution of healthy and regenerated periodontium. Histomorphometric study in dogs.

    PubMed

    Souza, Sérgio L S; Macedo, Guilherme O; Silveira E Souza, Adriana M M; Taba, Mário; Novaes, Arthur B; Oliver, Constance; Jamur, Maria C; Correa, Vani M A

    2013-10-01

    Previous studies have shown that there is a relationship between periodontal disease and the distribution of collagen fibers. This study evaluated the distribution of collagen types I and III in regenerated bone and periodontal ligament, comparing them to the tissues near the regenerated area and to the healthy periodontium. In the third (P3) and fourth (P4) mandibular premolars of 5 healthy mongrel dogs, bilaterally, buccal class 2 furcation lesions were surgically created and chronified for 3 weeks. After that, full flaps were elevated and expanded polytetrafluoroethylene (e-PTFE) membranes were adapted, sutured and recovered by the flaps. Two weeks after surgery, two membranes on the same side were removed and the other membranes were removed four weeks after surgery. The dogs were euthanized at 12 weeks following placement of the e-PTFE membranes. P3 and P4 teeth as well as the second premolars (healthy control teeth) and their periodontal tissues were removed and histologically processed for Collagen Quantification (COLQ). The amount of type III collagen was higher in native bone compared to the regenerated area. For periodontal ligament, COLQ for type I collagen showed statistically significant differences (Tukeys's Multiple Comparison, p⟨0.05) between the regenerated groups and the control group. These differences were not found for type III COLQ. There are significant differences in collagen distribution among the regenerated, native and control tissues. Membrane removal 2 or 4 weeks postoperatively did not influence the collagen composition.

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

    NASA Astrophysics Data System (ADS)

    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.

  3. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo

    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,more » 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

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

    PubMed Central

    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

  5. Mechanical properties and solubility in water of corn starch-collagen composite films: Effect of starch type and concentrations.

    PubMed

    Wang, Kun; Wang, Wenhang; Ye, Ran; Liu, Anjun; Xiao, Jingdong; Liu, Yaowei; Zhao, Yana

    2017-02-01

    This study investigated the possibility of enhancing the properties of collagen with three different maize starches: waxy maize starch, normal starch, and high amylose starch. Scanning electron microscopy images revealed that starch-collagen films had a rougher surface compared to pure collagen films which became smoother upon heating. Amylose starch and normal starch increased the tensile strength of unheated collagen films in both dry and wet states, while all starches increased tensile strength of collagen film by heating. Depending upon the amylose content and starch concentrations, film solubility in water decreased with the addition of starch. DSC thermograms demonstrated that addition of all starches improved the thermal stability of the collagen film. Moreover, X-ray diffraction results indicated that except for high amylose starch, the crystallinity of both starch and collagen was significantly decreased when subject to heating. FTIR spectra indicated that intermolecular interactions between starch and collagen were enhanced upon heating. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    PubMed Central

    Miller, Benjamin F; Olesen, Jens L; Hansen, Mette; Døssing, Simon; Crameri, Regina M; Welling, Rasmus J; Langberg, Henning; Flyvbjerg, Allan; Kjaer, Michael; Babraj, John A; Smith, Kenneth; Rennie, Michael J

    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 over 72 h after 1 h of one-legged kicking exercise at 67% of maximum workload (Wmax). To label tissue proteins in muscle and tendon primed, constant infusions of [1-13C]leucine or [1-13C]valine and flooding doses of [15N] or [13C]proline were given intravenously, with estimation of labelling in target proteins by gas chromatography–mass spectrometry. Patellar tendon and quadriceps biopsies were taken in exercised and rested legs at 6, 24, 42 or 48 and 72 h after exercise. The fractional synthetic rates of all proteins were elevated at 6 h and rose rapidly to peak at 24 h post exercise (tendon 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 in human tendon and muscle. The similar time course of changes of protein synthetic rates in different cell types supports the idea of coordinated musculotendinous adaptation. PMID:16002437

  7. In Vitro Analysis of Cartilage Regeneration Using a Collagen Type I Hydrogel (CaReS) in the Bovine Cartilage Punch Model.

    PubMed

    Horbert, Victoria; Xin, Long; Foehr, Peter; Brinkmann, Olaf; Bungartz, Matthias; Burgkart, Rainer H; Graeve, T; Kinne, Raimund W

    2018-02-01

    Objective Limitations of matrix-assisted autologous chondrocyte implantation to regenerate functional hyaline cartilage demand a better understanding of the underlying cellular/molecular processes. Thus, the regenerative capacity of a clinically approved hydrogel collagen type I implant was tested in a standardized bovine cartilage punch model. Methods Cartilage rings (outer diameter 6 mm; inner defect diameter 2 mm) were prepared from the bovine trochlear groove. Collagen implants (± bovine chondrocytes) were placed inside the cartilage rings and cultured up to 12 weeks. Cartilage-implant constructs were analyzed by histology (hematoxylin/eosin; safranin O), immunohistology (aggrecan, collagens 1 and 2), and for protein content, RNA expression, and implant push-out force. Results Cartilage-implant constructs revealed vital morphology, preserved matrix integrity throughout culture, progressive, but slight proteoglycan loss from the "host" cartilage or its surface and decreasing proteoglycan release into the culture supernatant. In contrast, collagen 2 and 1 content of cartilage and cartilage-implant interface was approximately constant over time. Cell-free and cell-loaded implants showed (1) cell migration onto/into the implant, (2) progressive deposition of aggrecan and constant levels of collagens 1 and 2, (3) progressively increased mRNA levels for aggrecan and collagen 2, and (4) significantly augmented push-out forces over time. Cell-loaded implants displayed a significantly earlier and more long-lasting deposition of aggrecan, as well as tendentially higher push-out forces. Conclusion Preserved tissue integrity and progressively increasing cartilage differentiation and push-out forces for up to 12 weeks of cultivation suggest initial cartilage regeneration and lateral bonding of the implant in this in vitro model for cartilage replacement materials.

  8. Biological effect of hydrolyzed collagen on bone metabolism.

    PubMed

    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.

  9. Collagen matrix as a tool in studying fibroblastic cell behavior.

    PubMed

    Kanta, Jiří

    2015-01-01

    Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes.

  10. Collagen matrix as a tool in studying fibroblastic cell behavior

    PubMed Central

    Kanta, Jiří

    2015-01-01

    Type I collagen is a fibrillar protein, a member of a large family of collagen proteins. It is present in most body tissues, usually in combination with other collagens and other components of extracellular matrix. Its synthesis is increased in various pathological situations, in healing wounds, in fibrotic tissues and in many tumors. After extraction from collagen-rich tissues it is widely used in studies of cell behavior, especially those of fibroblasts and myofibroblasts. Cells cultured in a classical way, on planar plastic dishes, lack the third dimension that is characteristic of body tissues. Collagen I forms gel at neutral pH and may become a basis of a 3D matrix that better mimics conditions in tissue than plastic dishes. PMID:25734486

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

    PubMed

    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

  12. A novel collagen hydrogel cross-linked by gamma-ray irradiation in acidic pH conditions.

    PubMed

    Inoue, Naoki; Bessho, Masahiko; Furuta, Masakazu; Kojima, Takao; Okuda, Shuichi; Hara, Masayuki

    2006-01-01

    We made a new type of collagen gel by gamma-ray irradiation of an acidic solution of type-I collagen, and performed comparative studies on a conventional gel and the new type of gel. The neutral gel, a conventional 0.3% (w/v) collagen gel, was formed at neutral pH and then irradiated by gamma-rays. The acidic gel, a 0.3% (w/v) collagen gel, was formed directly from the acidic solution of collagen by y-ray irradiation. Both types of gel were prepared, swollen in water and then dried for the measurement of specific water content. The neutral gel showed a relatively high specific water content and shrunk moderately, depending on the dose, while the acidic gel showed lower specific water content and shrunk clearly by y-ray irradiation. A three-dimensional tangled network of microfibrils was clearly observed in the neutral gels by scanning electron microscopy, but not in the acidic gels. From these results, we concluded that the acidic gel was quite different from a conventional collagen gel. Sodium dodecylsulfate-polyacrylamide gel electrophoresis showed that the alpha1 subunit and alpha2 subunit of the collagen molecule were cross-linked. The triple-helical structure of collagen was only partially perturbed, but not denatured completely, because the circular dichroism spectrum of the collagen solution irradiated at 1.3 kGy was similar to that of native collagen solution. Amino-acid analysis revealed that tyrosine, phenylalanine and histidine decreased by irradiation in the neutral gel. In the case of the acidic gel, these three amino acids and methionine decreased. We considered that these amino acids were cross-linking points between the collagen subunits during the gamma-ray irradiation.

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

    PubMed

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

    2016-08-01

    Fish collagen has recently been reported to be a novel biomaterial for cell and tissue culture as an alternative to conventional mammalian collagens such as bovine and porcine collagens. Fish collagen could overcome the risk of zoonosis, such as from bovine spongiform encephalopathy. Among fish collagens, tilapia collagen, the denaturing temperature of which is near 37°C, is appropriate for cell and tissue culture. In this study, we investigated chondrogenic differentiation of human mesenchymal stem cells (hMSCs) cultured on tilapia scale collagen fibrils compared with porcine collagen and non-coated dishes. The collagen fibrils were observed using a scanning electronic microscope. Safranin O staining, glycosaminoglycans (GAG) expression, and real-time PCR were examined to evaluate chondrogenesis of hMSCs on each type of collagen fibril. The results showed that hMSCs cultured on tilapia scale collagen showed stronger Safranin O staining and higher GAG expression at day 6. Results of real-time PCR indicated that hMSCs cultured on tilapia collagen showed earlier SOX9 expression on day 4 and higher AGGRECAN and COLLAGEN II expression on day 6 compared with on porcine collagen and non-coated dishes. Furthermore, low mRNA levels of bone gamma-carboxyglutamate, a specific marker of osteogenesis, showed that tilapia collagen fibrils specifically enhanced chondrogenic differentiation of hMSCs in chondrogenic medium, as well as porcine collagen. Accordingly, tilapia scale collagen may provide an appropriate collagen source for hMSC chondrogenesis in vitro. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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

    PubMed

    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.

  15. Isolation, Characterization and Biological Evaluation of Jellyfish Collagen for Use in Biomedical Applications

    PubMed Central

    Addad, Sourour; Exposito, Jean-Yves; Faye, Clément; Ricard-Blum, Sylvie; Lethias, Claire

    2011-01-01

    Fibrillar collagens are the more abundant extracellular proteins. They form a metazoan-specific family, and are highly conserved from sponge to human. Their structural and physiological properties have been successfully used in the food, cosmetic, and pharmaceutical industries. On the other hand, the increase of jellyfish has led us to consider this marine animal as a natural product for food and medicine. Here, we have tested different Mediterranean jellyfish species in order to investigate the economic potential of their collagens. We have studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using Rhizostoma pulmo oral arms and the pepsin extraction method (2–10 mg collagen/g of wet tissue). Although a significant yield was obtained with Cotylorhiza tuberculata (0.45 mg/g), R. pulmo was used for further experiments, this jellyfish being considered as harmless to humans and being an abundant source of material. Then, we compared the biological properties of R. pulmo collagen with mammalian fibrillar collagens in cell cytotoxicity assays and cell adhesion. There was no statistical difference in cytotoxicity (p > 0.05) between R. pulmo collagen and rat type I collagen. However, since heparin inhibits cell adhesion to jellyfish-native collagen by 55%, the main difference is that heparan sulfate proteoglycans could be preferentially involved in fibroblast and osteoblast adhesion to jellyfish collagens. Our data confirm the broad harmlessness of jellyfish collagens, and their biological effect on human cells that are similar to that of mammalian type I collagen. Given the bioavailability of jellyfish collagen and its biological properties, this marine material is thus a good candidate for replacing bovine or human collagens in selected biomedical applications. PMID:21747742

  16. Aspartic Acid Racemization and Collagen Degradation Markers Reveal an Accumulation of Damage in Tendon Collagen That Is Enhanced with Aging*

    PubMed Central

    Thorpe, Chavaunne T.; Streeter, Ian; Pinchbeck, Gina L.; Goodship, Allen E.; Clegg, Peter D.; Birch, Helen L.

    2010-01-01

    Little is known about the rate at which protein turnover occurs in living tendon and whether the rate differs between tendons with different physiological roles. In this study, we have quantified the racemization of aspartic acid to calculate the age of the collagenous and non-collagenous components of the high strain injury-prone superficial digital flexor tendon (SDFT) and low strain rarely injured common digital extensor tendon (CDET) in a group of horses with a wide age range. In addition, the turnover of collagen was assessed indirectly by measuring the levels of collagen degradation markers (collagenase-generated neoepitope and cross-linked telopeptide of type I collagen). The fractional increase in d-Asp was similar (p = 0.7) in the SDFT (5.87 × 10−4/year) and CDET (5.82 × 10−4/year) tissue, and d/l-Asp ratios showed a good correlation with pentosidine levels. We calculated a mean (±S.E.) collagen half-life of 197.53 (±18.23) years for the SDFT, which increased significantly with horse age (p = 0.03) and was significantly (p < 0.001) higher than that for the CDET (34.03 (±3.39) years). Using similar calculations, the half-life of non-collagenous protein was 2.18 (±0.41) years in the SDFT and was significantly (p = 0.04) lower than the value of 3.51 (±0.51) years for the CDET. Collagen degradation markers were higher in the CDET and suggested an accumulation of partially degraded collagen within the matrix with aging in the SDFT. We propose that increased susceptibility to injury in older individuals results from an inability to remove partially degraded collagen from the matrix leading to reduced mechanical competence. PMID:20308077

  17. Reevaluation of the role of the polar groups of collagen in the platelet-collagen interaction.

    PubMed Central

    Chesney, C. M.; Pifer, D. D.; Crofford, L. J.; Huch, K. M.

    1983-01-01

    Chemical modification of collagen is a tool for exploring the platelet-collagen interaction. Since collagen must polymerize prior to the initiation of platelet aggregation and secretion, modification must be shown to affect platelet-collagen interaction and not collagen-collagen interaction. To address this point, the authors carried out the following chemical modifications on soluble monomeric collagen and preformed fibrillar collagen in parallel: 1) N-and O-acetylation, 2) esterification of the carboxyl groups, 3) succinylation of the free amino groups, 4) esterification of succinylated collagen. Intrinsic viscosity studies of the modified soluble collagens were consistent with normal triple helix conformation. Electron microscopy revealed all modified fibrillar collagen to maintain a fibrillar structure. Platelet aggregation and secretion of 14C-serotonin and platelet factor 4 by soluble and fibrillar collagen, respectively, were studied in human platelet-rich plasma. Neutralization of polar groups by 1) totally abolished aggregation and secretion by both collagens, while blocking acidic groups 2) resulted in enhanced aggregation and secretion by both soluble and fibrillar collagen. Blockage of amino groups by 3) abolished aggregation and secretion by both collagens. Esterified succinylated collagen 4) caused aggregation and secretion at relatively high collagen concentrations. These data support the theory that positive groups of collagen are important in platelet-collagen interaction. Images Figure 1 PMID:6881287

  18. Collagen-Proteoglycan Relationships in Epiphyseal Cartilage

    PubMed Central

    Eisenstein, Reuben; Larsson, Sven-Erik; Sorgente, Nino; Kuettner, Klaus E.

    1973-01-01

    Columnar and hypertrophic zones of calf scapular cartilage were studied before and after extraction with 3 M guanidinium chloride (GuCl) and digestion with enzymes which degrade various components of the extracellular matrix. Morphologic and chemical analysis suggests that there are at least two anatomic pools of proteoglycan in this tissue. One, which resides between collagen fibrils, is extractable with GuCl. Another appears attached to collagen by strong bonds and is apparently not extractable with GuCl. This type of collagen-proteoglycan relationship is possibly restricted to epiphyseal cartilage. The morphology of the lacuna is different in the columnar and hypertrophic zones. Proteoglycans in the distal hypertrophic zone are less resistant to GuCl extraction. ImagesFig 9Fig 10Fig 11Fig 1Fig 2Fig 3Fig 4Fig 5Fig 6Fig 7Fig 8 PMID:4357177

  19. Identification of Pyridinoline Trivalent Collagen Cross-Links by Raman Microspectroscopy.

    PubMed

    Gamsjaeger, Sonja; Robins, Simon P; Tatakis, Dimitris N; Klaushofer, Klaus; Paschalis, Eleftherios P

    2017-06-01

    Intermolecular cross-linking of bone collagen is intimately related to the way collagen molecules are arranged in a fibril, imparts certain mechanical properties to the fibril, and may be involved in the initiation of mineralization. Raman microspectroscopy allows the analysis of minimally processed bone blocks and provides simultaneous information on both the mineral and organic matrix (mainly type I collagen) components, with a spatial resolution of ~1 μm. The aim of the present study was to validate Raman spectroscopic parameters describing one of the major mineralizing type I trivalent cross-links, namely pyridinoline (PYD). To achieve this, a series of collagen cross-linked peptides with known PYD content (as determined by HPLC analysis), human bone, porcine skin, predentin and dentin animal model tissues were analyzed by Raman microspectroscopy. The results of the present study confirm that it is feasible to monitor PYD trivalent collagen cross-links by Raman spectroscopic analysis in mineralized tissues, exclusively through a Raman band ~1660 wavenumbers. This allows determination of the relative PYD content in undecalcified bone tissues with a spatial resolution of ~1 μm, thus enabling correlations with histologic and histomorphometric parameters.

  20. Lysyl oxidases regulate fibrillar collagen remodelling in idiopathic pulmonary fibrosis.

    PubMed

    Tjin, Gavin; White, Eric S; Faiz, Alen; Sicard, Delphine; Tschumperlin, Daniel J; Mahar, Annabelle; Kable, Eleanor P W; Burgess, Janette K

    2017-11-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung with few effective therapeutic options. Structural remodelling of the extracellular matrix [i.e. collagen cross-linking mediated by the lysyl oxidase (LO) family of enzymes (LOX, LOXL1-4)] might contribute to disease pathogenesis and represent a therapeutic target. This study aimed to further our understanding of the mechanisms by which LO inhibitors might improve lung fibrosis. Lung tissues from IPF and non-IPF subjects were examined for collagen structure (second harmonic generation imaging) and LO gene (microarray analysis) and protein (immunohistochemistry and western blotting) levels. Functional effects (collagen structure and tissue stiffness using atomic force microscopy) of LO inhibitors on collagen remodelling were examined in two models, collagen hydrogels and decellularized human lung matrices. LOXL1 / LOXL2 gene expression and protein levels were increased in IPF versus non-IPF. Increased collagen fibril thickness in IPF versus non-IPF lung tissues correlated with increased LOXL1/LOXL2, and decreased LOX, protein expression. β-Aminoproprionitrile (β-APN; pan-LO inhibitor) but not Compound A (LOXL2-specific inhibitor) interfered with transforming growth factor-β-induced collagen remodelling in both models. The β-APN treatment group was tested further, and β-APN was found to interfere with stiffening in the decellularized matrix model. LOXL1 activity might drive collagen remodelling in IPF lungs. The interrelationship between collagen structural remodelling and LOs is disrupted in IPF lungs. Inhibition of LO activity alleviates fibrosis by limiting fibrillar collagen cross-linking, thereby potentially impeding the formation of a pathological microenvironment in IPF. © 2017. Published by The Company of Biologists Ltd.

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

    PubMed Central

    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

  2. Quantification of three-dimensional cell-mediated collagen remodeling using graph theory.

    PubMed

    Bilgin, Cemal Cagatay; Lund, Amanda W; Can, Ali; Plopper, George E; Yener, Bülent

    2010-09-30

    Cell cooperation is a critical event during tissue development. We present the first precise metrics to quantify the interaction between mesenchymal stem cells (MSCs) and extra cellular matrix (ECM). In particular, we describe cooperative collagen alignment process with respect to the spatio-temporal organization and function of mesenchymal stem cells in three dimensions. We defined two precise metrics: Collagen Alignment Index and Cell Dissatisfaction Level, for quantitatively tracking type I collagen and fibrillogenesis remodeling by mesenchymal stem cells over time. Computation of these metrics was based on graph theory and vector calculus. The cells and their three dimensional type I collagen microenvironment were modeled by three dimensional cell-graphs and collagen fiber organization was calculated from gradient vectors. With the enhancement of mesenchymal stem cell differentiation, acceleration through different phases was quantitatively demonstrated. The phases were clustered in a statistically significant manner based on collagen organization, with late phases of remodeling by untreated cells clustering strongly with early phases of remodeling by differentiating cells. The experiments were repeated three times to conclude that the metrics could successfully identify critical phases of collagen remodeling that were dependent upon cooperativity within the cell population. Definition of early metrics that are able to predict long-term functionality by linking engineered tissue structure to function is an important step toward optimizing biomaterials for the purposes of regenerative medicine.

  3. Collagen structure of tendon relates to function.

    PubMed

    Franchi, Marco; Trirè, Alessandra; Quaranta, Marilisa; Orsini, Ester; Ottani, Victoria

    2007-03-30

    A tendon is a tough band of fibrous connective tissue that connects muscle to bone, designed to transmit forces and withstand tension during muscle contraction. Tendon may be surrounded by different structures: 1) fibrous sheaths or retinaculae; 2) reflection pulleys; 3) synovial sheaths; 4) peritendon sheaths; 5) tendon bursae. Tendons contain a) few cells, mostly represented by tenoblasts along with endothelial cells and some chondrocytes; b) proteoglycans (PGs), mainly decorin and hyaluronan, and c) collagen, mostly type I. Tendon is a good example of a high ordered extracellular matrix in which collagen molecules assemble into filamentous collagen fibrils (formed by microfibrils) which aggregate to form collagen fibers, the main structural components. It represents a multihierarchical structure as it contains collagen molecules arranged in fibrils then grouped in fibril bundles, fascicles and fiber bundles that are almost parallel to the long axis of the tendon, named as primary, secondary and tertiary bundles. Collagen fibrils in tendons show prevalently large diameter, a D-period of about 67 nm and appear built of collagen molecules lying at a slight angle (< 5 degrees). Under polarized light microscopy the collagen fiber bundles appear crimped with alternative dark and light transverse bands. In recent studies tendon crimps observed via SEM and TEM show that the single collagen fibrils suddenly changing their direction contain knots. These knots of collagen fibrils inside each tendon crimp have been termed "fibrillar crimps", and even if they show different aspects they all may fulfil the same functional role. As integral component of musculoskeletal system, the tendon acts to transmit muscle forces to the skeletal system. There is no complete understanding of the mechanisms in transmitting/absorbing tensional forces within the tendon; however it seems likely that a flattening of tendon crimps may occur at a first stage of tendon stretching. Increasing

  4. Modeling pulmonary fibrosis by abnormal expression of telomerase/apoptosis/collagen V in experimental usual interstitial pneumonia

    PubMed Central

    Parra, E.R.; Pincelli, M.S.; Teodoro, W.R.; Velosa, A.P.P.; Martins, V.; Rangel, M.P.; Barbas-Filho, J.V.; Capelozzi, V.L.

    2014-01-01

    Limitations on tissue proliferation capacity determined by telomerase/apoptosis balance have been implicated in pathogenesis of idiopathic pulmonary fibrosis. In addition, collagen V shows promise as an inductor of apoptosis. We evaluated the quantitative relationship between the telomerase/apoptosis index, collagen V synthesis, and epithelial/fibroblast replication in mice exposed to butylated hydroxytoluene (BHT) at high oxygen concentration. Two groups of mice were analyzed: 20 mice received BHT, and 10 control mice received corn oil. Telomerase expression, apoptosis, collagen I, III, and V fibers, and hydroxyproline were evaluated by immunohistochemistry, in situ detection of apoptosis, electron microscopy, immunofluorescence, and histomorphometry. Electron microscopy confirmed the presence of increased alveolar epithelial cells type 1 (AEC1) in apoptosis. Immunostaining showed increased nuclear expression of telomerase in AEC type 2 (AEC2) between normal and chronic scarring areas of usual interstitial pneumonia (UIP). Control lungs and normal areas from UIP lungs showed weak green birefringence of type I and III collagens in the alveolar wall and type V collagen in the basement membrane of alveolar capillaries. The increase in collagen V was greater than collagens I and III in scarring areas of UIP. A significant direct association was found between collagen V and AEC2 apoptosis. We concluded that telomerase, collagen V fiber density, and apoptosis evaluation in experimental UIP offers the potential to control reepithelization of alveolar septa and fibroblast proliferation. Strategies aimed at preventing high rates of collagen V synthesis, or local responses to high rates of cell apoptosis, may have a significant impact in pulmonary fibrosis. PMID:24919172

  5. Preclinical evaluation of collagen type I scaffolds, including gelatin-collagen microparticles and loaded with a hydroglycolic Calendula officinalis extract in a lagomorph model of full-thickness skin wound.

    PubMed

    Millán, D; Jiménez, R A; Nieto, L E; Linero, I; Laverde, M; Fontanilla, M R

    2016-02-01

    Previously, we have developed collagen type I scaffolds including microparticles of gelatin-collagen type I (SGC) that are able to control the release of a hydroglycolic extract of the Calendula officinalis flower. The main goal of the present work was to carry out the preclinical evaluation of SGC alone or loaded with the C. officinalis extract (SGC-E) in a lagomorph model of full-thickness skin wound. A total of 39 rabbits were distributed in three groups, of 13 animals each. The first group was used to compare wound healing by secondary intention (control) with wound healing observed when wounds were grafted with SGC alone. Comparison of control wounds with wounds grafted with SGC-E was performed in the second group, and comparison of wounds grafted with SGC with wounds grafted with SGC-E was performed in the third group. Clinical follow-ups were carried in all animals after surgery, and histological and histomorphometric analyses were performed on tissues taken from the healed area and healthy surrounding tissue. Histological and histomorphometric results indicate that grafting of SGC alone favors wound healing and brings a better clinical outcome than grafting SGC-E. In vitro collagenase digestion data suggested that the association of the C. officinalis extract to SGC increased the SGC-E cross-linking, making it difficult to degrade and affecting its biocompatibility.

  6. Doxycycline alters collagen composition following ventral hernia repair.

    PubMed

    Tharappel, Job C; Harris, Jennifer W; Totten, Crystal; Zwischenberger, Brittany A; Roth, John S

    2017-04-01

    Doxycycline, a nonspecific metalloproteinase (MMP) inhibitor, has been demonstrated to impact the strength of the polypropylene (PP) mesh-repaired hernia with an increase in the deposition of collagen type 1. The impact of doxycycline with porcine acellular dermal matrices (PADM) is unknown; therefore, we evaluated the impact of doxycycline administration upon hernia repair with PP and PADM mesh. Sprague-Dawley rats weighing ~400 g underwent laparotomy with creation of a midline ventral hernia. After a 27-day recovery, animals were randomly assigned to four groups of eight and underwent intraperitoneal underlay hernia repair with either PP or PADM. Groups were assigned to daily normal saline (S) or daily doxycycline in normal saline 10 mg/kg (D) via oral gavage for 8 weeks beginning 24 h preoperatively. Animals were euthanized at 8 weeks and underwent tensiometric testing of the abdominal wall and western blot analyses for collagen subtypes and MMPs. Thirty-two animals underwent successful hernia creation and repair with either PADM or PP. At 8 weeks, 15 of 16 PP-implanted animals survived with only 12 of 16 PADM-implanted animals surviving. There were no differences in the mesh to fascial interface tensiometric strength between groups. Densitometric counts in the PADM-D group demonstrated increased collagen type 1 compared to PP-S (PADM-D [1286.5], PADM-S [906.9], PP-S [700.4], p = 0.037) and decreased collagen type 3 compared to PP-S (PADM-D [7446.9], PADM-S [8507.6], PP-S [11,297.1], p = 0.01). MMP-9 levels were increased in PADM-D (PP-S vs. PADM-D, p = 0.04), while MMP-2 levels were similar between PADM-D and PADM-S, respectively. Collagen type 1 deposition at the mesh to fascial interface is enhanced following administration of doxycycline in ventral hernia repairs with porcine acellular dermal matrices. Doxycycline administration may have implications for enhancing hernia repair outcomes using biologic mesh.

  7. Local delivery of a collagen-binding FGF-1 chimera to smooth muscle cells in collagen scaffolds for vascular tissue engineering.

    PubMed

    Pang, Yonggang; Wang, Xiaoli; Ucuzian, Areck A; Brey, Eric M; Burgess, Wilson H; Jones, Kathryn J; Alexander, Thomas D; Greisler, Howard P

    2010-02-01

    We investigated the delivery of R136K-CBD (a collagen-binding mutant chimera of fibroblast growth factor-1) with a type I collagen scaffold as the delivery vehicle to smooth muscle cells (SMCs) for vascular tissue engineering. The binding affinity of R136K-CBD to 3-D collagen scaffolds was investigated both in the presence and absence of cells and/or salts. 2-D and 3-D visualization of delivery of R136K-CBD into SMCs were accomplished by combined fluorescent and reflection confocal microscopy. The mitogenic effect of collagen-immobilized R136K-CBD on SMCs in 3-D collagen was studied by Cyquant assay at different time intervals. In the group devoid of salt and cells, no detectable release of R136K-CBD into overlying culture media was found, compared with burst-and-continuous release of R136K and FGF-1 over a 14-day period in all other groups. The release rate of R136K-CBD was 1.7 and 1.6-fold less than R-136K and FGF-1 when media was supplemented with 2m salt (P<0.0001), and 2.6 and 2.5-fold less in cell-populated collagen hydrogels (P<0.0001), respectively. R136K-CBD showed essentially uniform binding to collagen and its distribution was dependent on that of the collagen scaffold. Internalization of R136K-CBD into SMCs was documented by confocal microscopy. 3-D local delivery of collagen-immobilized R136K-CBD increased the proliferation of SMCs in the collagen matrix to significantly greater levels and for a significantly greater duration than R136K or FGF-1, with 2.0 and 2.1-fold more mitogenicity than R136K and FGF-1 respectively (P<0.0001) at day 7. The results suggest that our collagen-binding fusion protein is an effective strategy for growth factor delivery for vascular tissue engineering.

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

    PubMed

    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.

  9. Development of biomimetic tilapia collagen nanofibers for skin regeneration through inducing keratinocytes differentiation and collagen synthesis of dermal fibroblasts.

    PubMed

    Zhou, Tian; Wang, Nanping; Xue, Yang; Ding, Tingting; Liu, Xin; Mo, Xiumei; Sun, Jiao

    2015-02-11

    In this study, tilapia skin collagen sponge and electrospun nanofibers were developed for wound dressing. The collagen sponge was composed of at least two α-peptides, and its denaturation temperature was 44.99 °C. It did not change the number of spleen-derived lymphocytes in BALB/c mice, the ratio of CD4+/CD8+ lymphocytes, and the level of IgG or IgM in Sprague-Dawley rat. The contact angle, tensile strength, and weight loss temperature of collagen nanofibers were 21.2°, 6.72±0.44 MPa, and 300 °C, respectively. The nanofibers could promote the viabilities of human keratinocytes (HaCaTs) and human dermal fibroblasts (HDFs), inducing epidermal differentiation through the gene expression of involucrin, filaggrin, and type I transglutaminase of HaCaTs, and they could also accelerate migration of HaCaTs with the expression of matrix metalloproteinase-9 and transforming growth factor-β1 (TGF-β1). Besides, the nanofibers could upregulate the protien level of Col-I in HDFs both via a direct effect and TGF-β1 secreted from HaCaTs, thus facilitating the formation of collagen fibers. Furthermore, the collagen nanofibers stimulated the skin regeneration rapidly and effectively in vivo. These biological effects could be explained as the contributions from the biomimic extracellular cell matrix structure, hydrophilicity, and the multiple amino acids of the collagen nanofibers.

  10. Moisture absorption and retention properties, and activity in alleviating skin photodamage of collagen polypeptide from marine fish skin.

    PubMed

    Hou, Hu; Li, Bafang; Zhang, Zhaohui; Xue, Changhu; Yu, Guangli; Wang, Jingfeng; Bao, Yuming; Bu, Lin; Sun, Jiang; Peng, Zhe; Su, Shiwei

    2012-12-01

    Collagen polypeptides were prepared from cod skin. Moisture absorption and retention properties of collagen polypeptides were determined at different relative humidities. In addition, the protective effects of collagen polypeptide against UV-induced damage to mouse skin were evaluated. Collagen polypeptides had good moisture absorption and retention properties and could alleviate the damage induced by UV radiation. The action mechanisms of collagen polypeptide mainly involved enhancing immunity, reducing the loss of moisture and lipid, promoting anti-oxidative properties, inhibiting the increase of glycosaminoglycans, repairing the endogenous collagen and elastin protein fibres, and maintaining the ratio of type III to type I collagen. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Collagen Self-Assembly on Orthopedic Magnesium Biomaterials Surface and Subsequent Bone Cell Attachment

    PubMed Central

    Zhao, Nan; Zhu, Donghui

    2014-01-01

    Magnesium (Mg) biomaterials are a new generation of biodegradable materials and have promising potential for orthopedic applications. After implantation in bone tissues, these materials will directly interact with extracellular matrix (ECM) biomolecules and bone cells. Type I collagen, the major component of bone ECM, forms the architecture scaffold that provides physical support for bone cell attachment. However, it is still unknown how Mg substrate affects collagen assembly on top of it as well as subsequent cell attachment and growth. Here, we studied the effects of collagen monomer concentration, pH, assembly time, and surface roughness of two Mg materials (pure Mg and AZ31) on collagen fibril formation. Results showed that formation of fibrils would not initiate until the monomer concentration reached a certain level depending on the type of Mg material. The thickness of collagen fibril increased with the increase of assembly time. The structures of collagen fibrils formed on semi-rough surfaces of Mg materials have a high similarity to that of native bone collagen. Next, cell attachment and growth after collagen assembly were examined. Materials with rough surface showed higher collagen adsorption but compromised bone cell attachment. Interestingly, surface roughness and collagen structure did not affect cell growth on AZ31 for up to a week. Findings from this work provide some insightful information on Mg-tissue interaction at the interface and guidance for future surface modifications of Mg biomaterials. PMID:25303459

  12. Collagenous gastritis and collagenous colitis: a report with sequential histological and ultrastructural findings

    PubMed Central

    Pulimood, A; Ramakrishna, B; Mathan, M

    1999-01-01

    The case is reported of a young adult man with collagenous gastritis, an extremely rare disorder with only three case reports in the English literature, who subsequently presented with collagenous colitis. Sequential gastric biopsies showed a notable increase in thickness of the subepithelial collagen band. Ultrastructural study of gastric and rectal mucosa showed the characteristic subepithelial band composed of haphazardly arranged collagen fibres, prominent degranulating eosinophils, and activated pericryptal fibroblasts.


Keywords: collagenous gastritis; collagenous colitis; stomach; colon PMID:10323893

  13. Fluorescence, aggregation properties and FT-IR microspectroscopy of elastin and collagen fibers.

    PubMed

    Vidal, Benedicto de Campos

    2014-10-01

    Histological and histochemical observations support the hypothesis that collagen fibers can link to elastic fibers. However, the resulting organization of elastin and collagen type complexes and differences between these materials in terms of macromolecular orientation and frequencies of their chemical vibrational groups have not yet been solved. This study aimed to investigate the macromolecular organization of pure elastin, collagen type I and elastin-collagen complexes using polarized light DIC-microscopy. Additionally, differences and similarities between pure elastin and collagen bundles (CB) were investigated by Fourier transform-infrared (FT-IR) microspectroscopy. Although elastin exhibited a faint birefringence, the elastin-collagen complex aggregates formed in solution exhibited a deep birefringence and formation of an ordered-supramolecular complex typical of collagen chiral structure. The FT-IR study revealed elastin and CB peptide NH groups involved in different types of H-bonding. More energy is absorbed in the vibrational transitions corresponding to CH, CH2 and CH3 groups (probably associated with the hydrophobicity demonstrated by 8-anilino-1-naphtalene sulfonic acid sodium salt [ANS] fluorescence), and to νCN, δNH and ωCH2 groups of elastin compared to CB. It is assumed that the α-helix contribution to the pure elastin amide I profile is 46.8%, whereas that of the B-sheet is 20% and that unordered structures contribute to the remaining percentage. An FT-IR profile library reveals that the elastin signature within the 1360-1189cm(-1) spectral range resembles that of Conex-Toray aramid fibers. Copyright © 2014 Elsevier GmbH. All rights reserved.

  14. Quantitative characterization of collagen in the fibrotic capsule surrounding implanted polymeric microparticles through second harmonic generation imaging

    DOE PAGES

    Akilbekova, Dana; Bratlie, Kaitlin M.; Abraham, Thomas

    2015-06-30

    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. Variationmore » 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.« less

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

    PubMed Central

    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. PMID:26125551

  16. Quantitative characterization of collagen in the fibrotic capsule surrounding implanted polymeric microparticles through second harmonic generation imaging

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Akilbekova, Dana; Bratlie, Kaitlin M.; Abraham, Thomas

    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. Variationmore » 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.« less

  17. Structure to function: Spider silk and human collagen

    NASA Astrophysics Data System (ADS)

    Rabotyagova, Olena S.

    , morphological features and assembly. Aside from fundamental perspectives, we anticipate that these results will provide a blueprint for the design of precise materials for a range of potential applications such as controlled release devices, functional coatings, components of tissue regeneration materials and environmentally friendly polymers in future studies. In the second part of this work, human collagen type I was studied as another representative of the family of fibrous proteins. 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 a complex structural hierarchy, organized at different length scales, including the characteristic triple helical feature. In the present study we assessed the relationship between collagen structure (native vs. denatured) and sensitivity to UV radiation with a focus on changes in the primary structure, conformation, microstructure and material properties. Free radical reactions are involved in collagen degradation and a mechanism for UV-induced collagen degradation related to structure was proposed. The results from this study demonstrated the role of collagen supramolecular organization (triple helix) in the context of the effects of electromagnetic radiation on extracellular matrices. Owing to the fact that both silks and collagens are proteins that have found widespread interest for biomaterial related needs, we anticipate that the current studies will serve as a foundation for future biomaterial designs with controlled properties. Furthermore, fundamental insight into self-assembly and environmentally-2mediated degradation, will build a foundation for fundamental understanding of the remodeling and functions of these types of fibrous proteins in vivo and in vitro. This type of insight is essential for many areas of scientific inquiry, from drug delivery, to scaffolds for tissue engineering, and to the stability of

  18. Substitutions of aspartic acid for glycine-220 and of arginine for glycine-664 in the triple helix of the pro alpha 1(I) chain of type I procollagen produce lethal osteogenesis imperfecta and disrupt the ability of collagen fibrils to incorporate crystalline hydroxyapatite.

    PubMed Central

    Culbert, A A; Lowe, M P; Atkinson, M; Byers, P H; Wallis, G A; Kadler, K E

    1995-01-01

    We identified two infants with lethal (type II) osteogenesis imperfecta (OI) who were heterozygous for mutations in the COL1A1 gene that resulted in substitutions of aspartic acid for glycine at position 220 and arginine for glycine at position 664 in the product of one COL1A1 allele in each individual. In normal age- and site-matched bone, approximately 70% (by number) of the collagen fibrils were encrusted with plate-like crystallites of hydroxyapatite. In contrast, approximately 5% (by number) of the collagen fibrils in the probands' bone contained crystallites. In contrast with normal bone, the c-axes of hydroxyapatite crystallites were sometimes poorly aligned with the long axis of fibrils obtained from OI bone. Chemical analysis showed that the OI samples contained normal amounts of calcium. The probands' bone samples contained type I collagen, overmodified type I collagen and elevated levels of type III and V collagens. On the basis of biochemical and morphological data, the fibrils in the OI samples were co-polymers of normal and mutant collagen. The results are consistent with a model of fibril mineralization in which the presence of abnormal type I collagen prevents normal collagen in the same fibril from incorporating hydroxyapatite crystallites. Images Figure 1 Figure 2 Figure 3 PMID:7487936

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

    PubMed

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

    2013-01-01

    Collagen-based dressings are of great interest in wound care. However, evidence supporting their mechanism of action is scanty. This work provides first results from a preclinical swine model of excisional wounds, elucidating the mechanism of action of a modified collagen gel (MCG) dressing. Following wounding, wound-edge tissue was collected at specific time intervals (3, 7, 14, and 21 days postwounding). On day 7, histological analysis showed significant increase in the length of rete ridges, suggesting improved biomechanical properties of the healing wound tissue. Rapid and transient mounting of inflammation is necessary for efficient healing. MCG significantly accelerated neutrophil and macrophage recruitment to the wound site on day 3 and day 7 with successful resolution of inflammation on day 21. MCG induced monocyte chemotactic protein-1 expression in neutrophil-like human promyelocytic leukemia-60 cells in vitro. In vivo, MCG-treated wound tissue displayed elevated vascular endothelial growth factor expression. Consistently, MCG-treated wounds displayed significantly higher abundance of endothelial cells with increased blood flow to the wound area indicating improved vascularization. This observation was explained by the finding that MCG enhanced proliferation of wound-site endothelial cells. In MCG-treated wound tissue, Masson's trichrome and picrosirius red staining showed higher abundance of collagen and increased collagen type I:III ratio. This work presents first evidence from a preclinical setting explaining how a collagen-based dressing may improve wound closure by targeting multiple key mechanisms. The current findings warrant additional studies to determine whether the responses to the MCG are different from other collagen-based products used in clinical setting. © 2013 by the Wound Healing Society.

  20. Production of reactive oxygen species by withaferin A causes loss of type collagen expression and COX-2 expression through the PI3K/Akt, p38, and JNK pathways in rabbit articular chondrocytes.

    PubMed

    Yu, Seon-Mi; Kim, Song-Ja

    2013-11-01

    Withaferin A (WFA) is a major chemical constituent of Withania somnifera, also known as Indian ginseng. Many recent reports have provided evidence of its anti-tumor, anti-inflammation, anti-oxidant, and immune modulatory activities. Although the compound appears to have a large number of effects, its defined mechanisms of action have not yet been determined. We investigated the effects of WFA on loss of type collagen expression and inflammation in rabbit articular chondrocytes. WFA increased the production of reactive oxygen species, suggesting the induction of oxidative stress, in a dose-dependent manner. Also, we confirmed that WFA causes loss of type collagen expression and inflammation as determined by a decrease of type II collagen expression and an increase of cyclooxygenase-2 (COX-2) expression via western blot analysis in a dose- and time- dependent manner. WFA also reduced the synthesis of sulfated proteoglycan via Alcian blue staining and caused the synthesis of prostaglandin E2 (PGE2) via assay kit in dose- and time-dependent manners. Treatment with N-acetyl-L-cysteine (NAC), an antioxidant, inhibited WFA-induced loss of type II collagen expression and increase in COX-2 expression, accompanied by inhibition of reactive oxygen species production. WFA increased phosphorylation of both Akt and p38. Inhibition of PI3K/Akt, p38, and JNK with LY294002 (LY), SB203580 (SB), or SP600125 (SP) in WFA-treated cells rescued the expression of type II collagen and suppressed the expression of COX-2. These results demonstrate that WFA induces loss of type collagen expression and inflammation via PI3K/Akt, p38, and JNK by generating reactive oxygen species in rabbit articular chondrocytes. © 2013 Published by Elsevier Inc.

  1. A comparative study of the properties and self-aggregation behavior of collagens from the scales and skin of grass carp (Ctenopharyngodon idella).

    PubMed

    Liu, Yaowen; Ma, Donghui; Wang, Yihao; Qin, Wen

    2018-01-01

    Collagens were extracted from the scales and skin of Ctenopharyngodon idella (C. idella) as raw materials using an acid-enzyme hybrid method. The structural properties of the extracted collagens were compared using ultraviolet-visible spectrophotometry, Fourier transform infrared spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and differential scanning calorimetry. Additionally, the in vitro self-aggregation behaviors of the two types of collagens (fish skin- and scale-derived collagens) were compared using turbidimetric assays, aggregation assays, and scanning electron microscopy (SEM). The results showed that both types of extracted collagen were typical type I collagen with two α chains and intact triple-helical structures. The denaturation temperatures of the collagens from fish scales and skin were 34.99°C and 39.75°C, respectively. Both types of collagens were capable of self-aggregation in neutral salt solution at 30°C, with aggregation degrees of 28% and 27.33% for the scale and skin collagens, respectively. SEM analysis revealed that both types of collagens could self-aggregate into interwoven fibers, and the fish scale-derived collagen had a more pronounced reticular fiber structure with a striped periodic D-band pattern of collagen fibrils, whereas the collagen fibers from the self-aggregation of fish skin-derived collagen had a certain degree of disruption without any D-band pattern. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Mechanical Properties of Single Collagen Fibrils Revealed by Force Spectroscopy

    NASA Astrophysics Data System (ADS)

    Graham, John; Phillips, Charlotte; Grandbois, Michel

    2004-03-01

    In the field of biomechanics, collagen fibrils are believed to be robust mechanical structures characterized by a low extensibility. Until very recently, information on the mechanical properties of collagen fibrils could only be derived from ensemble measurements performed on complete tissues such as bone, skin and tendon. Here we measure force-elongation/relaxation profiles of single collagen fibrils using atomic force microscopy-based force spectroscopy. The elongation profiles indicate that in vitro assembled heterotrimeric type I collagen fibrils are characterized by a large extensibility. Numerous discontinuities and a plateau in the force profile indicate major reorganization occurs within the fibrils in the 1.5 -- 4.5 nN range. Our study demonstrates that newly assembled collagen fibrils are robust structures with a significant reserve of elasticity that could play a determinant role in cellular motion in the context of tissue growth and morphogenesis. In contrast, homotrimeric collagen fibrils corresponding to osteogenesis imperfecta pathology exhibit a marked difference in their elasticity profile.

  3. Stabilization and Anomalous Hydration of Collagen Fibril under Heating

    PubMed Central

    Gevorkian, Sasun G.; Allahverdyan, Armen E.; Gevorgyan, David S.; Simonian, Aleksandr L.; Hu, Chin-Kun

    2013-01-01

    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. PMID:24244320

  4. Cutaneous Collagenous Vasculopathy

    PubMed Central

    Ortleb, Melanie; Boyd, Alan S.; Powers, Jennifer

    2015-01-01

    Cutaneous collagenous vasculopathy is a rare microangiopathy of dermal blood vessels. Clinically indistinguishable from generalized essential telangiectasia, this condition is diagnosed by its unique histological appearance. In contrast to other primary telangiectatic processes, cutaneous collagenous vasculopathy has dilated vascular structures that contain deposits of eosinophilic hyaline material within the vessel walls. To date, cutaneous collagenous vasculopathy has been described in a total of 19 cases in the medical literature. The first several cases were described exclusively in middle-aged to elderly men. Though it has now been described in both men and women, cutaneous collagenous vasculopathy is still most often described in middle-aged to older adults. No particular disease or medication has been linked to the development of cutaneous collagenous vasculopathy, and the etiology remains unknown. In this case series, the authors present three additional patients diagnosed with cutaneous collagenous vasculopathy and discuss their clinical and histopathologic features. PMID:26705441

  5. Biological Safety of Fish (Tilapia) Collagen

    PubMed Central

    Yamamoto, Kohei; Sugimoto, Kouji; Yoshizawa, Yuu; Yanagiguchi, Kajiro

    2014-01-01

    Marine collagen derived from fish scales, skin, and bone has been widely investigated for application as a scaffold and carrier due to its bioactive properties, including excellent biocompatibility, low antigenicity, and high biodegradability and cell growth potential. Fish type I collagen is an effective material as a biodegradable scaffold or spacer replicating the natural extracellular matrix, which serves to spatially organize cells, providing them with environmental signals and directing site-specific cellular regulation. This study was conducted to confirm the safety of fish (tilapia) atelocollagen for use in clinical application. We performed in vitro and in vivo biological studies of medical materials to investigate the safety of fish collagen. The extract of fish collagen gel was examined to clarify its sterility. All present sterility tests concerning bacteria and viruses (including endotoxin) yielded negative results, and all evaluations of cell toxicity, sensitization, chromosomal aberrations, intracutaneous reactions, acute systemic toxicity, pyrogenic reactions, and hemolysis were negative according to the criteria of the ISO and the Ministry of Health, Labour and Welfare of Japan. The present study demonstrated that atelocollagen prepared from tilapia is a promising biomaterial for use as a scaffold in regenerative medicine. PMID:24809058

  6. Biological safety of fish (tilapia) collagen.

    PubMed

    Yamamoto, Kohei; Igawa, Kazunari; Sugimoto, Kouji; Yoshizawa, Yuu; Yanagiguchi, Kajiro; Ikeda, Takeshi; Yamada, Shizuka; Hayashi, Yoshihiko

    2014-01-01

    Marine collagen derived from fish scales, skin, and bone has been widely investigated for application as a scaffold and carrier due to its bioactive properties, including excellent biocompatibility, low antigenicity, and high biodegradability and cell growth potential. Fish type I collagen is an effective material as a biodegradable scaffold or spacer replicating the natural extracellular matrix, which serves to spatially organize cells, providing them with environmental signals and directing site-specific cellular regulation. This study was conducted to confirm the safety of fish (tilapia) atelocollagen for use in clinical application. We performed in vitro and in vivo biological studies of medical materials to investigate the safety of fish collagen. The extract of fish collagen gel was examined to clarify its sterility. All present sterility tests concerning bacteria and viruses (including endotoxin) yielded negative results, and all evaluations of cell toxicity, sensitization, chromosomal aberrations, intracutaneous reactions, acute systemic toxicity, pyrogenic reactions, and hemolysis were negative according to the criteria of the ISO and the Ministry of Health, Labour and Welfare of Japan. The present study demonstrated that atelocollagen prepared from tilapia is a promising biomaterial for use as a scaffold in regenerative medicine.

  7. Inhibition of Glycoprotein VI Clustering by Collagen as a Mechanism of Inhibiting Collagen-Induced Platelet Responses: The Example of Losartan

    PubMed Central

    Jiang, Peng; Loyau, Stéphane; Tchitchinadze, Maria; Ropers, Jacques; Jondeau, Guillaume; Jandrot-Perrus, Martine

    2015-01-01

    Exposure of platelets to collagen triggers the formation of a platelet clot. Pharmacological agents capable of inhibiting platelet activation by collagen are thus of potential therapeutic interest. Thrombus formation is initiated by the interaction of the GPIb-V-IX complex with collagen-bound vWF, while GPVI interaction with collagen triggers platelet activation that is reinforced by ADP and thromboxane A2. Losartan is an angiotensin II (Ang II) type I receptor (AT1R) antagonist proposed to have an antiplatelet activity via the inhibition of both the thromboxane A2 (TXA2) receptor (TP) and the glycoprotein VI (GPVI). Here, we characterized in vitro the effects of losartan at different doses on platelet responses: losartan inhibited platelet aggregation and secretion induced by 1 μg.mL-1 and 10 μg.mL-1 of collagen with an IC50 of ~ 6 μM. Losartan inhibited platelet responses induced by the GPVI specific collagen related peptide but not by the α2β1 specific peptide. However, losartan did not inhibit the binding of recombinant GPVI to collagen, which is not in favor of a simple competition. Indeed, the clustering of GPVI observed in flow cytometry and using the Duolink methodology, was inhibited by losartan. The impact of a therapeutic dose of losartan (100 mg/day) on platelet responses was analyzed ex vivo in a double blind study. No statistically significant differences were observed between losartan-treated (n=25) and non-treated (n=30) patients in terms of collagen and U46619-induced platelet activation. These data indicate that in treated patients, losartan does not achieve a measurable antiplatelet effect but provide the proof of concept that inhibiting collagen-induced GPVI clustering is of pharmacological interest to obtain an antithrombotic efficacy. Trial Registration ClinicalTrials.gov NCT00763893 PMID:26052700

  8. Hindlimb unloading induces a collagen isoform shift in the soleus muscle of the rat.

    PubMed

    Miller, T A; Lesniewski, L A; Muller-Delp, J M; Majors, A K; Scalise, D; Delp, M D

    2001-11-01

    To determine whether hindlimb unloading (HU) alters the extracellular matrix of skeletal muscle, male Sprague-Dawley rats were subjected to 0 (n = 11), 1 (n = 11), 14 (n = 13), or 28 (n = 11) days of unloading. Remodeling of the soleus and plantaris muscles was examined biochemically for collagen abundance via measurement of hydroxyproline, and the percentage of cross-sectional area of collagen was determined histologically with picrosirius red staining. Total hydroxyproline content in the soleus and plantaris muscles was unaltered by HU at any time point. However, the relative proportions of type I collagen in the soleus muscle decreased relative to control (Con) with 14 and 28 days HU (Con 68 +/- 5%; 14 days HU 53 +/- 4%; 28 days HU 53 +/- 7%). Correspondingly, type III collagen increased in soleus muscle with 14 and 28 days HU (Con 32 +/- 5%; 14 days HU 47 +/- 4%; 28 days HU 48 +/- 7%). The proportion of type I muscle fibers in soleus muscle was diminished with HU (Con 96 +/- 2%; 14 days HU 86 +/- 1%; 28 days HU 83 +/- 1%), and the proportion of hybrid type I/IIB fibers increased (Con 0%; 14 days HU 8 +/- 2%; 28 days HU 14 +/- 2%). HU had no effect on the proportion of type I and III collagen or muscle fiber composition in plantaris muscle. The data demonstrate that HU induces a shift in the relative proportion of collagen isoform (type I to III) in the antigravity soleus muscle, which occurs concomitantly with a slow-to-fast myofiber transformation.

  9. Hindlimb unloading induces a collagen isoform shift in the soleus muscle of the rat

    NASA Technical Reports Server (NTRS)

    Miller, T. A.; Lesniewski, L. A.; Muller-Delp, J. M.; Majors, A. K.; Scalise, D.; Delp, M. D.

    2001-01-01

    To determine whether hindlimb unloading (HU) alters the extracellular matrix of skeletal muscle, male Sprague-Dawley rats were subjected to 0 (n = 11), 1 (n = 11), 14 (n = 13), or 28 (n = 11) days of unloading. Remodeling of the soleus and plantaris muscles was examined biochemically for collagen abundance via measurement of hydroxyproline, and the percentage of cross-sectional area of collagen was determined histologically with picrosirius red staining. Total hydroxyproline content in the soleus and plantaris muscles was unaltered by HU at any time point. However, the relative proportions of type I collagen in the soleus muscle decreased relative to control (Con) with 14 and 28 days HU (Con 68 +/- 5%; 14 days HU 53 +/- 4%; 28 days HU 53 +/- 7%). Correspondingly, type III collagen increased in soleus muscle with 14 and 28 days HU (Con 32 +/- 5%; 14 days HU 47 +/- 4%; 28 days HU 48 +/- 7%). The proportion of type I muscle fibers in soleus muscle was diminished with HU (Con 96 +/- 2%; 14 days HU 86 +/- 1%; 28 days HU 83 +/- 1%), and the proportion of hybrid type I/IIB fibers increased (Con 0%; 14 days HU 8 +/- 2%; 28 days HU 14 +/- 2%). HU had no effect on the proportion of type I and III collagen or muscle fiber composition in plantaris muscle. The data demonstrate that HU induces a shift in the relative proportion of collagen isoform (type I to III) in the antigravity soleus muscle, which occurs concomitantly with a slow-to-fast myofiber transformation.

  10. Collagen remodeling after myocardial infarction in the rat heart.

    PubMed Central

    Cleutjens, J. P.; Verluyten, M. J.; Smiths, J. F.; Daemen, M. J.

    1995-01-01

    In this study changes in the amount and distribution of types I and III collagen mRNA and protein were investigated in the rat heart after induction of a left ventricular myocardial infarction (MI). Sham operated rats served as controls. The animals were sacrificed at different time intervals after operation. Northern blotting of cardiac RNA and hybridization with cDNA probes for types I and III procollagen revealed a 5- to 15-fold increase in the infarcted left ventricle. Type III procollagen mRNA levels were already increased at day 2 after MI, whereas type I procollagen mRNA followed this response at day 4 after MI. This increase was sustained for at least 21 days in the infarcted left ventricle for type III procollagen mRNA, whereas type 1 procollagen mRNA levels were still elevated at 90 days after MI. In the noninfarcted right ventricle a 5- to 7-fold increase was observed for both type I and type III procollagen mRNA levels, but only at day 4 after MI. In the non-infarcted septum a transient increase was observed for type I procollagen mRNA from day 7-21 (4- to 5-fold increase) and a decline to sham levels thereafter. In the septum type III procollagen mRNA levels were only elevated at 7 days after MI (4- to 5-fold increase) compared with sham operated controls. In situ hybridization with the same types I and III procollagen probes showed procollagen mRNA-producing cells in the infarcted area around necrotic cardiomyocytes, and in the interstitial cells in the non-infarcted part of the myocardium. No labeling was detected above cardiomyocytes. Combined in situ hybridization and immunohistochemistry showed that the collagen mRNA producing cells have a myofibroblast-like phenotype in the infarcted myocardium and are fibroblasts in the noninfarcted septum and right ventricle. The increase in types I and III procollagen mRNA in both infarcted and non-infarcted myocardium was followed by an increased collagen deposition, measured by computerized morphometry on

  11. Collagen remodeling after myocardial infarction in the rat heart.

    PubMed

    Cleutjens, J P; Verluyten, M J; Smiths, J F; Daemen, M J

    1995-08-01

    In this study changes in the amount and distribution of types I and III collagen mRNA and protein were investigated in the rat heart after induction of a left ventricular myocardial infarction (MI). Sham operated rats served as controls. The animals were sacrificed at different time intervals after operation. Northern blotting of cardiac RNA and hybridization with cDNA probes for types I and III procollagen revealed a 5- to 15-fold increase in the infarcted left ventricle. Type III procollagen mRNA levels were already increased at day 2 after MI, whereas type I procollagen mRNA followed this response at day 4 after MI. This increase was sustained for at least 21 days in the infarcted left ventricle for type III procollagen mRNA, whereas type 1 procollagen mRNA levels were still elevated at 90 days after MI. In the noninfarcted right ventricle a 5- to 7-fold increase was observed for both type I and type III procollagen mRNA levels, but only at day 4 after MI. In the non-infarcted septum a transient increase was observed for type I procollagen mRNA from day 7-21 (4- to 5-fold increase) and a decline to sham levels thereafter. In the septum type III procollagen mRNA levels were only elevated at 7 days after MI (4- to 5-fold increase) compared with sham operated controls. In situ hybridization with the same types I and III procollagen probes showed procollagen mRNA-producing cells in the infarcted area around necrotic cardiomyocytes, and in the interstitial cells in the non-infarcted part of the myocardium. No labeling was detected above cardiomyocytes. Combined in situ hybridization and immunohistochemistry showed that the collagen mRNA producing cells have a myofibroblast-like phenotype in the infarcted myocardium and are fibroblasts in the noninfarcted septum and right ventricle. The increase in types I and III procollagen mRNA in both infarcted and non-infarcted myocardium was followed by an increased collagen deposition, measured by computerized morphometry on

  12. Species Identification of Bovine, Ovine and Porcine Type 1 Collagen; Comparing Peptide Mass Fingerprinting and LC-Based Proteomics Methods.

    PubMed

    Buckley, Mike

    2016-03-24

    Collagen is one of the most ubiquitous proteins in the animal kingdom and the dominant protein in extracellular tissues such as bone, skin and other connective tissues in which it acts primarily as a supporting scaffold. It has been widely investigated scientifically, not only as a biomedical material for regenerative medicine, but also for its role as a food source for both humans and livestock. Due to the long-term stability of collagen, as well as its abundance in bone, it has been proposed as a source of biomarkers for species identification not only for heat- and pressure-rendered animal feed but also in ancient archaeological and palaeontological specimens, typically carried out by peptide mass fingerprinting (PMF) as well as in-depth liquid chromatography (LC)-based tandem mass spectrometric methods. Through the analysis of the three most common domesticates species, cow, sheep, and pig, this research investigates the advantages of each approach over the other, investigating sites of sequence variation with known functional properties of the collagen molecule. Results indicate that the previously identified species biomarkers through PMF analysis are not among the most variable type 1 collagen peptides present in these tissues, the latter of which can be detected by LC-based methods. However, it is clear that the highly repetitive sequence motif of collagen throughout the molecule, combined with the variability of the sites and relative abundance levels of hydroxylation, can result in high scoring false positive peptide matches using these LC-based methods. Additionally, the greater alpha 2(I) chain sequence variation, in comparison to the alpha 1(I) chain, did not appear to be specific to any particular functional properties, implying that intra-chain functional constraints on sequence variation are not as great as inter-chain constraints. However, although some of the most variable peptides were only observed in LC-based methods, until the range of

  13. Diabetes Alters Mechanical Properties and Collagen Fiber Re-Alignment in Multiple Mouse Tendons

    PubMed Central

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

    2014-01-01

    Tendons function to transfer load from muscle to bone through their complex composition and hierarchical structure, consisting mainly of type I collagen. Recent evidence suggests that type II diabetes may cause alterations in collagen structure, such as irregular fibril morphology and density, which could play a role in the mechanical function of tendons. Using the db/db mouse model of type II diabetes, the diabetic skin was found to have impaired biomechanical properties when compared to the non-diabetic group. The purpose of this study was to assess the effect of diabetes on biomechanics, collagen fiber re-alignment, and biochemistry in three functionally different tendons (Achilles, supraspinatus, patellar) using the db/db mouse model. Results showed that cross-sectional area and stiffness, but not modulus, were significantly reduced in all three tendons. However, the tendon response to load (transition strain, collagen fiber re-alignment) occurred earlier in the mechanical test, contrary to expectations. In addition, the patellar tendon had an altered response to diabetes when compared to the other two tendons, with no changes in fiber realignment and decreased collagen content at the midsubstance of the tendon. Overall, type II diabetes alters tendon mechanical properties and the dynamic response to load. PMID:24833253

  14. Aerosolized polymerized type I collagen reduces airway inflammation and remodelling in a guinea pig model of allergic asthma.

    PubMed

    Moreno-Alvarez, Paola; Sánchez-Guerrero, Edgar; Martínez-Cordero, Erasmo; Hernández-Pando, Rogelio; Campos, María G; Cetina, Lucely; Bazán-Perkins, Blanca

    2010-04-01

    Collagen-polyvinylpyrrolidone (Collagen-PVP) has been demonstrated to elicit immunomodulatory properties in different chronic inflammatory diseases. Nevertheless, its effects on asthma are still unknown. We have evaluated whether collagen-PVP could modulate airway inflammation and remodelling in a guinea pig model of allergic asthma. Sensitized guinea pigs were challenged with the allergen (ovalbumin) six times (at 10-day intervals). From the third challenge on, animals were treated every 5 days with saline aerosols containing 0.16, 0.33, or 0.66 mg/ml of collagen-PVP (n = 5, respectively). Some guinea pigs, sensitized and challenged with saline as well as treated with 0 or 0.66 mg/ml collagen-PVP, were included in the study as control (n = 7) and sham groups (n = 5), respectively. From the first challenge on, ovalbumin induced a transient airway obstruction, measured by barometric plethysmography, which was not modified by collagen-PVP treatments. After the last allergen challenge, guinea pigs were anesthetized to obtain bronchoalveolar lavage (BAL) and the left lung caudal lobe. As expected, BAL cell count from allergen-challenged guinea pigs showed abundant neutrophils and eosinophils, as well as numerous tumor necrosis factor (TNF)-alpha-expressing granulocytes and macrophages in airway wall (determined by immunohistochemical assay). Neutrophilia and TNF-alpha-expressing leukocytes, from collagen-PVP treated animals, diminished from 0.16 mg/ml, and eosinophilia from 0.66 mg/ml of collagen-PVP doses. Histological changes induced by allergen challenges include thickening of connective tissue below airway epithelium and vascular wall widening of airway adjacent vessels; these changes were reduced by collagen-PVP treatment. Collagen-PVP seems to have anti-inflammatory and antifibrotic properties in this guinea pig asthma model.

  15. Role of integrin-linked kinase in regulating phosphorylation of Akt and fibroblast survival in type I collagen matrices through a beta1 integrin viability signaling pathway.

    PubMed

    Nho, Richard Seonghun; Xia, Hong; Kahm, Judy; Kleidon, Jill; Diebold, Deanna; Henke, Craig A

    2005-07-15

    A beta1 integrin phosphatidylinositol 3-kinase/Akt pathway regulates fibroblast survival in collagen matrices. When fibroblasts attach to collagen, Akt becomes phosphorylated, providing a survival signal. In contrast, in response to mechanical forces generated during collagen contraction, Akt is dephosphorylated and fibroblasts undergo apoptosis. The kinase(s) responsible for regulating Akt phosphorylation in response to matrix-derived mechanical signals are unclear. Integrin-linked kinase (ILK) is associated with the beta1 integrin in the focal adhesion complex and as such is a candidate kinase that may regulate Akt phosphorylation and fibroblast viability. Nevertheless, there is no direct evidence that matrix-derived mechanical forces regulate cell viability by modulating ILK activity. Here, we show that ILK activity decreased in response to collagen matrix contraction, which correlated with Akt dephosphorylation and induction of fibroblast apoptosis. In contrast, enforced activation of beta1 integrin by activating antibody preserved ILK and Akt activity during collagen matrix contraction, and this is associated with protection from collagen contraction-induced apoptosis. Knock-down of ILK by small, interfering RNA (siRNA) attenuated Akt phosphorylation in response to ligation of beta1 integrin by collagen or activating antibody and enhanced fibroblast apoptosis in response to collagen contraction. Kinase dead ILK attenuated Akt phosphorylation and enhanced fibroblast apoptosis, whereas hyperactive and wild type ILK augmented Akt phosphorylation and protected fibroblasts from apoptosis. Constitutively active Akt preserved Akt activity and rescued ILK siRNA-treated fibroblasts from collagen contraction-induced apoptosis. These data establish that matrix-derived mechanical forces sensed by beta1 integrin are capable of modulating ILK activity which regulates fibroblast viability via an Akt-dependent mechanism.

  16. Minor Type IV Collagen α5 Chain Promotes Cancer Progression through Discoidin Domain Receptor-1

    PubMed Central

    Xiao, Qian; Jiang, Yan; Liu, Qingbo; Yue, Jiao; Liu, Chunying; Zhao, Xiaotong; Qiao, Yuemei; Ji, Hongbin; Chen, Jianfeng; Ge, Gaoxiang

    2015-01-01

    Type IV collagens (Col IV), components of basement membrane, are essential in the maintenance of tissue integrity and proper function. Alteration of Col IV is related to developmental defects and diseases, including cancer. Col IV α chains form α1α1α2, α3α4α5 and α5α5α6 protomers that further form collagen networks. Despite knowledge on the functions of major Col IV (α1α1α2), little is known whether minor Col IV (α3α4α5 and α5α5α6) plays a role in cancer. It also remains to be elucidated whether major and minor Col IV are functionally redundant. We show that minor Col IV α5 chain is indispensable in cancer development by using α5(IV)-deficient mouse model. Ablation of α5(IV) significantly impeded the development of KrasG12D-driven lung cancer without affecting major Col IV expression. Epithelial α5(IV) supports cancer cell proliferation, while endothelial α5(IV) is essential for efficient tumor angiogenesis. α5(IV), but not α1(IV), ablation impaired expression of non-integrin collagen receptor discoidin domain receptor-1 (DDR1) and downstream ERK activation in lung cancer cells and endothelial cells. Knockdown of DDR1 in lung cancer cells and endothelial cells phenocopied the cells deficient of α5(IV). Constitutively active DDR1 or MEK1 rescued the defects of α5(IV)-ablated cells. Thus, minor Col IV α5(IV) chain supports lung cancer progression via DDR1-mediated cancer cell autonomous and non-autonomous mechanisms. Minor Col IV can not be functionally compensated by abundant major Col IV. PMID:25992553

  17. Establishment of a simple and quantitative immunospot assay for detecting anti-type II collagen antibody using an infrared fluorescence imaging system (IFIS).

    PubMed

    Ota, Shusuke; Kanazawa, Satoshi; Kobayashi, Masaaki; Otsuka, Takanobu; Okamoto, Takashi

    2005-04-01

    Antibodies to type II collagen (col II) have been detected in patients with rheumatoid arthritis and in animal models of collagen induced arthritis. Here, we describe a novel method to detect anti-col II antibodies using an immunospot assay with an infrared fluorescence imaging system. This method showed very high sensitivity and specificity, and was simple, with low background levels. It also showed higher reproducibility and linearity, with a dynamic range of approximately 500-fold, than the conventional immunospot assay with enhanced chemiluminescence detection. Using this method we were able to demonstrate the antibody affinity maturation process in mice immunized with col II. In these immunized mice, although cross-reactive antibodies reacting with other collagen species were detected in earlier stages of immunization, the titers of cross-reactive antibodies rapidly diminished after the antigen boost, concomitantly with the elevation of the anti-col II antibody. The method and its possible applications are discussed.

  18. Collagen type XI alpha1 may be involved in the structural plasticity of the vertebral column in Atlantic salmon (Salmo salar L.).

    PubMed

    Wargelius, A; Fjelldal, P G; Nordgarden, U; Grini, A; Krossøy, C; Grotmol, S; Totland, G K; Hansen, T

    2010-04-01

    Atlantic salmon (Salmo salar L.) vertebral bone displays plasticity in structure, osteoid secretion and mineralization in response to photoperiod. Other properties of the vertebral bone, such as mineral content and mechanical strength, are also associated with common malformations in farmed Atlantic salmon. The biological mechanisms that underlie these changes in bone physiology are unknown, and in order to elucidate which factors might be involved in this process, microarray assays were performed on vertebral bone of Atlantic salmon reared under natural or continuous light. Eight genes were upregulated in response to continuous light treatment, whereas only one of them was upregulated in a duplicate experiment. The transcriptionally regulated gene was predicted to code for collagen type XI alpha1, a protein known to be involved in controlling the diameter of fibrillar collagens in mammals. Furthermore, the gene was highly expressed in the vertebrae, where spatial expression was found in trabecular and compact bone osteoblasts and in the chordoblasts of the notochordal sheath. When we measured the expression level of the gene in the tissue compartments of the vertebrae, the collagen turned out to be 150 and 25 times more highly expressed in the notochord and compact bone respectively, relative to the expression in the trabecular bone. Gene expression was induced in response to continuous light, and reduced in compressed vertebrae. The downregulation in compressed vertebrae was due to reduced expression in the compact bone, while expression in the trabecular bone and the notochord was unaffected. These data support the hypothesis that this gene codes for a presumptive collagen type XI alpha1, which may be involved in the regulatory pathway leading to structural adaptation of the vertebral architecture.

  19. Collagen Quantification in Tissue Specimens.

    PubMed

    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.

  20. Lysine hydroxylation of collagen in a fibroblast cell culture system

    NASA Technical Reports Server (NTRS)

    Uzawa, Katsuhiro; Yeowell, Heather N.; Yamamoto, Kazushi; Mochida, Yoshiyuki; Tanzawa, Hideki; Yamauchi, Mitsuo

    2003-01-01

    The lysine (Lys) hydroxylation pattern of type I collagen produced by human fibroblasts in culture was analyzed and compared. Fibroblasts were cultured from normal human skin (NSF), keloid (KDF), fetal skin (FDF), and skin tissues of Ehlers-Danlos syndrome type VIA and VIB patients (EDS-VIA and -VIB). The type I collagen alpha chains with or without non-helical telopeptides were purified from the insoluble matrix and analyzed. In comparison with NSFs, KDF and FDF showed significantly higher Lys hydroxylation, particularly in the telopeptide domains of both alpha chains. Both EDS-VIA and -VIB showed markedly lower Lys hydroxylation in the helical domains of both alpha chains whereas that in the telopeptides was comparable with those of NSFs. A similar profile was observed in the tissue sample of the EDS-VIB patient. These results demonstrate that the Lys hydroxylation pattern is domain-specific within the collagen molecule and that this method is useful to characterize the cell phenotypes in normal/pathological connective tissues.

  1. Postnatal changes and sexual dimorphism in collagen expression in mouse skin

    PubMed Central

    Arai, Koji Y.; Hara, Takuya; Nagatsuka, Toyofumi; Kudo, Chikako; Tsuchiya, Sho; Nomura, Yoshihiro; Nishiyama, Toshio

    2017-01-01

    To investigate sexual dimorphism and postnatal changes in skin collagen expression, mRNA levels of collagens and their regulatory factors in male and female skin were examined during the first 120 days of age by quantitative realtime PCR. Levels of mRNAs encoding extracellular matrices did not show any differences between male and female mice until day 15. Col1a1 and Col1a2 mRNAs noticeably increased at day 30 and remained at high levels until day 120 in male mice, while those in female mice remained at low levels during the period. Consistent with the mRNA expression, pepsin-soluble type I collagen contents in skin was very high in mature male as compared to female. Col3a1 mRNA in male mice also showed significantly high level at day 120 as compared to female. On the other hand, expression of mRNAs encoding TGF-ßs and their receptors did not show apparent sexual dimorphism although small significant differences were observed at some points. Castration at 60 days of age resulted in a significant decrease in type I collagen mRNA expression within 3 days, and noticeably decreased expression of all fibril collagen mRNAs examined within 14 days, while administration of testosterone tube maintained the mRNA expression at high levels. Despite the in vivo effect of testosterone, administration of physiological concentrations of testosterone did not affect fibril collagen mRNA expression in either human or mouse skin fibroblasts in vitro, suggesting that testosterone does not directly affect collagen expression in fibroblasts. In summary, present study demonstrated dynamic postnatal changes in expression of collagens and their regulatory factors, and suggest that testosterone and its effects on collagen expression are responsible for the skin sexual dimorphism but the effects of testosterone is not due to direct action on dermal fibroblasts. PMID:28494009

  2. Development, characterization, and applications of self-assembling, photocrosslinkable collagen-based hydrogels

    NASA Astrophysics Data System (ADS)

    Gaudet, Ian Daniel

    Development of functional soft-tissue engineered constructs for use in regenerative medicine is currently limited by homogeneity within scaffolds that fails to recapitulate the complex architecture that supports normal function in healthy tissues. Additionally, recent breakthroughs in our understanding the biomechanical cell-matrix interface have provided insight into the role of substrate compliance during development and in the pathophysiological environment. This thesis is the result of investigation into using type-I collagen as a base material for creating dynamic, self-assembling, mechanically and biochemically tunable 3D hydrogel scaffolds into which instructive cellular cues can be imparted anisotropically via the directed application of light. This overarching goal was approached by (1) evaluating extant methods for photonically manipulating type I collagen mechanical properties, which led us to the conclusion that published methods were inadequate for our purposes. Following this realization, we (2) developed a novel process for derivatizing free amines on collagen amino acid residues to reactive methacrylamide moieties, allowing robust spatiotemporal control of mechanical properties through photocrosslinking with long-wave UV light and the water-soluble photoinitiator Irgacure 2959. Thorough characterization of this material, collagen methacrylamide (CMA), provided the basis for multiple applications in the field of soft tissue engineering. Additionally, (3) CMA was used in conjunction with synthetic photopolymers in an effort to create a hybrid natural/synthetic hydrogel material. CMA was also (4) employed as a dynamic hydrogel scaffold which we showed could be used to culture a number of neurogenic stem and progenitor cell types with a focus on using photomodulation to impart instructive heterogeneity to the mechanical and biochemical microenvironment. Finally, (5) we used a computational modeling approach to explain interesting yet poorly understood

  3. In vivo magnetic resonance imaging of type I collagen scaffold in rat: improving visualization of bladder and subcutaneous implants.

    PubMed

    Sun, Yi; Geutjes, Paul; Oosterwijk, Egbert; Heerschap, Arend

    2014-12-01

    Noninvasive monitoring of implanted scaffolds is important to understand their behavior and role in tissue engineering, in particular to follow their degradation and interaction with host tissue. Magnetic resonance imaging (MRI) is well suited for this goal, but its application is often hampered by the low contrast of scaffolds that are prepared from biomaterials such as type I collagen. The aim of this study was to test iron oxide particles incorporation in improving their MRI contrasts, and to follow their degradation and tissue interactions. Scaffolds with and without iron oxide particles were implanted either subcutaneously or on the bladder of rats. At predetermined time points, in vivo MRI were obtained and tissues were then harvested for histology analysis and transmission electron microscopy. The result showed that the incorporation of iron oxide particles improved MRI contrast of the implants, providing information on their location, shapes, and degradation. Second, the host tissue reaction to the type I collagen implants could be observed in both MRI and histology. Finally, MRI also revealed that the degradation and host tissue reaction of iron particles-loaded scaffolds differed between subcutaneous and bladder implantation, which was substantiated by histology.

  4. Effect of intrinsic and extrinsic factors on the simulated D-band length of type I collagen.

    PubMed

    Varma, Sameer; Botlani, Mohsen; Hammond, Jeff R; Scott, H Larry; Orgel, Joseph P R O; Schieber, Jay D

    2015-10-01

    A signature feature of collagen is its axial periodicity visible in TEM as alternating dark and light bands. In mature, type I collagen, this repeating unit, D, is 67 nm long. This periodicity reflects an underlying packing of constituent triple-helix polypeptide monomers wherein the dark bands represent gaps between axially adjacent monomers. This organization is visible distinctly in the microfibrillar model of collagen obtained from fiber diffraction. However, to date, no atomistic simulations of this diffraction model under zero-stress conditions have reported a preservation of this structural feature. Such a demonstration is important as it provides the baseline to infer response functions of physiological stimuli. In contrast, simulations predict a considerable shrinkage of the D-band (11-19%). Here we evaluate systemically the effect of several factors on D-band shrinkage. Using force fields employed in previous studies we find that irrespective of the temperature/pressure coupling algorithms, assumed salt concentration or hydration level, and whether or not the monomers are cross-linked, the D-band shrinks considerably. This shrinkage is associated with the bending and widening of individual monomers, but employing a force field whose backbone dihedral energy landscape matches more closely with our computed CCSD(T) values produces a small D-band shrinkage of < 3%. Since this force field also performs better against other experimental data, it appears that the large shrinkage observed in earlier simulations is a force-field artifact. The residual shrinkage could be due to the absence of certain atomic-level details, such as glycosylation sites, for which we do not yet have suitable data. © 2015 Wiley Periodicals, Inc.

  5. Potential in two types of collagen scaffolds for urological tissue engineering applications - Are there differences in growth behaviour of juvenile and adult vesical cells?

    PubMed

    Leonhäuser, D; Vogt, M; Tolba, R H; Grosse, J O

    2016-02-01

    The aging society has a deep impact on patient care in urology. The number of patients in need of partial or whole bladder wall replacement is increasing simultaneously with the number of cancer incidents. Therefore, urological research requires a model of bladder wall replacement in adult and elderly people. Two types of porcine collagen I/III scaffolds were used in vitro for comparison of cell growth of two different pig breeds at different growth stages. Scaffolds were characterised with scanning electron and laser scanning microscopy. Urothelial and detrusor smooth muscle cells were isolated from 15 adult Göttingen minipigs and 15 juvenile German Landrace pigs. Growth behaviour was examined in cell culture and seeded onto the collagen scaffolds via immunohistochemistry, two-photon laser scanning microscopy and a viability assay. The collagen scaffolds showed different structured surfaces which are appropriate for seeding of the two different cell types. Moisturisation of the scaffolds resulted in a change of the structure. Cell growth of German Landrace urothelial cells and smooth muscle cells was significantly higher than cell growth of the Göttingen minipig cells. Seeding of scaffolds with both cell types from both pig races was possible which could be shown by immunohistochemistry and two-photon laser scanning microscopy. Growth behaviour on the scaffolds was significantly increased for the German Landrace compared to Göttingen minipig. Nevertheless, seeding with the adult Göttingen minipig cells resulted in a closed layer on the surface and urothelial cells and smooth muscle cells showed increasing growth until day 14. The results show that these collagen scaffolds are adequate for the seeding with vesical cells. Moreover, they seem appropriate for the use as an in vitro model for the adult or elderly as the cells of the adult Göttingen minipig too, show good growth behaviour. © The Author(s) 2015.

  6. Quantification of nitrogenous bases, DNA and Collagen type I for the estimation of the postmortem interval in bone remains.

    PubMed

    Pérez-Martínez, Cristina; Pérez-Cárceles, María D; Legaz, Isabel; Prieto-Bonete, Gemma; Luna, Aurelio

    2017-12-01

    Estimating the postmortem interval (PMI) is an important goal in forensic medicine and continues to be one of the most difficult tasks of the forensic investigator. Few accurate methods exist to determine the time since death of skeletonized human remains due to the great number of intrinsic and external factors that may alter the normal course of postmortem change. The purpose of this research was to assess the usefulness of various biochemical parameters, such as nitrogenous bases (adenine, guanine, purines, cytosine, thymine, pyrimidines, hypoxanthine and xanthine), DNA and Collagen Type I peptides to estimate PMI. These parameters were analysed in cortical bone for the establishment of data in a total of 80 long bones of 80 corpses (50 males, 30 females) with a mean age of 68.31 years (S.D.=18.021, range=20-97). The bones were removed from the cement niches of a cemetery in Murcia (south-eastern Spain), where they had lain for between 5 and 47 years (mean time 23.83 years, S.D.=10.85). Our results show a significant decrease in adenine (p=0.0004), guanine (p=0.0001), purines (p=0.0001), cytosine (p=0.0001), thymine (p=0.0226), pyrimidines (p=0.0002) and the number of peptides of Collagen type I (p=0.0053) in those with a PMI≥20 years. In a curvilinear regression analysis the results show that 30.6% of the variable PMI could be explained by guanine concentration, in bones with a PMI<20 years, while in cases of a PMI≥20 years, the variable that best explained membership of this group was adenine (38.0%). In the discriminant analysis applied to the all the variables as a function of PMI when two groups were established, 86.7% of the cases were correctly classified. These results show that the quantification of Collagen type I proteins and nitrogenous bases could be used as a complementary tool, together with other analyses, in the estimation of PMI. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Collagen morphology and texture analysis: from statistics to classification

    PubMed Central

    Mostaço-Guidolin, Leila B.; Ko, Alex C.-T.; Wang, Fei; Xiang, Bo; Hewko, Mark; Tian, Ganghong; Major, Arkady; Shiomi, Masashi; Sowa, Michael G.

    2013-01-01

    In this study we present an image analysis methodology capable of quantifying morphological changes in tissue collagen fibril organization caused by pathological conditions. Texture analysis based on first-order statistics (FOS) and second-order statistics such as gray level co-occurrence matrix (GLCM) was explored to extract second-harmonic generation (SHG) image features that are associated with the structural and biochemical changes of tissue collagen networks. Based on these extracted quantitative parameters, multi-group classification of SHG images was performed. With combined FOS and GLCM texture values, we achieved reliable classification of SHG collagen images acquired from atherosclerosis arteries with >90% accuracy, sensitivity and specificity. The proposed methodology can be applied to a wide range of conditions involving collagen re-modeling, such as in skin disorders, different types of fibrosis and muscular-skeletal diseases affecting ligaments and cartilage. PMID:23846580

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

    PubMed

    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.

  9. Identification of Collagen-Derived Hydroxyproline (Hyp)-Containing Cyclic Dipeptides with High Oral Bioavailability: Efficient Formation of Cyclo(X-Hyp) from X-Hyp-Gly-Type Tripeptides by Heating.

    PubMed

    Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

    2017-11-01

    Cyclic dipeptides (2,5-diketopiperazines) are present in a variety of foods and are reported to demonstrate antioxidant, antidepressant, and other beneficial effects. We recently developed a novel collagen hydrolysate characterized by a high content of X-hydroxyproline (Hyp)-Gly-type tripeptides using ginger protease. In the present study, we found that, through heating, X-Hyp-Gly can be easily converted into Hyp-containing cyclic dipeptides. After heating for 3 h at 85 °C and pH 4.8, Ala-Hyp-Gly was almost completely cyclized to cyclo(Ala-Hyp), in contrast to a slight cyclization of Ala-Hyp. The contents of cyclo(Ala-Hyp) and cyclo(Leu-Hyp) reached 0.5-1% (w/w) each in the ginger-degraded collagen hydrolysate under the heating conditions. Oral administration experiments using mice revealed that cyclo(Ala-Hyp) and cyclo(Leu-Hyp) were absorbed into the blood at markedly higher efficiencies compared to collagenous oligopeptides, including Pro-Hyp. The high productivity and oral bioavailability of the collagen-specific cyclic dipeptides suggest significant health benefits of the heat-treated ginger-degraded collagen hydrolysate.

  10. FTIR spectro-imaging of collagen scaffold formation during glioma tumor development.

    PubMed

    Noreen, Razia; Chien, Chia-Chi; Chen, Hsiang-Hsin; Bobroff, Vladimir; Moenner, Michel; Javerzat, Sophie; Hwu, Yeukuang; Petibois, Cyril

    2013-11-01

    Evidence has recently emerged that solid and diffuse tumors produce a specific extracellular matrix (ECM) for division and diffusion, also developing a specific interface with microvasculature. This ECM is mainly composed of collagens and their scaffolding appears to drive tumor growth. Although collagens are not easily analyzable by UV-fluorescence means, FTIR imaging has appeared as a valuable tool to characterize collagen contents in tissues, specially the brain, where ECM is normally devoid of collagen proteins. Here, we used FTIR imaging to characterize collagen content changes in growing glioma tumors. We could determine that C6-derived solid tumors presented high content of triple helix after 8-11 days of growth (typical of collagen fibrils formation; 8/8 tumor samples; 91 % of total variance), and further turned to larger α-helix (days 12-15; 9/10 of tumors; 94 % of variance) and β-turns (day 18-21; 7/8 tumors; 97 % of variance) contents, which suggest the incorporation of non-fibrillar collagen types in ECM, a sign of more and more organized collagen scaffold along tumor progression. The growth of tumors was also associated to the level of collagen produced (P < 0.05). This study thus confirms that collagen scaffolding is a major event accompanying the angiogenic shift and faster tumor growth in solid glioma phenotypes.

  11. SIRT1 deacetylates RFX5 and antagonizes repression of collagen type I (COL1A2) transcription in smooth muscle cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xia, Jun; Department of Respiratory Medicine, Jiangsu Provincial Hospital of Chinese Traditional Medicine; Wu, Xiaoyan

    Highlights: Black-Right-Pointing-Pointer SIRT1 interacts with and deacetylates RFX5. Black-Right-Pointing-Pointer SIRT1 activation attenuates whereas SIRT1 inhibition enhances collagen repression by RFX5 in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 promotes cytoplasmic localization and proteasomal degradation of RFX5 and cripples promoter recruitment of RFX5. Black-Right-Pointing-Pointer IFN-{gamma} represses SIRT1 expression in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 agonist alleviates collagen repression by IFN-{gamma} in vascular smooth muscle cells. -- Abstract: Decreased expression of collagen by vascular smooth muscle cells (SMCs) within the atherosclerotic plaque contributes to the thinning of the fibrous cap and poses a great threat to plaque rupture. Elucidation of the mechanismmore » underlying repressed collagen type I (COL1A2) gene would potentially provide novel solutions that can prevent rupture-induced complications. We have previously shown that regulatory factor for X-box (RFX5) binds to the COL1A2 transcription start site and represses its transcription. Here we report that SIRT1, an NAD-dependent, class III deacetylase, forms a complex with RFX5. Over-expression of SIRT1 or NAMPT, which synthesizes NAD+ to activate SIRT1, or treatment with the SIRT1 agonist resveratrol decreases RFX5 acetylation and disrupts repression of the COL1A2 promoter activity by RFX5. On the contrary, knockdown of SIRT1 or treatment with SIRT1 inhibitors induces RFX5 acetylation and enhances the repression of collagen transcription. SIRT1 antagonizes RFX5 activity by promoting its nuclear expulsion and proteasomal degradation hence dampening its binding to the COL1A2 promoter. The pro-inflammatory cytokine IFN-{gamma} represses COL1A2 transcription by down-regulating SIRT1 expression in SMCs. Therefore, our data have identified as novel pathway whereby SIRT1 maintains collagen synthesis in SMCs by modulating RFX5 activity.« less

  12. Evaluation of nanostructural, mechanical, and biological properties of collagen-nanotube composites.

    PubMed

    Tan, Wei; Twomey, John; Guo, Dongjie; Madhavan, Krishna; Li, Min

    2010-06-01

    Collagen I is an essential structural and mechanical building block of various tissues, and it is often used as tissue-engineering scaffolds. However, collagen-based constructs reconstituted in vitro often lacks robust fiber structure, mechanical stability, and molecule binding capability. To enhance these performances, the present study developed 3-D collagen-nanotube composite constructs with two types of functionalized carbon nanotubes, carboxylated nanotubes and covalently functionalized nanotubes (CFNTs). The influences of nanotube functionalization and loading concentration on the collagen fiber structure, mechanical property, biocompatibility, and molecule binding were examined. Results revealed that surface modification and loading concentration of nanotubes determined the interactions between nanotubes and collagen fibrils, thus altering the structure and property of nanotube-collagen composites. Scanning electron microscopy and confocal microscopy revealed that the incorporation of CFNT in collagen-based constructs was an effective means of restructuring collagen fibrils because CFNT strongly bound to collagen molecules inducing the formation of larger fibril bundles. However, increased nanotube loading concentration caused the formation of denser fibril network and larger aggregates. Static stress-strain tests under compression showed that the addition of nanotube into collagen-based constructs did not significantly increase static compressive moduli. Creep/recovery testing under compression revealed that CFNT-collagen constructs showed improved mechanical stability under continuous loading. Testing with endothelial cells showed that biocompatibility was highly dependent on nanotube loading concentration. At a low loading level, CFNT-collagen showed higher endothelial coverage than the other tested constructs or materials. Additionally, CFNT-collagen showed capability of binding to other biomolecules to enhance the construct functionality. In conclusion

  13. Osteoblast Differentiation on Collagen Scaffold with Immobilized Alkaline Phosphatase.

    PubMed

    Jafary, F; Hanachi, P; Gorjipour, K

    2017-01-01

    In tissue engineering, scaffold characteristics play an important role in the biological interactions between cells and the scaffold. Cell adhesion, proliferation, and activation depend on material properties used for the fabrication of scaffolds. In the present investigation, we used collagen with proper characteristics including mechanically stability, biodegradability and low antigenicity. Optimization of the scaffold was done by immobilization of alkaline phosphatase on the collagen surface via cross-linking method, because this enzyme is one of the most important markers of osteoblast, which increases inorganic phosphate concentration and promote mineralization of bone formation. Alkaline phosphatase was immobilized on a collagen surface by 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride, as a reagent. Then, rat mesenchymal stem cells were cultured in osteogenic medium in control and treated groups. The osteogenesis-related genes were compared between treatments (differentiated cells with immobilized alkaline phosphatase/collagen scaffold) and control groups (differentiated cells on collagen surface without alkaline phosphatase) on days 3 and 7 by quantitative real-time PCR (QIAGEN software). Several genes, including alkaline phosphatase, collagen type I and osteocalcine associated with calcium binding and mineralization, showed upregulation in expression during the first 3 days, whereas tumor necrosis factor-α, acting as an inhibitor of differentiation, was down-regulated during osteogenesis. Collagen scaffold with immobilized alkaline phosphatase can be utilized as a good candidate for enhancing the differentiation of osteoblasts from mesenchymal stem cells.

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

    PubMed

    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

  15. Lack of Cyclophilin B in Osteogenesis Imperfecta with Normal Collagen Folding

    PubMed Central

    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.

    2011-01-01

    SUMMARY 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. PMID:20089953

  16. Developmental changes in skin collagen biosynthesis pathway in posthatch male and female chickens

    NASA Technical Reports Server (NTRS)

    Pines, M.; Schickler, M.; Hurwitz, S.; Yamauchi, M.

    1996-01-01

    The developmental changes in skin collagen biosynthesis pathway in male and female chickens were evaluated. Concentration of collagen, levels of mRNA for collagen type I subunits and for lysyl hydroxylase, and the level of three lysyl oxidase-derived cross-links: dehydro-dihydroxylysinonorleucine (DHLNL), dehydro-hydroxylysinonorleucine (HLNL), and dehydro-histidinohydroxymerodesmosine (HHMD) were determined during 4 wk posthatching. Skin collagen content increased with age and was higher in males than in females. In both sexes, the expression of the genes coding for alpha 1 and alpha 2 of collagen type I decreased with age: alpha 1(I) gene expression decreased from Day 3 onwards, whereas the reduction in alpha 2(I) gene expression started 1 wk later. At all ages examined, the expression of both genes was higher in male than in female skin. Males and females lysyl hydroxylase gene expression remained low until Day 16, after which an increase in the enzyme gene expression was observed. An increase in skin HLNL content was observed from Day 3 in both sexes reaching a peak in males at Day 9 and in females 1 wk later. The DHLNL content, which was higher in males than in females at all ages tested, dramatically decreased in both male and female skin from 3 d of age, reaching its lowest level at Day 16, and remained at that low level thereafter. The skin content of HHMD in males and females followed an oscillatory behavior with higher peaks in the male skin. The results suggest that the higher tensile strength of male skin than female skin may be due to the elevated skin collagen content that resulted from increased expression in collagen type I genes on the one hand, and from the higher amounts of various collagen cross-links on the other.

  17. Secretome profiling of oral squamous cell carcinoma-associated fibroblasts reveals organization and disassembly of extracellular matrix and collagen metabolic process signatures.

    PubMed

    Bagordakis, Elizabete; Sawazaki-Calone, Iris; Macedo, Carolina Carneiro Soares; Carnielli, Carolina M; de Oliveira, Carine Ervolino; Rodrigues, Priscila Campioni; Rangel, Ana Lucia C A; Dos Santos, Jean Nunes; Risteli, Juha; Graner, Edgard; Salo, Tuula; Paes Leme, Adriana Franco; Coletta, Ricardo D

    2016-07-01

    An important role has been attributed to cancer-associated fibroblasts (CAFs) in the tumorigenesis of oral squamous cell carcinoma (OSCC), the most common tumor of the oral cavity. Previous studies demonstrated that CAF-secreted molecules promote the proliferation and invasion of OSCC cells, inducing a more aggressive phenotype. In this study, we searched for differences in the secretome of CAFs and normal oral fibroblasts (NOF) using mass spectrometry-based proteomics and biological network analysis. Comparison of the secretome profiles revealed that upregulated proteins involved mainly in extracellular matrix organization and disassembly and collagen metabolism. Among the upregulated proteins were fibronectin type III domain-containing 1 (FNDC1), serpin peptidase inhibitor type 1 (SERPINE1), and stanniocalcin 2 (STC2), the upregulation of which was validated by quantitative PCR and ELISA in an independent set of CAF cell lines. The transition of transforming growth factor beta 1 (TGF-β1)-mediating NOFs into CAFs was accompanied by significant upregulation of FNDC1, SERPINE1, and STC2, confirming the participation of these proteins in the CAF-derived secretome. Type I collagen, the main constituent of the connective tissue, was also associated with several upregulated biological processes. The immunoexpression of type I collagen N-terminal propeptide (PINP) was significantly correlated in vivo with CAFs in the tumor front and was associated with significantly shortened survival of OSCC patients. Presence of CAFs in the tumor stroma was also an independent prognostic factor for OSCC disease-free survival. These results demonstrate the value of secretome profiling for evaluating the role of CAFs in the tumor microenvironment and identify potential novel therapeutic targets such as FNDC1, SERPINE1, and STC2. Furthermore, type I collagen expression by CAFs, represented by PINP levels, may be a prognostic marker of OSCC outcome.

  18. Biomimetic mineralization of recombinant collagen type I derived protein to obtain hybrid matrices for bone regeneration.

    PubMed

    Ramírez-Rodríguez, Gloria Belén; Delgado-López, José Manuel; Iafisco, Michele; Montesi, Monica; Sandri, Monica; Sprio, Simone; Tampieri, Anna

    2016-11-01

    Understanding the mineralization mechanism of synthetic protein has recently aroused great interest especially in the development of advanced materials for bone regeneration. Herein, we propose the synthesis of composite materials through the mineralization of a recombinant collagen type I derived protein (RCP) enriched with RGD sequences in the presence of magnesium ions (Mg) to closer mimic bone composition. The role of both RCP and Mg ions in controlling the precipitation of the mineral phase is in depth evaluated. TEM and X-ray powder diffraction reveal the crystallization of nanocrystalline apatite (Ap) in all the evaluated conditions. However, Raman spectra point out also the precipitation of amorphous calcium phosphate (ACP). This amorphous phase is more evident when RCP and Mg are at work, indicating the synergistic role of both in stabilizing the amorphous precursor. In addition, hybrid matrices are prepared to tentatively address their effectiveness as scaffolds for bone tissue engineering. SEM and AFM imaging show an homogeneous mineral distribution on the RCP matrix mineralized in presence of Mg, which provides a surface roughness similar to that found in bone. Preliminary in vitro tests with pre-osteoblast cell line show good cell-material interaction on the matrices prepared in the presence of Mg. To the best of our knowledge this work represents the first attempt to mineralize recombinant collagen type I derived protein proving the simultaneous effect of the organic phase (RCP) and Mg on ACP stabilization. This study opens the possibility to engineer, through biomineralization process, advanced hybrid matrices for bone regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2013-01-01

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

  20. Quantification of collagen contraction in three-dimensional cell culture.

    PubMed

    Kopanska, Katarzyna S; Bussonnier, Matthias; Geraldo, Sara; Simon, Anthony; Vignjevic, Danijela; Betz, Timo

    2015-01-01

    Many different cell types including fibroblasts, smooth muscle cells, endothelial cells, and cancer cells exert traction forces on the fibrous components of the extracellular matrix. This can be observed as matrix contraction both macro- and microscopically in three-dimensional (3D) tissues models such as collagen type I gels. The quantification of local contraction at the micron scale, including its directionality and speed, in correlation with other parameters such as cell invasion, local protein or gene expression, can provide useful information to study wound healing, organism development, and cancer metastasis. In this article, we present a set of tools to quantify the flow dynamics of collagen contraction, induced by cells migrating out of a multicellular cancer spheroid into a three-dimensional (3D) collagen matrix. We adapted a pseudo-speckle technique that can be applied to bright-field and fluorescent microscopy time series. The image analysis presented here is based on an in-house written software developed in the Matlab (Mathworks) programming environment. The analysis program is freely available from GitHub following the link: http://dx.doi.org/10.5281/zenodo.10116. This tool provides an automatized technique to measure collagen contraction that can be utilized in different 3D cellular systems. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Effect of structural modification on second harmonic generation in collagen

    NASA Astrophysics Data System (ADS)

    Stoller, Patrick C.; Reiser, Karen M.; Celliers, Peter M.; Rubenchik, Alexander M.

    2003-07-01

    The effects of structural perturbation on second harmonic generation in collagen were investigated. Type I collagen fascicles obtained from rat tails were structurally modified by increasing nonenzymatic cross-linking, by thermal denaturation, by collagenase digestion, or by dehydration. Changes in polarization dependence were observed in the dehydrated samples. Surprisingly, no changes in polarization dependence were observed in highly crosslinked samples, despite significant alterations in packing structure. Complete thermal denaturation and collagenase digestion produced samples with no detectable second harmonic signal. Prior to loss of signal, no change in polarization dependence was observed in partially heated or digested collagen.

  2. Incidence and specificity of antibodies to types I, II, III, IV, and V collagen in rheumatoid arthritis and other rheumatic diseases as measured by 125I-radioimmunoassay

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Stuart, J.M.; Huffstutter, E.H.; Townes, A.S.

    1983-07-01

    Antibodies to human native and denatured types I, II, III, IV, and V collagens were measured using 125I-radioimmunoassay. Mean levels of binding by sera from 30 rheumatoid arthritis patients were significantly higher than those from 20 normal subjects against all of the collagens tested. The relative antibody concentration was higher in synovial fluid than in simultaneously obtained serum. Many patients with gout or various other rheumatic diseases also had detectable anticollagen antibodies. With a few notable exceptions, the majority of the reactivity detected in all patient groups was directed against covalent structural determinants present on all of the denatured collagens,more » suggesting a secondary reaction to tissue injury.« less

  3. Tumor Cell Invasion Can Be Blocked by Modulators of Collagen Fibril Alignment That Control Assembly of the Extracellular Matrix.

    PubMed

    Grossman, Moran; Ben-Chetrit, Nir; Zhuravlev, Alina; Afik, Ran; Bassat, Elad; Solomonov, Inna; Yarden, Yosef; Sagi, Irit

    2016-07-15

    Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECM-associated pathologies. Cancer Res; 76(14); 4249-58. ©2016 AACR. ©2016 American Association for Cancer Research.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Orgel, Joseph P.R.O.; Eid, Aya; Antipova, Olga

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

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

    PubMed

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

    2009-09-15

    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.

  6. Molecular structure, mechanical behavior and failure mechanism of the C-terminal cross-link domain in type I collagen.

    PubMed

    Uzel, Sebastien G M; Buehler, Markus J

    2011-02-01

    Collagen is a key constituent in structural materials found in biology, including bone, tendon, skin and blood vessels. Here we report a first molecular level model of an entire overlap region of a C-terminal cross-linked type I collagen assembly and carry out a nanomechanical characterization based on large-scale molecular dynamics simulation in explicit water solvent. Our results show that the deformation mechanism and strength of the structure are greatly affected by the presence of the cross-link, and by the specific loading condition of how the stretching is applied. We find that the presence of a cross-link results in greater strength during deformation as complete intermolecular slip is prevented, and thereby particularly affects larger deformation levels. Conversely, the lack of a cross-link results in the onset of intermolecular sliding during deformation and as a result an overall weaker structure is obtained. Through a detailed analysis of the distribution of deformation by calculating the molecular strain we show that the location of largest strains does not occur around the covalent bonding region, but is found in regions further away from this location. The insight developed from understanding collagenous materials from a fundamental molecular level upwards could play a role in advancing our understanding of physiological and disease states of connective tissues, and also enable the development of new scaffolding material for applications in regenerative medicine and biologically inspired materials. Copyright © 2011. Elsevier Ltd. All rights reserved.

  7. Fish scale-derived collagen patch promotes growth of blood and lymphatic vessels in vivo.

    PubMed

    Wang, Jun Kit; Yeo, Kim Pin; Chun, Yong Yao; Tan, Timothy Thatt Yang; Tan, Nguan Soon; Angeli, Véronique; Choong, Cleo

    2017-11-01

    In this study, Type I collagen was extracted from fish scales asa potential alternative source of collagen for tissue engineering applications. Since unmodified collagen typically has poor mechanical and degradation stability both in vitro and in vivo, additional methylation modification and 1,4-butanediol diglycidyl ether (BDE) crosslinking steps were used to improve the physicochemical properties of fish scale-derived collagen. Subsequently, in vivo studies using a murine model demonstrated the biocompatibility of the different fish scale-derived collagen patches. In general, favorable integration of the collagen patches to the surrounding tissues, with good infiltration of cells, blood vessels (BVs) and lymphatic vessels (LVs) were observed under growth factor-free conditions. Interestingly, significantly higher (p<0.05) number of LVs was found to be more abundant around collagen patches with methylation modification and BDE crosslinking. Overall, we have demonstrated the potential application of fish scale-derived collagen as a promising scaffolding material for various biomedical applications. Currently the most common sources of collagen are of bovine and porcine origins, although the industrial use of collagen obtained from non-mammalian species is growing in importance, particularly since they have a lower risk of disease transmission and are not subjected to any cultural or religious constraints. However, unmodified collagen typically has poor mechanical and degradation stability both in vitro and in vivo. Hence, in this study, Type I collagen was successfully extracted from fish scales and chemically modified and crosslinked. In vitro studies showed overall improvement in the physicochemical properties of the material, whilst in vivo implantation studies showed improvements in the growth of blood and lymphatic host vessels in the vicinity of the implants. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  8. Decreased expression of microRNA-29 family in leiomyoma contributes to increased major fibrillar collagen production.

    PubMed

    Marsh, Erica E; Steinberg, Marissa L; Parker, J Brandon; Wu, Ju; Chakravarti, Debabrata; Bulun, Serdar E

    2016-09-01

    To determine the expression and function of the microRNA-29 family (miRNA-29a, miRNA-29b, miRNA-29c) in human leiomyoma and myometrium. Basic science experimental design. Academic medical center. Women undergoing surgery for symptomatic uterine fibroids. Overexpression and knockdown of miRNA-29a, miRNA-29b, and miRNA-29c in primary leiomyoma and myometrial cells. [1] Expression of the miRNA-29 family members in vivo in leiomyoma versus myometrium; [2] Major fibrillar collagen (I, II, III) expression in leiomyoma and myometrial cells with manipulation of miRNA-29 species. Members of the miRNA-29 family (29a, 29b, 29c) are all down-regulated in leiomyoma versus myometrium in vivo. The expression of the miRNA-29 family can be successfully modulated in primary leiomyoma and myometrial cells. Overexpression of the miRNA-29 family in leiomyoma cells results in down-regulation of the major fibrillar collagens. Down-regulation of the miRNA-29 species in myometrium results in an increase in collagen type III deposition. The miRNA-29 family is consistently down-regulated in leiomyoma compared to matched myometrial tissue. This down-regulation contributes to the increased collagen seen in leiomyomas versus myometrium. When miRNA-29 members are overexpressed in leiomyoma cells, protein levels of all of the major fibrillar collagens decrease. The miRNA-29 members are potential therapeutic targets in this highly prevalent condition. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  9. Mechanical properties of a collagen fibril under simulated degradation.

    PubMed

    Malaspina, David C; Szleifer, Igal; Dhaher, Yasin

    2017-11-01

    Collagen fibrils are a very important component in most of the connective tissue in humans. An important process associated with several physiological and pathological states is the degradation of collagen. Collagen degradation is usually mediated by enzymatic and non-enzymatic processes. In this work we use molecular dynamics simulations to study the influence of simulated degradation on the mechanical properties of the collagen fibril. We applied tensile stress to the collagen fiber at different stages of degradation. We compared the difference in the fibril mechanical priorities due the removal of enzymatic crosslink, surface degradation and volumetric degradation. As anticipated, our results indicated that, regardless of the degradation scenario, fibril mechanical properties is reduced. The type of degradation mechanism (crosslink, surface or volumetric) expressed differential effect on the change in the fibril stiffness. Our simulation results showed dramatic change in the fibril stiffness with a small amount of degradation. This suggests that the hierarchical structure of the fibril is a key component for the toughness and is very sensitive to changes in the organization of the fibril. The overall results are intended to provide a theoretical framework for the understanding the mechanical behavior of collagen fibrils under degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Collagen Membranes Adsorb the Transforming Growth Factor-β Receptor I Kinase-Dependent Activity of Enamel Matrix Derivative.

    PubMed

    Stähli, Alexandra; Miron, Richard J; Bosshardt, Dieter D; Sculean, Anton; Gruber, Reinhard

    2016-05-01

    Enamel matrix derivative (EMD) and collagen membranes (CMs) are simultaneously applied in regenerative periodontal surgery. The aim of this study is to evaluate the ability of two CMs and a collagen matrix to adsorb the activity intrinsic to EMD that provokes transforming growth factor (TGF)-β signaling in oral fibroblasts. Three commercially available collagen products were exposed to EMD or recombinant TGF-β1, followed by vigorous washing. Oral fibroblasts were either seeded directly onto collagen products or were incubated with the respective supernatant. Expression of TGF-β target genes interleukin (IL)-11 and proteoglycan 4 (PRG4) was evaluated by real time polymerase chain reaction. Proteomic analysis was used to study the fraction of EMD proteins binding to collagen. EMD or TGF-β1 provoked a significant increase of IL-11 and PRG4 expression of oral fibroblasts when seeded onto collagen products and when incubated with the respective supernatant. Gene expression was blocked by the TGF-β receptor I kinase inhibitor SB431542. Amelogenin bound most abundantly to gelatin-coated culture dishes. However, incubation of palatal fibroblasts with recombinant amelogenin did not alter expression of IL-11 and PRG4. These in vitro findings suggest that collagen products adsorb a TGF-β receptor I kinase-dependent activity of EMD and make it available for potential target cells.

  11. T Cell Post-Transcriptional miRNA-mRNA Interaction Networks Identify Targets Associated with Susceptibility/Resistance to Collagen-induced Arthritis

    PubMed Central

    Macedo, Claudia; Cunha, Thiago M.; Nascimento, Daniele C. B.; Sakamoto-Hojo, Elza T.; Donadi, Eduardo A.; Cunha, Fernando Q.; Passos, Geraldo A.

    2013-01-01

    Background Due to recent studies indicating that the deregulation of microRNAs (miRNAs) in T cells contributes to increased severity of rheumatoid arthritis, we hypothesized that deregulated miRNAs may interact with key mRNA targets controlling the function or differentiation of these cells in this disease. Methodology/Principal Findings To test our hypothesis, we used microarrays to survey, for the first time, the expression of all known mouse miRNAs in parallel with genome-wide mRNAs in thymocytes and naïve and activated peripheral CD3+ T cells from two mouse strains the DBA-1/J strain (MHC-H2q), which is susceptible to collagen induced arthritis (CIA), and the DBA-2/J strain (MHC-H2d), which is resistant. Hierarchical clustering of data showed the several T cell miRNAs and mRNAs differentially expressed between the mouse strains in different stages of immunization with collagen. Bayesian statistics using the GenMir++ algorithm allowed reconstruction of post-transcriptional miRNA-mRNA interaction networks for target prediction. We revealed the participation of miR-500, miR-202-3p and miR-30b*, which established interactions with at least one of the following mRNAs: Rorc, Fas, Fasl, Il-10 and Foxo3. Among the interactions that were validated by calculating the minimal free-energy of base pairing between the miRNA and the 3′UTR of the mRNA target and luciferase assay, we highlight the interaction of miR-30b*-Rorc mRNA because the mRNA encodes a protein implicated in pro-inflammatory Th17 cell differentiation (Rorγt). FACS analysis revealed that Rorγt protein levels and Th17 cell counts were comparatively reduced in the DBA-2/J strain. Conclusions/Significance This result showed that the miRNAs and mRNAs identified in this study represent new candidates regulating T cell function and controlling susceptibility and resistance to CIA. PMID:23359619

  12. T cell post-transcriptional miRNA-mRNA interaction networks identify targets associated with susceptibility/resistance to collagen-induced arthritis.

    PubMed

    Donate, Paula B; Fornari, Thais A; Macedo, Claudia; Cunha, Thiago M; Nascimento, Daniele C B; Sakamoto-Hojo, Elza T; Donadi, Eduardo A; Cunha, Fernando Q; Passos, Geraldo A

    2013-01-01

    Due to recent studies indicating that the deregulation of microRNAs (miRNAs) in T cells contributes to increased severity of rheumatoid arthritis, we hypothesized that deregulated miRNAs may interact with key mRNA targets controlling the function or differentiation of these cells in this disease. To test our hypothesis, we used microarrays to survey, for the first time, the expression of all known mouse miRNAs in parallel with genome-wide mRNAs in thymocytes and naïve and activated peripheral CD3(+) T cells from two mouse strains the DBA-1/J strain (MHC-H2q), which is susceptible to collagen induced arthritis (CIA), and the DBA-2/J strain (MHC-H2d), which is resistant. Hierarchical clustering of data showed the several T cell miRNAs and mRNAs differentially expressed between the mouse strains in different stages of immunization with collagen. Bayesian statistics using the GenMir(++) algorithm allowed reconstruction of post-transcriptional miRNA-mRNA interaction networks for target prediction. We revealed the participation of miR-500, miR-202-3p and miR-30b*, which established interactions with at least one of the following mRNAs: Rorc, Fas, Fasl, Il-10 and Foxo3. Among the interactions that were validated by calculating the minimal free-energy of base pairing between the miRNA and the 3'UTR of the mRNA target and luciferase assay, we highlight the interaction of miR-30b*-Rorc mRNA because the mRNA encodes a protein implicated in pro-inflammatory Th17 cell differentiation (Rorγt). FACS analysis revealed that Rorγt protein levels and Th17 cell counts were comparatively reduced in the DBA-2/J strain. This result showed that the miRNAs and mRNAs identified in this study represent new candidates regulating T cell function and controlling susceptibility and resistance to CIA.

  13. Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide.

    PubMed

    Hwang, Jeongmin; San, Boi Hoa; Turner, Neill J; White, Lisa J; Faulk, Denver M; Badylak, Stephen F; Li, Yang; Yu, S Michael

    2017-04-15

    Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold's regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cell-removing detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology. Preservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules (e.g., collagen) during decellularization can strongly influence the cellular response

  14. Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide

    PubMed Central

    Hwang, Jeongmin; San, Boi Hoa; Turner, Neill J.; White, Lisa J.; Faulk, Denver M.; Badylak, Stephen F.; Li, Yang; Yu, S. Michael

    2017-01-01

    Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold’s regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cellremoving detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propa nesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology. Statement of Significance Preservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules (e.g., collagen) during decellularization can strongly

  15. Target-type probability combining algorithms for multisensor tracking

    NASA Astrophysics Data System (ADS)

    Wigren, Torbjorn

    2001-08-01

    Algorithms for the handing of target type information in an operational multi-sensor tracking system are presented. The paper discusses recursive target type estimation, computation of crosses from passive data (strobe track triangulation), as well as the computation of the quality of the crosses for deghosting purposes. The focus is on Bayesian algorithms that operate in the discrete target type probability space, and on the approximations introduced for computational complexity reduction. The centralized algorithms are able to fuse discrete data from a variety of sensors and information sources, including IFF equipment, ESM's, IRST's as well as flight envelopes estimated from track data. All algorithms are asynchronous and can be tuned to handle clutter, erroneous associations as well as missed and erroneous detections. A key to obtain this ability is the inclusion of data forgetting by a procedure for propagation of target type probability states between measurement time instances. Other important properties of the algorithms are their abilities to handle ambiguous data and scenarios. The above aspects are illustrated in a simulations study. The simulation setup includes 46 air targets of 6 different types that are tracked by 5 airborne sensor platforms using ESM's and IRST's as data sources.

  16. Halogens are key cofactors in building of collagen IV scaffolds outside the cell.

    PubMed

    Brown, Kyle L; Hudson, Billy G; Voziyan, Paul A

    2018-05-01

    The purpose of this review is to highlight recent advances in understanding the molecular assembly of basement membranes, as exemplified by the glomerular basement membrane (GBM) of the kidney filtration apparatus. In particular, an essential role of halogens in the basement membrane formation has been discovered. Extracellular chloride triggers a molecular switch within non collagenous domains of collagen IV that induces protomer oligomerization and scaffold assembly outside the cell. Moreover, bromide is an essential cofactor in enzymatic cross-linking that reinforces the stability of scaffolds. Halogenation and halogen-induced oxidation of the collagen IV scaffold in disease states damage scaffold function. Halogens play an essential role in the formation of collagen IV scaffolds of basement membranes. Pathogenic damage of these scaffolds by halogenation and halogen-induced oxidation is a potential target for therapeutic interventions.

  17. [Morphology of collagen matrices for tissue engineering (biocompatibility, biodegradation, tissue response)].

    PubMed

    Shekhter, A B; Guller, A E; Istranov, L P; Istranova, E V; Butnaru, D V; Vinarov, A Z; Zakharkina, O L; Kurkov, A V; Kantimerov, D F; Antonov, E N; Marisov, L V; Glybochko, P V

    2015-01-01

    to perform a comparative morphological study of biocompatibility, biodegradation, and tissue response to implantation of collagen matrices (scaffolds) for tissue engineering in urology and other areas of medicine. Nine matrix types, such as porous materials reconstructed from collagen solution; a collagen sponge-vicryl mesh composite; decellularized and freeze-dried bovine, equine, and fish dermis; small intestinal submucosa, decellularized bovine dura mater; and decellularized human femoral artery, were implanted subcutaneously in 225 rats. The tissues at the implantation site were investigated for a period of 5 to 90 days. Classical histology and nonlinear optical microscopy (NLOM) were applied. The investigations showed no rejection of all the collagen materials. The period of matrix bioresorption varied from 10 days for collagen sponges to 2 months for decellularized and freeze-dried vessels and vicryl meshes. Collagen was prone to macrophage resorption and enzymatic lysis, being replaced by granulation tissue and then fibrous tissue, followed by its involution. NLOM allowed the investigators to study the number, density, interposition, and spatial organization of collagen structures in the matrices and adjacent tissues, and their change over time during implantation. The performed investigation could recommend three matrices: hybrid collagen/vicryl composite; decellularized bovine dermis; and decellularized porcine small intestinal submucosa, which are most adequate for tissue engineering in urology. These and other collagen matrices may be used in different areas of regenerative medicine.

  18. Extracellular post-translational modifications of collagen are major determinants of biomechanical properties of fetal bovine cortical bone.

    PubMed

    Garnero, Patrick; Borel, Olivier; Gineyts, Evelyne; Duboeuf, Francois; Solberg, Helene; Bouxsein, Mary L; Christiansen, Claus; Delmas, Pierre D

    2006-03-01

    Mechanical behavior of bone depends on its mass and architecture, and on the material properties of the matrix, which is composed of a mineral phase and an organic component mainly constituted of type I collagen. Mineral accounts largely for the stiffness of bone, whereas type I collagen provides bone its ductility and toughness, i.e., its ability to undergo deformation and absorb energy after it begins to yield. The molecular mechanisms underlying the effect of alterations in type I collagen on bone mechanical properties are unclear. We used an in vitro model of fetal bovine cortical bone specimens (n = 44), where the extent of type I collagen cross-linking was modified by incubation at 37 degrees C for 0, 60, 90 and 120 days, keeping constant the architecture and the mineral content. At each incubation time, the following parameters were determined: (1) the bone concentration of enzymatic (pyridinoline; PYD and deoxypyridinoline, DPD) and non-enzymatic (pentosidine) crosslinks by HPLC, (2) the extent of aspartic acid isomerization of the type I collagen C-telopeptide (CTX) by ELISA of native (alpha CTX) and isomerized (beta CTX) forms, (3) the mineral density by DXA, (4) the porosity by micro-computed tomography and (5) the bending and compressive mechanical properties. Incubation of bone specimens at 37 degrees C for 60 days increased the level (per molecule of collagen) of PYD (+98%, P = 0.005), DPD (+42%, P = 0.013), pentosidine (+55-fold, P = 0.005), and the degree of type I collagen C-telopeptide isomerization (+4.9-fold, P = 0.005). These biochemical changes of collagen were associated with a 30% decrease in bending and compressive yield stress and a 2.5-fold increase in compressive post-yield energy absorption (P < 0.02 for all), with no significant change of bone stiffness. In multivariate analyses, the level of collagen cross-linking was associated with yield stress and post-yield energy absorption independently of bone mineral density, explaining up to

  19. Collagen IV Diseases: A Focus on the Glomerular Basement Membrane in Alport Syndrome

    PubMed Central

    Cosgrove, Dominic; Liu, Shiguang

    2016-01-01

    Alport syndrome is the result of mutations in any of three type IV collagen genes, COL4A3, COL4A4, or COL4A5. Because the three collagen chains form heterotrimers, there is an absence of all three proteins in the basement membranes where they are expressed. In the glomerulus, the mature glomerular basement membrane type IV collagen network, normally comprised of two separate networks, α3(IV)/α4(IV)/α5(IV) and α1(IV)/α2(IV), is comprised entirely of collagen α1(IV)/α2. This review addresses the current state of our knowledge regarding the consequence of this change in basement membrane composition, including both the direct, via collagen receptor binding, and indirect, regarding influences on glomerular biomechanics. The state of our current understanding regarding mechanisms of glomerular disease initiation and progression will be examined, as will the current state of the art regarding emergent therapeutic approaches to slow or arrest glomerular disease in Alport patients. PMID:27576055

  20. The downregulation of microRNA let-7a contributes to the excessive expression of type I collagen in systemic and localized scleroderma.

    PubMed

    Makino, Katsunari; Jinnin, Masatoshi; Hirano, Ayaka; Yamane, Keitaro; Eto, Mitsuhiko; Kusano, Takamitsu; Honda, Noritoshi; Kajihara, Ikko; Makino, Takamitsu; Sakai, Keisuke; Masuguchi, Shinichi; Fukushima, Satoshi; Ihn, Hironobu

    2013-04-15

    Systemic and localized scleroderma (SSc and LSc) is characterized by excessive deposition of collagen and tissue fibrosis in the skin. Although they have fundamental common characteristics including autoimmunity, little is known about the exact mechanism that mediates the excessive collagen expression in these disorders. In the current study, we tried to evaluate the possibility that microRNAs (miRNAs) play some roles in the pathogenesis of fibrosis seen in these diseases. miRNA expression patterns were evaluated by miRNA array analysis, real-time PCR, and in situ hybridization. The function of miRNAs in dermal fibroblasts was assessed using miRNA inhibitors, precursors, or protectors. In the mouse model of bleomycin-induced dermal sclerosis, the overexpression of miRNAs was performed by i.p. miRNA injection. We demonstrated let-7a expression was downregulated in SSc and LSc skin both in vivo and in vitro, compared with normal or keloid skin. The inhibition or overexpression of let-7a in human or mouse skin fibroblasts affected the protein expression of type I collagen or luciferase activity of collagen 3'-untranslated region. Also, we found let-7a was detectable and quantitative in the serum and investigated serum let-7a levels in patients with SSc or LSc. let-7a concentration was significantly decreased in these patients, especially in LSc patients. Moreover, we revealed that the intermittent overexpression of let-7a in the skin by i.p. miRNA injection improved the skin fibrosis induced by bleomycin in mice. Investigation of more detailed mechanisms of miRNA-mediated regulation of collagen expression may lead to new therapeutic approaches against SSc and LSc.

  1. Neurotensin-loaded collagen dressings reduce inflammation and improve wound healing in diabetic mice.

    PubMed

    Moura, Liane I F; Dias, Ana M A; Suesca, Edward; Casadiegos, Sergio; Leal, Ermelindo C; Fontanilla, Marta R; Carvalho, Lina; de Sousa, Hermínio C; Carvalho, Eugénia

    2014-01-01

    Impaired wound healing is an important clinical problem in diabetes mellitus and results in failure to completely heal diabetic foot ulcers (DFUs), which may lead to lower extremity amputations. In the present study, collagen based dressings were prepared to be applied as support for the delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. The performance of NT alone and NT-loaded collagen matrices to treat wounds in streptozotocin (STZ) diabetic induced mice was evaluated. Results showed that the prepared dressings were not-cytotoxic up to 72h after contact with macrophages (Raw 264.7) and human keratinocyte (HaCaT) cell lines. Moreover, those cells were shown to adhere to the collagen matrices without noticeable change in their morphology. NT-loaded collagen dressings induced faster healing (17% wound area reduction) in the early phases of wound healing in diabetic wounded mice. In addition, they also significantly reduced inflammatory cytokine expression namely, TNF-α (p<0.01) and IL-1β (p<0.01) and decreased the inflammatory infiltrate at day 3 post-wounding (inflammatory phase). After complete healing, metalloproteinase 9 (MMP-9) is reduced in diabetic skin (p<0.05) which significantly increased fibroblast migration and collagen (collagen type I, alpha 2 (COL1A2) and collagen type III, alpha 1 (COL3A1)) expression and deposition. These results suggest that collagen-based dressings can be an effective support for NT release into diabetic wound enhancing the healing process. Nevertheless, a more prominent scar is observed in diabetic wounds treated with collagen when compared to the treatment with NT alone. © 2013.

  2. Human collagen produced in plants: more than just another molecule.

    PubMed

    Shoseyov, Oded; Posen, Yehudit; Grynspan, Frida

    2014-01-01

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

  3. Collagen V expression is crucial in regional development of the supraspinatus tendon.

    PubMed

    Connizzo, Brianne K; Adams, Sheila M; Adams, Thomas H; Birk, David E; Soslowsky, Louis J

    2016-12-01

    Manipulations in cell culture and mouse models have demonstrated that reduction of collagen V results in altered fibril structure and matrix assembly. A tissue-dependent role for collagen V in determining mechanical function was recently established, but its role in determining regional properties has not been addressed. The objective of this study was to define the role(s) of collagen V expression in establishing the site-specific properties of the supraspinatus tendon. The insertion and midsubstance of tendons from wild type, heterozygous and tendon/ligament-specific null mice were assessed for crimp morphology, fibril morphology, cell morphology, as well as total collagen and pyridinoline cross-link (PYD) content. Fibril morphology was altered at the midsubstance of both groups with larger, but fewer, fibrils and no change in cell morphology or collagen compared to the wild type controls. In contrast, a significant disruption of fibril assembly was observed at the insertion site of the null group with the presence of structurally aberrant fibrils. Alterations were also present in cell density and PYD content. Altogether, these results demonstrate that collagen V plays a crucial role in determining region-specific differences in mouse supraspinatus tendon structure. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2154-2161, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  4. Dynamics of Cancer Cell near Collagen Fiber Chain

    NASA Astrophysics Data System (ADS)

    Kim, Jihan; Sun, Bo

    Cell migration is an integrated process that is important in life. Migration is essential for embryonic development as well as homeostatic processes such as wound healing and immune responses. When cell migrates through connective extracellular matrix (ECM), it applies cellular traction force to ECM and senses the rigidity of their local environment. We used human breast cancer cell (MDA-MB-231) which is highly invasive and applies strong traction force to ECM. As cancer cell applies traction force to type I collage-based ECM, it deforms collagen fibers near the surface. Patterns of deforming collagen fibers are significantly different with pairs of cancer cells compared to a single cancer cell. While a pair of cancer cells within 60 um creates aligned collagen fiber chains between them permanently, a single cancer cell does not form any fiber chains. In this experiment we measured a cellular response and an interaction between a pair of cells through the chain. Finally, we analyzed correlation of directions between cancer cell migration and the collagen chain alignment.

  5. Oxidized Regenerated Cellulose/Collagen Dressings: Review of Evidence and Recommendations.

    PubMed

    Wu, Stephanie; Applewhite, Andrew J; Niezgoda, Jeffrey; Snyder, Robert; Shah, Jayesh; Cullen, Breda; Schultz, Gregory; Harrison, Janis; Hill, Rosemary; Howell, Melania; Speyrer, Marcus; Utra, Howard; de Leon, Jean; Lee, Wayne; Treadwell, Terry

    2017-11-01

    Healthcare systems are being challenged to manage increasing numbers of nonhealing wounds. Wound dressings are one of the first lines of defense in wound management, and numerous options exist. The oxidized regenerated cellulose (ORC)/collagen dressing may offer healthcare providers a robust and cost-effective tool for use in a variety of wounds. A multidisciplinary panel meeting was convened to discuss the use of ORC/collagen dressings in wound care and provide practice recommendations. A literature search was conducted to provide a brief review of the peer-reviewed studies published between January 2000 and March 2016 to inform the meeting. A 2-day panel meeting convened in February 2017. Healthcare providers with experience using ORC/collagen dressings. This multidisciplinary panel of 15 experts in wound healing included podiatrists, wound care specialists (doctors, certified wound care nurses, and research scientists), and an orthopedist. The literature search identified 58 articles, a majority of which were low levels of evidence (69.3% were level 3 or lower). Panel members identified wound types, such as abrasions, burns, stalled wounds, diabetic foot ulcers, and pressure injuries, where ORC/collagen dressing use could be beneficial. Panel members then provided recommendations and technical pearls for the use of ORC/collagen dressings in practice. Barriers to ORC/collagen dressing use were discussed, and potential resolutions were offered. An ORC/collagen dressing can be a critical tool for clinicians to help manage a variety of wounds. Clinical and economic studies comparing standard-of-care dressings and plain collagen dressings to ORC/collagen dressings are needed.

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

    PubMed Central

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

    2013-01-01

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

  7. Use of an activated beta-catenin to identify Wnt pathway target genes in caenorhabditis elegans, including a subset of collagen genes expressed in late larval development.

    PubMed

    Jackson, Belinda M; Abete-Luzi, Patricia; Krause, Michael W; Eisenmann, David M

    2014-04-16

    The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

  8. Collagen and the myocardium: fibrillar structure, biosynthesis and degradation in relation to hypertrophy and its regression.

    PubMed

    Eghbali, M; Weber, K T

    1990-07-17

    The extracellular matrix of the myocardium contains an elaborate structural matrix composed mainly of fibrillar types I and III collagen. This matrix is responsible for the support and alignment of myocytes and capillaries. Because of its alignment, location, configuration and tensile strength, relative to cardiac myocytes, the collagen matrix represents a major determinant of myocardial stiffness. Cardiac fibroblasts, not myocytes, contain the mRNA for these fibrillar collagens. In the hypertrophic remodeling of the myocardium that accompanies arterial hypertension, a progressive structural and biochemical remodeling of the matrix follows enhanced collagen gene expression. The resultant significant accumulation of collagen in the interstitium and around intramyocardial coronary arteries, or interstitial and perivascular fibrosis, represents a pathologic remodeling of the myocardium that compromises this normally efficient pump. This report reviews the structural nature, biosynthesis and degradation of collagen in the normal and hypertrophied myocardium. It suggests that interstitial heart disease, or the disproportionate growth of the extracellular matrix relative to myocyte hypertrophy, is an entity that merits greater understanding, particularly the factors regulating types I and III collagen gene expression and their degradation.

  9. Red grape (Vitis vinifera L.) flavonoids down-regulate collagen type III expression after UV-A in primary human dermal blood endothelial cells.

    PubMed

    Di Francesco, Serena; Savio, Monica; Bloise, Nora; Borroni, Giovanni; Stivala, Lucia Anna; Borroni, Riccardo G

    2018-05-09

    Red grape (Vitis vinifera L.) flavonoids including flavan-3-ols (e.g., catechin and epicatechin), flavonols (e.g., quercetin) and anthocyanins (e.g., malvidin) exert anti-inflammatory and antioxidant activities. In the skin they also have a photoprotective action, and their effects have been extensively investigated in keratinocytes, melanocytes and fibroblasts. Despite their known effects also on blood vasculature, little is known on their activities on human dermal blood endothelial cells (HDBECs), which are critically involved in skin homeostasis as well as in the pathogenesis of neoplastic and inflammatory skin diseases. We sought to study the biological effects of selected red grape flavonoids in preventing the consequences of ultraviolet (UV)-A irradiation in vitro. Our results show that red grape flavonoids prevent UV-A-induced sICAM-1 release in HDBECs, suggesting that this cell type could represent an additional target of the anti-inflammatory activity of flavonoids. In addition, flavonoids effectively inhibited UV-A-induced synthesis of collagen type III at both RNA and protein level, indicating that dermal blood microvasculature could be actively involved in ECM remodelling as a consequence of skin photo-ageing, and that this can be prevented by red grape flavonoids. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  10. Collagen V haploinsufficiency in a murine model of classic Ehlers-Danlos syndrome is associated with deficient structural and mechanical healing in tendons.

    PubMed

    Johnston, Jessica M; Connizzo, Brianne K; Shetye, Snehal S; Robinson, Kelsey A; Huegel, Julianne; Rodriguez, Ashley B; Sun, Mei; Adams, Sheila M; Birk, David E; Soslowsky, Louis J

    2017-12-01

    Classic Ehlers-Danlos syndrome (EDS) patients suffer from connective tissue hyperelasticity, joint instability, skin hyperextensibility, tissue fragility, and poor wound healing due to heterozygous mutations in COL5a1 or COL5a2 genes. This study investigated the roles of collagen V in establishing structure and function in uninjured patellar tendons as well as in the injury response using a Col5a1 +/- mouse, a model for classic EDS. These analyses were done comparing tendons from a classic EDS model (Col5a1 +/- ) with wild-type controls. Tendons were subjected to mechanical testing, histological, and fibril analysis before injury as well as 3 and 6 weeks after injury. We found that Col5a1 +/- tendons demonstrated diminished recovery of mechanical competency after injury as compared to normal wild-type tendons, which recovered their pre-injury values by 6 weeks post injury. Additionally, the Col5a1 +/- tendons demonstrated altered fibril morphology and diameter distributions compared to the wild-type tendons. This study indicates that collagen V plays an important role in regulating collagen fibrillogenesis and the associated recovery of mechanical integrity in tendons after injury. In addition, the dysregulation with decreased collagen V expression in EDS is associated with a diminished injury response. The results presented herein have the potential to direct future targeted therapeutics for classic EDS patients. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2707-2715, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  11. Collagen membranes: a review.

    PubMed

    Bunyaratavej, P; Wang, H L

    2001-02-01

    Collagen materials have been utilized in medicine and dentistry because of their proven biocompatability and capability of promoting wound healing. For guided tissue regeneration (GTR) procedures, collagen membranes have been shown to be comparable to non-absorbable membranes with regard to probing depth reduction, clinical attachment gain, and percent of bone fill. Although these membranes are absorbable, collagen membranes have been demonstrated to prevent epithelial down-growth along the root surfaces during the early phase of wound healing. The use of grafting material in combination with collagen membranes seems to improve clinical outcomes for furcation, but not intrabony, defects when compared to the use of membranes alone. Recently, collagen materials have also been applied in guided bone regeneration (GBR) and root coverage procedures with comparable success rates to non-absorbable expanded polytetrafluoroethylene (ePTFE) membranes and conventional subepithelial connective tissue grafts, respectively. Long-term clinical trials are still needed to further evaluate the benefits of collagen membranes in periodontal and peri-implant defects. This article will review the rationale for each indication and its related literature, both in vitro and in vivo studies. The properties that make collagen membranes attractive for use in regenerative therapy will be addressed. In addition, varieties of cross-linking techniques utilized to retard the degradation rate of collagen membranes will be discussed.

  12. Effects of Cysteine Proteases on the Structural and Mechanical Properties of Collagen Fibers*

    PubMed Central

    Panwar, Preety; Du, Xin; Sharma, Vidhu; Lamour, Guillaume; Castro, Mickael; Li, Hongbin; Brömme, Dieter

    2013-01-01

    Excessive cathepsin K (catK)-mediated turnover of fibrillar type I and II collagens in bone and cartilage leads to osteoporosis and osteoarthritis. However, little is known about how catK degrades compact collagen macromolecules. The present study is aimed to explore the structural and mechanical consequences of collagen fiber degradation by catK. Mouse tail type I collagen fibers were incubated with either catK or non-collagenase cathepsins. Methods used include scanning electron microscopy, protein electrophoresis, atomic force microscopy, and tensile strength testing. Our study revealed evidence of proteoglycan network degradation, followed by the progressive disassembly of macroscopic collagen fibers into primary structural elements by catK. Proteolytically released GAGs are involved in the generation of collagenolytically active catK-GAG complexes as shown by AFM. In addition to their structural disintegration, a decrease in the tensile properties of fibers was observed due to the action of catK. The Young's moduli of untreated collagen fibers versus catK-treated fibers in dehydrated conditions were 3.2 ± 0.68 GPa and 1.9 ± 0.65 GPa, respectively. In contrast, cathepsin L, V, B, and S revealed no collagenase activity, except the disruption of proteoglycan-GAG interfibrillar bridges, which slightly decreased the tensile strength of fibers. PMID:23297404

  13. Effects of ingestion of collagen peptide on collagen fibrils and glycosaminoglycans in the dermis.

    PubMed

    Matsuda, Naoya; Koyama, Yoh-ichi; Hosaka, Yoshinao; Ueda, Hiromi; Watanabe, Takafumi; Araya, Takayuki; Irie, Shinkichi; Takehana, Kazushige

    2006-06-01

    In order to investigate the effects of collagen peptide ingestion on fibroblasts and the extracellular matrix in the dermis, collagen peptide was administered orally to pigs at 0.2 g/kg body weight/d for 62 d, and its effects were compared with those of lactalbumin and water controls. Fibroblast density, and diameter and density of collagen fibrils were significantly larger in the collagen peptide group than in the lactalbumin and water control groups. The two major components of dermal glycosaminoglycans, hyaluronic acid and dermatan sulfate, which are present in the inter-fibrillar space, did not differ significantly among the three groups. However, the ratio of dermatan sulfate, which is derived from fibril-bound decorin, was largest in the collagen peptide group. These results suggest that ingestion of collagen peptide induces increased fibroblast density and enhances formation of collagen fibrils in the dermis in a protein-specific manner.

  14. Bone resorption analysis of platelet-derived growth factor type BB application on collagen for bone grafts secured by titanium mesh over a pig jaw defect model

    PubMed Central

    Herford, Alan Scott; Cicciù, Marco

    2012-01-01

    Purpose: The aim of this investigation was to evaluate whether the addition of the platelet derived growth factor type BB (PDGF-BB) to a collagen matrix applied on a titanium mesh would favor healing and resorption onto the grafted bone. A histologic and radiographic study of two different groups (test and control) was performed. Designs: A surgical procedure was performed on 8 pigs to obtain 16 bilateral mandibular alveolar defects. All the defects were then reconstructed with a mixture of autogenous bovine bone using titanium mesh positioning. Two groups, with a total of 16 defects were created: The first to study collagen sponge and PDGF-BB and the second to control collagen only. The collagen matrix was positioned directly over the mesh and soft tissue was closed without tensions onto both groups without attempting to obtain primary closure. Possible exposure of the titanium mesh as well as the height and volume of the new bone was recorded. Results: New bone formation averaged about 6.68 mm in the test group studied; the control group had less regenerated bone at 4.62 mm. Conclusion: PDGF-BB addition to the collagen matrix induced a strong increase in hard and soft tissue healing and favored bone formation, reducing bone resorption even if the mesh was exposed. PMID:23833493

  15. ECM turnover-stimulated gene delivery through collagen-mimetic peptide-plasmid integration in collagen.

    PubMed

    Urello, Morgan A; Kiick, Kristi L; Sullivan, Millicent O

    2017-10-15

    Gene therapies have great potential in regenerative medicine; however, clinical translation has been inhibited by low stability and limited transfection efficiencies. Herein, we incorporate collagen-mimetic peptide (CMP)-linked polyplexes in collagen scaffolds to increase DNA stability by up to 400% and enable tailorable in vivo transgene expression at 100-fold higher levels and 10-fold longer time periods. These improvements were directly linked to a sustained interaction between collagen and polyplexes that persisted during cellular remodeling, polyplex uptake, and intracellular trafficking. Specifically, incorporation of CMPs into polyethylenimine (PEI) polyplexes preserved serum-exposed polyplex-collagen activity over a period of 14days, with 4 orders-of-magnitude more intact DNA present in CMP-modified polyplex-collagen relative to unmodified polyplex-collagen after a 10day incubation under cell culture conditions. CMP-modification also altered endocytic uptake, as indicated by gene silencing studies showing a nearly 50% decrease in transgene expression in response to caveolin-1 silencing in modified samples versus only 30% in unmodified samples. Furthermore, cellular internalization studies demonstrated that polyplex-collagen association persisted within cells in CMP polyplexes, but not in unmodified polyplexes, suggesting that CMP linkage to collagen regulates intracellular transport. Moreover, experiments in an in vivo repair model showed that CMP modification enabled tailoring of transgene expression from 4 to 25days over a range of concentrations. Overall, these findings demonstrate that CMP decoration provides substantial improvements in gene retention, altered release kinetics, improved serum-stability, and improved gene activity in vivo. This versatile technique has great potential for multiple applications in regenerative medicine. In this work, we demonstrate a novel approach for stably integrating DNA into collagen scaffolds to exploit the natural

  16. The fibrillar collagen family.

    PubMed

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

    2010-01-28

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

  17. Development and in vitro evaluation of expandable gastroretentive dosage forms based on compressed collagen sponges.

    PubMed

    Gröning, R; Cloer, C; Müller, R S

    2006-07-01

    The objective of this study was to develop and evaluate new collagen gastroretentive dosage forms (GRDFs) which expand in the stomach after contact with gastric fluids. The GRDFs should remain in the stomach for a prolonged period of time due to their size. The dosage forms were prepared from collagen sponges. The sponges were manufactured by freeze-drying a riboflavin-containing collagen solution. A computer controlled material supply was constructed to transport precompressed collagen into a tablet machine. A second type of tablet was manufactured by combining compressed collagen sponges with hydrophilic matrix layers of hydroxypropylmethylcellulose. Matrix layers containing captopril or aciclovir were developed. In vitro experiments were performed with both types of dosage forms. The collagen tablets expand within a few minutes after contact with artificial gastric juice and form a drug delivery system with a size of 8 mm x 18 mm x 60 mm. Riboflavin is released over 16 h. If two layer tablets are used, the release of aciclovir or captopril can be controlled by the composition of the sustained release layer.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-10-06

    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. © 2015 The Authors.

  1. Collagenous gastritis.

    PubMed

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

    2013-09-01

    In the present paper, we report a case of rare collagenous gastritis. The patient was a 25-year-old man who had experienced nausea, abdominal distention and epigastralgia since 2005. Esophagogastroduodenoscopy (EGD) carried out at initial examination by the patient's local doctor revealed an extensively discolored depression from the upper gastric body to the lower gastric body, mainly including the greater curvature, accompanied by residual mucosa with multiple islands and nodularity with a cobblestone appearance. Initial biopsies sampled from the nodules and accompanying atrophic mucosa were diagnosed as chronic gastritis. In August, 2011, the patient was referred to Tohoku University Hospital for observation and treatment. EGD at our hospital showed the same findings as those by the patient's local doctor. Pathological findings included a membranous collagen band in the superficial layer area of the gastric mucosa, which led to a diagnosis of collagenous gastritis. Collagenous gastritis is an extremely rare disease, but it is important to recognize its characteristic endoscopic findings to make a diagnosis. © 2012 The Authors. Digestive Endoscopy © 2012 Japan Gastroenterological Endoscopy Society.

  2. Imaging Prostate Cancer Microenvironment by Collagen Hybridization

    DTIC Science & Technology

    2016-12-01

    2: Evaluation of 18F-DCFPyL labelling and tracking of PSMA+ CAR T cells Title: PSMA Directed Imaging of Prostate Cancer Focus on Androgen Receptor ... receptors to treat cancer Title: Plasmid Selection and Characterisation Time Commitments: 1.20 calendar months Supporting Agency: Cancer Targeting...AWARD NUMBER: W81XWH-12-1-0556 TITLE: Imaging Prostate Cancer Microenvironment by Collagen Hybridization PRINCIPAL INVESTIGATOR: Martin

  3. Formation of Highly Aligned Collagen Nanofibers by Continuous Cyclic Stretch of a Collagen Hydrogel Sheet.

    PubMed

    Nam, Eunryel; Lee, Won Chul; Takeuchi, Shoji

    2016-07-01

    A collagen sheet with highly aligned collagen fibers is fabricated by continuous cyclic stretch. The rearrangement of the collagen fibers depends on the different process parameters of the cyclic stretch, including magnitude, frequency, and period of stretch. The collagen fibers are aligned perpendicularly to the direction of the stretch. Corneal stromal cells and smooth muscle cells cultivated on the highly aligned collagen sheet show alignment along the collagen fibers without the stretch during culture. Thus, the sheet can be a suitable scaffold for use in regenerative medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. The effects of ethanol on the size-exclusion characteristics of type I dentin collagen to adhesive resin monomers.

    PubMed

    Chiba, A; Zhou, J; Nakajima, M; Tan, J; Tagami, J; Scheffel, D L S; Hebling, J; Agee, K A; Breschi, L; Grégoire, G; Jang, S S; Tay, F R; Pashley, D H

    2016-03-01

    During dentin bonding with etch-and-rinse adhesive systems, phosphoric acid etching of mineralized dentin solubilizes the mineral crystallites and replaces them with bound and unbound water. During the infiltration phase of dentin bonding, solvated adhesive resin comonomers are supposed to replace all of the unbound collagen water and polymerize into copolymers. A recently published review suggested that dental monomers are too large to enter and displace water from tightly-packed collagen molecules. Conversely, recent work from the authors' laboratory demonstrated that HEMA and TEGDMA freely equilibrate with water-saturated dentin matrices. However, because adhesive blends are solvated in organic solvents, those solvents may remove enough free water to allow collagen molecules to come close enough to exclude adhesive monomer permeation. The present study analyzed the size-exclusion characteristics of dentin collagen, using a gel permeation-like column chromatography technique, filled with dentin powder instead of Sephadex beads as the stationary phase. The elution volumes of different sized test molecules, including adhesive resin monomers, studied in both water-saturated dentin, and again in ethanol-dehydrated dentin powder, showed that adhesive resin monomers can freely diffuse into both hydrated and dehydrated collagen molecules. Under these in vitro conditions, all free and some of the loosely-bound water seems to have been removed by ethanol. These results validate the concept that adhesive resin monomers can permeate tightly-bound water in ethanol-saturated collagen molecules during infiltration by etch-and-rinse adhesives. It has been reported that collagen molecules in dentin matrices are packed too close together to allow permeation of adhesive monomers between them. Resin infiltration, in this view, would be limited to extrafibrillar spaces. Our work suggests that monomers equilibrate with collagen water in both water and ethanol-saturated dentin

  5. Effect of nicotinamide on amino acids content in bone collagen depending on biological availability of vitamins in diabetic rats.

    PubMed

    Guzyk, M M; Sergiichuk, Iu T; Dyakun, K O; Yanitska, L V; Kuchmerovska, T M

    2014-01-01

    Connective tissue is highly susceptible to imbalances induced by diabetes. Diabetes-related osteopenia, decreased bone strength etc. may be associated with altered metabolism of various collagens: Although it is assumed that alterations in collagen amino acids (AA) may strongly affect protein properties andphysiological functions, however, very limited evidences are present at the moment regarding AA composition of bone type I collagen and its relevance to abnormal availability of vitamins which are necessary for collagen synthesis in diabetes. We have tested whether nicotinamide (NAm) can influence type Icollagen formation and AA composition as well as vitamins availability in diabetes. After 4 weeks of STZ-induced diabetes (60 mg/ kg) male Wistar rats were injected for 2 weeks with/without NAm (200 mg/kg b. w). Acid extraction of type I collagen from the bones was performed with following stepwise salting out. The content of type I collagen after its acid extraction from the bones was estimated by the amounts of hydroxyproline. Amino acids were assayed by cation exchange chromatography Diabetes-associated changes in AA composition of type I collagen mainly affect those amino acids which are known to be involved in helix formation and cross-linking of the molecules. Diabetes was found to significantly reduce bone collagen contents of o-Pro, Gly, Ala, o-Lys and Pro, whereas Lys, His, Arg, Glu, Thr, Leu, Phe contents were elevated (P < 0.05). NAm treatment was able to partially normalise AA contents. In diabetes, blood serum and hepatic vitamin C and B3 contents were shown to be significantly lowered, whereas a-tocopherol was slightly increased compared with control (P < 0.05). Restoration of circulatory and liver vitamin C and B3 was observed. The data demonstrate the close relationship between the diabetes-associated decrease in type I collagen deposition, altered amino acids metabolism and impaired availability of vitamins, which are necessary for collagen

  6. Articular cartilage repair with recombinant human type II collagen/polylactide scaffold in a preliminary porcine study.

    PubMed

    Muhonen, Virpi; Salonius, Eve; Haaparanta, Anne-Marie; Järvinen, Elina; Paatela, Teemu; Meller, Anna; Hannula, Markus; Björkman, Mimmi; Pyhältö, Tuomo; Ellä, Ville; Vasara, Anna; Töyräs, Juha; Kellomäki, Minna; Kiviranta, Ilkka

    2016-05-01

    The purpose of this study was to investigate the potential of a novel recombinant human type II collagen/polylactide scaffold (rhCo-PLA) in the repair of full-thickness cartilage lesions with autologous chondrocyte implantation technique (ACI). The forming repair tissue was compared to spontaneous healing (spontaneous) and repair with a commercial porcine type I/III collagen membrane (pCo). Domestic pigs (4-month-old, n = 20) were randomized into three study groups and a circular full-thickness chondral lesion with a diameter of 8 mm was created in the right medial femoral condyle. After 3 weeks, the chondral lesions were repaired with either rhCo-PLA or pCo together with autologous chondrocytes, or the lesion was only debrided and left untreated for spontaneous repair. The repair tissue was evaluated 4 months after the second operation. Hyaline cartilage formed most frequently in the rhCo-PLA treatment group. Biomechanically, there was a trend that both treatment groups resulted in better repair tissue than spontaneous healing. Adverse subchondral bone reactions developed less frequently in the spontaneous group (40%) and the rhCo-PLA treated group (50%) than in the pCo control group (100%). However, no statistically significant differences were found between the groups. The novel rhCo-PLA biomaterial showed promising results in this proof-of-concept study, but further studies will be needed in order to determine its effectiveness in articular cartilage repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:745-753, 2016. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  7. TNF-alpha, but not IFN-gamma, regulates CCN2 (CTGF), collagen type I, and proliferation in mesangial cells: possible roles in the progression of renal fibrosis.

    PubMed

    Cooker, Laurinda A; Peterson, Darryl; Rambow, Joann; Riser, Melisa L; Riser, Rebecca E; Najmabadi, Feridoon; Brigstock, David; Riser, Bruce L

    2007-07-01

    Connective tissue growth factor (CCN2) is a profibrotic factor acting downstream and independently of TGF-beta to mediate renal fibrosis. Although inflammation is often involved in the initiation and/or progression of fibrosis, the role of inflammatory cytokines in regulation of glomerular CCN2 expression, cellular proliferation, and extracellular matrix accumulation is unknown. We studied two such cytokines, TNF-alpha and IFN-gamma, for their effects on cultured mesangial cells in the presence or absence of TGF-beta, as a model for progressive renal fibrosis. Short-term treatment with TNF-alpha, like TGF-beta, significantly increased secreted CCN2 per cell, but unlike TGF-beta inhibited cellular replication. TNF-alpha combined with TGF-beta further increased CCN2 secretion and mRNA levels and reduced proliferation. Surprisingly, however, TNF-alpha treatment decreased baseline collagen type I protein and mRNA levels and largely blocked their stimulation by TGF-beta. Long-term treatment with TGF-beta or TNF-alpha alone no longer increased CCN2 protein levels. However, the combination synergistically increased CCN2. IFN-gamma had no effect on either CCN2 or collagen activity and produced a mild inhibition of TGF-beta-induced collagen only at a high concentration (500 U/ml). In summary, we report a strong positive regulatory role for TNF-alpha, but not IFN-gamma, in CCN2 production and secretion, including that driven by TGF-beta. The stimulation of CCN2 release by TNF-alpha, unlike TGF-beta, is independent of cellular proliferation and not linked to increased collagen type I accumulation. This suggests that the paradigm of TGF-beta-driven CCN2 with subsequent collagen production may be overridden by an as yet undefined inhibitory mechanism acting either directly or indirectly on matrix metabolism.

  8. Biomarkers of the extracellular matrix and of collagen fragments.

    PubMed

    Chalikias, Georgios K; Tziakas, Dimitrios N

    2015-03-30

    A great body of evidence has shown that extracellular matrix (ECM) alterations are present in the major types of cardiac diseases: ischemic heart disease, heart disease associated with pressure overload, heart disease associated with volume overload, and intrinsic myocardial disease or cardiomyopathy. Collagen, type I and III, is the principal structural protein found in the myocardium and its pro- or telopeptides are released into the circulation during the course of cardiovascular diseases. Therefore, these peptides may reflect collagen synthesis and break-down and also represent a much more useful tool to address ECM changes from a distance. Clinical trials have been performed during recent years to examine the usage of these peptides as diagnostic or prognostic biomarkers in heart failure (HF) patients. This review aims to summarize published data concerning cardiac ECM and its circulating biomarkers. Studies that focused on collagen metabolism related biomarkers in patients with HF are analyzed. Finally, limitations associated with the clinical use of the aforementioned biomarkers are also discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Epitaxially grown collagen fibrils reveal diversity in contact guidance behavior among cancer cells.

    PubMed

    Wang, Juan; Petefish, Joseph W; Hillier, Andrew C; Schneider, Ian C

    2015-01-01

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

  10. Effect of laser soldering irradiation on covalent bonds of pure collagen.

    PubMed

    Constantinescu, Mihai A; Alfieri, Alex; Mihalache, George; Stuker, Florian; Ducray, Angélique; Seiler, Rolf W; Frenz, Martin; Reinert, Michael

    2007-03-01

    Laser tissue welding and soldering is being increasingly used in the clinical setting for defined surgical procedures. The exact induced changes responsible for tensile strength are not yet fully investigated. To further improve the strength of the bonding, a better understanding of the laser impact at the subcellular level is necessary. The goal of this study was to analyze whether the effect of laser irradiation on covalent bonding in pure collagen using irradiances typically applied for tissue soldering. Pure rabbit and equine type I collagen were subjected to laser irradiation. In the first part of the study, rabbit and equine collagen were compared using identical laser and irradiation settings. In the second part of the study, equine collagen was irradiated at increasing laser powers. Changes in covalent bonding were studied indirectly using the sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique. Tensile strengths of soldered membranes were measured with a calibrated tensile force gauge. In the first experiment, no differences between the species-specific collagen bands were noted, and no changes in banding were found on SDS-PAGE after laser irradiation. In the second experiment, increasing laser irradiation power showed no effect on collagen banding in SDS-PAGE. Finally, the laser tissue soldering of pure collagen membranes showed virtually no determinable tensile strength. Laser irradiation of pure collagen at typical power settings and exposure times generally used in laser tissue soldering does not induce covalent bonding between collagen molecules. This is true for both rabbit and equine collagen proveniences. Furthermore, soldering of pure collagen membranes without additional cellular components does not achieve the typical tensile strength reported in native, cell-rich tissues. This study is a first step in a better understanding of laser impact at the molecular level and might prove useful in engineering of combined

  11. 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.

    PubMed

    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

  12. Lamina propria of the human vocal fold: histomorphometric study of collagen fibers.

    PubMed

    Prades, Jean-Michel; Dumollard, Jean Marc; Duband, Sébastien; Timoshenko, Andrei; Richard, Céline; Dubois, Marie Dominique; Martin, Christian; Peoc'h, Michel

    2010-04-01

    Since the seminal work of M. Hirano, which defined the three-layered lamina propria of the human vocal fold, there has been confusion in the labeling of each layer. Recent studies described the composition of fibers and interstitial molecules within the lamina propria leading to various biomechanical properties. However, collagen fibers appear as the most important structure component. We used an optical analysis and the picrosirius-polarization method to describe collagen fibers from six adult and two fetal human larynges fixed in formalin and frontally sectioned in the middle part of the vocal fold. The deep layer of the lamina propria is the most densely organized band of collagen fibers penetrating the superficial muscle bundles of the vocal muscle. The mean thickness of this layer is about 36% of the lamina propria and shows a network of strongly birefringent fibers (collagen type I and III). The superficial layer of the lamina propria is a narrow band of collagen fibers immediately below the basement membrane of the epithelium. The mean thickness of this layer is about 13% of the lamina propria and shows strong birefringent fibers. The intermediate layer is the less densely organized band between the deep and superficial layers. The mean thickness of this layer is about 51% of the lamina propria and shows clear, green weakly birefringent fibers characterized as collagen type III. The fetal lamina propria contains only a monolayer distribution of loose collagen fibers between the epithelium and the vocal muscle. These results help describe the distribution of collagen fibers within the lamina propria of the human vocal fold and have implications to understand the cover-body theory of voice production both in the adult and newborn.

  13. Effects of the addition of mechanically deboned poultry meat and collagen fibers on quality characteristics of frankfurter-type sausages.

    PubMed

    Pereira, Anirene Galvão Tavares; Ramos, Eduardo Mendes; Teixeira, Jacyara Thaís; Cardoso, Giselle Pereira; Ramos, Alcinéia de Lemos Souza; Fontes, Paulo Rogério

    2011-12-01

    The effects of mechanically deboned poultry meat (MDPM) and levels of collagen fibers on comminuted, cooked sausage quality characteristics were investigated using the central composite rotatable design of response surface methodology (RSM). Use of collagen fiber as an additive affected the sausage characteristics, but the effect depended on the amount of the MDPM used. While MDPM additions resulted in higher cooking loss and darker and redder frankfurters, the addition of collagen fibers improved cooking yields and contributed to the lightness of the final product. Higher collagen fiber content was also accompanied by a significant increase in frankfurter hardness regardless of the MDPM content. Use of collagen fibers countered the negative effects of MDPM on sausage quality attributes, especially on cooking yields and final product color. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Haemorrhoids - a collagen disease?

    PubMed

    Willis, S; Junge, K; Ebrahimi, R; Prescher, A; Schumpelick, V

    2010-12-01

    The cause of haemorrhoidal disease is unknown, epidemiological data and histopathological findings support the hypothesis that reduced connective tissue stability is associated with the incidence of haemorrhoids. Therefore the aim of this study was to analyse the quantity and quality of collagen formation in the corpus cavernosum recti in patients with III°/IV° haemorrhoids in comparison with persons without haemorrhoids. Haemorrhoidectomy specimens of 31 patients with III°/IV° haemorrhoids were examined. The specimens of 20 persons who died a natural death and who had no haemorrhoidal disease served as the controls. The amount of collagen was estimated photometrically by calculating the collagen/protein ratio. The collagen I/III ratio served as parameter for the quality of collagen formation and was calculated using cross polarization spectroscopy. Patients with haemorrhoids had a significantly reduced collagen/protein ratio (42.2 ± 16.2μg/mg vs 72.5±31.0μg/mg; P= 0.02) and a significantly reduced collagen I/III ratio (2.0±0.1 vs 4.6±0.3; P<0.001) compared with persons without haemorrhoidal disease. There was no correlation with patients' age or gender.  There is a fundamental disorder of collagen metabolism in patients with haemorrhoidal disease. It remains unclear whether this is due to exogenous or endogenous influences. © 2010 The Authors. Colorectal Disease © 2010 The Association of Coloproctology of Great Britain and Ireland.

  15. Epidermolysis Bullosa Acquisita: Autoimmunity to Anchoring Fibril Collagen

    PubMed Central

    Chen, Mei; Kim, Gene H.; Prakash, Lori; Woodley, David T.

    2012-01-01

    Epidermolysis bullosa acquisita (EBA) is a rare and acquired autoimmune subepidermal bullous disease of the skin and mucosa. EBA includes various distinct clinical manifestations resembling Bullous Pemphigus, Brunsting-Perry pemphigoid, or cicatricial pemphigoid. These patients have autoantibodies against type VII collagen, an integral component of anchoring fibrils, which are responsible for attaching the dermis to the epidermis. Destruction or perturbation of the normally functioning anchoring fibrils clinically results in skin fragility, blisters, erosions, scars, milia and nail loss, all features reminiscent of genetic dystrophic epidermolysis bullosa. These anti-type VII collagen antibodies are “pathogenic” because when injected into a mouse, the mouse develops an EBA-like blistering disease. Currently treatment is often unsatisfactory, however some success has been achieved with colchichine, dapsone, photopheresis, plasmaphresis, infliximab, rituximab and IVIG. PMID:21955050

  16. Collagen vascular disease

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/001223.htm Collagen vascular disease To use the sharing features on ... were previously said to have "connective tissue" or "collagen vascular" disease. We now have names for many ...

  17. Fabrication of three-dimensional collagen scaffold using an inverse mould-leaching process.

    PubMed

    Ahn, SeungHyun; Lee, SuYeon; Cho, Youngseok; Chun, Wook; Kim, GeunHyung

    2011-09-01

    Natural biopolymers, such as collagen or chitosan, are considered ideal for biomedical scaffolds. However, low processability of the materials has hindered the fabrication of designed pore structures controlled by various solid freeform-fabrication methods. A new technique to fabricate a biomedical three-dimensional collagen scaffold, supplemented with a sacrificial poly(ethylene oxide) mould is proposed. The fabricated collagen scaffold shows a highly porous surface and a three-dimensional structure with high porosity as well as mechanically stable structure. To show its feasibility for biomedical applications, fibroblasts/keratinocytes were co-cultured on the scaffold, and the cell proliferation and cell migration of the scaffold was more favorable than that obtained with a spongy-type collagen scaffold.

  18. Analysis of Orientations of Collagen Fibers by Novel Fiber-Tracking Software

    NASA Astrophysics Data System (ADS)

    Wu, Jun; Rajwa, Bartlomiej; Filmer, David L.; Hoffmann, Christoph M.; Yuan, Bo; Chiang, Ching-Shoei; Sturgis, Jennie; Robinson, J. Paul

    2003-12-01

    Recent evidence supports the notion that biological functions of extracellular matrix (ECM) are highly correlated to not only its composition but also its structure. This article integrates confocal microscopy imaging and image-processing techniques to analyze the microstructural properties of ECM. This report describes a two- and three-dimensional fiber middle-line tracing algorithm that may be used to quantify collagen fibril organization. We utilized computer simulation and statistical analysis to validate the developed algorithm. These algorithms were applied to confocal images of collagen gels made with reconstituted bovine collagen type I, to demonstrate the computation of orientations of individual fibers.

  19. Oxidized Regenerated Cellulose/Collagen Dressings: Review of Evidence and Recommendations

    PubMed Central

    Wu, Stephanie; Applewhite, Andrew J.; Niezgoda, Jeffrey; Snyder, Robert; Shah, Jayesh; Cullen, Breda; Schultz, Gregory; Harrison, Janis; Hill, Rosemary; Howell, Melania; Speyrer, Marcus; Utra, Howard; de Leon, Jean; Lee, Wayne; Treadwell, Terry

    2017-01-01

    ABSTRACT OBJECTIVE: Healthcare systems are being challenged to manage increasing numbers of nonhealing wounds. Wound dressings are one of the first lines of defense in wound management, and numerous options exist. The oxidized regenerated cellulose (ORC)/collagen dressing may offer healthcare providers a robust and cost-effective tool for use in a variety of wounds. DESIGN: A multidisciplinary panel meeting was convened to discuss the use of ORC/collagen dressings in wound care and provide practice recommendations. A literature search was conducted to provide a brief review of the peer-reviewed studies published between January 2000 and March 2016 to inform the meeting. SETTING: A 2-day panel meeting convened in February 2017. PARTICIPANTS: Healthcare providers with experience using ORC/collagen dressings. This multidisciplinary panel of 15 experts in wound healing included podiatrists, wound care specialists (doctors, certified wound care nurses, and research scientists), and an orthopedist. RESULTS: The literature search identified 58 articles, a majority of which were low levels of evidence (69.3% were level 3 or lower). Panel members identified wound types, such as abrasions, burns, stalled wounds, diabetic foot ulcers, and pressure injuries, where ORC/collagen dressing use could be beneficial. Panel members then provided recommendations and technical pearls for the use of ORC/collagen dressings in practice. Barriers to ORC/collagen dressing use were discussed, and potential resolutions were offered. CONCLUSIONS: An ORC/collagen dressing can be a critical tool for clinicians to help manage a variety of wounds. Clinical and economic studies comparing standard-of-care dressings and plain collagen dressings to ORC/collagen dressings are needed. PMID:29049055

  20. The effect of oxygen partial pressure on protein synthesis and collagen hydroxylation by mature periodontal tissues maintained in organ cultures

    PubMed Central

    Yen, Edwin H. K.; Sodek, Jaro; Melcher, Antony H.

    1979-01-01

    Mature periodontal tissues from adult-mouse first mandibular molars were cultured in a continuous-flow organ-culture system which allowed the regulation of both ascorbic acid concentration and pO2 (oxygen partial pressure). Protein synthesis was measured by analysing the incorporation of [3H]proline into collagenous and non-collagenous proteins during the last 24h of a 2-day culture. At low pO2 [16.0kPa (approx. 120mmHg)] approx. 60% of protein-incorporated [3H]proline was found in collagenous proteins. However, it was evident that this collagen was considerably underhydroxylated. At high pO2 [56.0kPa (approx. 420mmHg)], both the amount of collagen deposited in the tissues and the degree of hydroxylation were increased considerably. In contrast, no significant effect on non-collagenous protein was observed. Tissues cultured at low pO2 for the first 48h were unable to respond to a subsequent increase in pO2 during the last 24h. Analysis of pepsin-solubilized collagen α-chains labelled with [14C]glycine demonstrated the synthesis of both type-I and type-III collagens by explants cultured for 48h at high pO2. Type-III collagen comprised 20–30% of the radioactivity in α-chains in both the periodontal ligament and the tissues of the alveolar process. The pattern of protein synthesis in the alveolar tissues at high pO2 was similar to that observed in these tissues in vivo. However, in the cultured periodontal ligament the proportions of non-collagenous proteins and type-III collagens were increased in comparison with the tissue in vivo. PMID:454369