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Sample records for human cartilage collagen

  1. Radiocarbon dating reveals minimal collagen turnover in both healthy and osteoarthritic human cartilage

    DEFF Research Database (Denmark)

    Heinemeier, Katja M; Schjerling, Peter; Heinemeier, Jan

    2016-01-01

    The poor regenerative capacity of articular cartilage presents a major clinical challenge and may relate to a limited turnover of the cartilage collagen matrix. However, the collagen turnover rate during life is not clear, and it is debated whether osteoarthritis (OA) can influence it. Using......, no replacement of the collagen matrix happened after skeletal maturity and that neither OA nor tissue damage, per se, influenced collagen turnover. Regional differences in (14)C content across the joint surface showed that cartilage collagen located centrally on the joint surface is formed several years earlier...

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

    Directory of Open Access Journals (Sweden)

    Riccardo Gottardi

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

  3. The type II collagen fragments Helix-II and CTX-II reveal different enzymatic pathways of human cartilage collagen degradation

    DEFF Research Database (Denmark)

    Charni-Ben Tabassi, N; Desmarais, S; Jensen, Anne-Christine Bay

    2008-01-01

    that they may be generated through different collagenolytic pathways. In this study we analyzed the release of Helix-II and CTX-II from human cartilage collagen by the proteinases reported to play a role in cartilage degradation. METHODS: In vitro, human articular cartilage extract was incubated with activated...... sections were then incubated for up to 84h in the presence or absence of E-64 and GM6001, inhibitors of cysteine proteases and MMPs, respectively. RESULTS: In vitro, Cats K, L and S generated large amount of Helix-II, but not CTX-II. Cat B generated CTX-II fragment, but destroyed Helix-II immunoreactivity...

  4. The minor collagens in articular cartilage

    DEFF Research Database (Denmark)

    Luo, Yunyun

    2017-01-01

    Articular cartilage is a connective tissue consisting of a specialized extracellular matrix (ECM) that dominates the bulk of its wet and dry weight. Type II collagen and aggrecan are the main ECM proteins in cartilage. However, little attention has been paid to less abundant molecular components......, especially minor collagens, including type IV, VI, IX, X, XI, XII, XIII, and XIV, etc. Although accounting for only a small fraction of the mature matrix, these minor collagens not only play essential structural roles in the mechanical properties, organization, and shape of articular cartilage, but also...... fulfil specific biological functions. Genetic studies of these minor collagens have revealed that they are associated with multiple connective tissue diseases, especially degenerative joint disease. The progressive destruction of cartilage involves the degradation of matrix constituents including...

  5. Biochemical markers of type II collagen breakdown and synthesis are positioned at specific sites in human osteoarthritic knee cartilage

    DEFF Research Database (Denmark)

    Jensen, Anne-Christine Bay; Levin Andersen, Thomas; Charni-Ben Tabassi, N

    2007-01-01

    sections were obtained from full-depth cartilage biopsies from 32 OA knees. Immunohistochemistry was performed for Helix-II and CTX-II, which are type II collagen fragments originating from the triple helix and the telopeptide region, respectively, and believed to reflect distinct breakdown events, as well......OBJECTIVE: To investigate whether type II collagen turnover markers used for osteoarthritis (OA) activity evaluation in body fluids can be detected at the level of specific histological features of OA cartilage tissue, as well as how they relate with each other at this level. METHODS: Adjacent...... as for type IIA N propeptide (PIIANP), a biochemical marker reflecting synthesis of type IIA collagen. RESULTS: Helix-II and CTX-II were detected in areas where collagen damage was reported previously, most frequently around chondrocytes, but also frequently in regions not previously investigated...

  6. Relationships Between Quantitative Pulse-Echo Ultrasound Parameters from the Superficial Zone of the Human Articular Cartilage and Changes in Surface Roughness, Collagen Content or Collagen Orientation Caused by Early Degeneration.

    Science.gov (United States)

    Kiyan, Wataru; Ito, Akira; Nakagawa, Yasuaki; Mukai, Shogo; Mori, Koji; Arai, Tatsuo; Uchino, Eiichiro; Okuno, Yasushi; Kuroki, Hiroshi

    2017-08-01

    We aimed to quantitatively investigate the relationship between amplitude-based pulse-echo ultrasound parameters and early degeneration of the knee articular cartilage. Twenty samples from six human femoral condyles judged as grade 0 or 1 according to International Cartilage Repair Society grading were assessed using a 15-MHz pulsed-ultrasound 3-D scanning system ex vivo. Surface roughness (R q ), average collagen content (A 1 ) and collagen orientation (A 12 ) in the superficial zone of the cartilage were measured via laser microscopy and Fourier transform infrared imaging spectroscopy. Multiple regression analysis with a linear mixed-effects model (LMM) revealed that a time-domain reflection coefficient at the cartilage surface (R c ) had a significant coefficient of determination with R q and A 12 (R LMMm 2 =0.79); however, R c did not correlate with A 1 . Concerning the collagen characteristic in the superficial zone, R c was found to be a sensitive indicator reflecting collagen disorganization, not collagen content, for the early degeneration samples. Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  7. Age-related accumulation of Maillard reaction products in human articular cartilage collagen

    NARCIS (Netherlands)

    Verzijl, N.; Degroot, J.; Oldehinkel, E.; Bank, R. A.; Thorpe, S. R.; Baynes, J. W.; Bayliss, M. T.; Bijlsma, J. W.; Lafeber, F. P.; TeKoppele, J. M.

    2000-01-01

    Non-enzymic modification of tissue proteins by reducing sugars, the so-called Maillard reaction, is a prominent feature of aging. In articular cartilage, relatively high levels of the advanced glycation end product (AGE) pentosidine accumulate with age. Higher pentosidine levels have been associated

  8. Deferoxamine Suppresses Collagen Cleavage and Protease, Cytokine, and COL10A1 Expression and Upregulates AMPK and Krebs Cycle Genes in Human Osteoarthritic Cartilage.

    Science.gov (United States)

    Tchetina, Elena V; Markova, Galina A; Poole, A Robin; Zukor, David J; Antoniou, John; Makarov, Sergey A; Kuzin, Aleksandr N

    2016-01-01

    This study reports the effects of the iron chelator deferoxamine (DFO) on collagen cleavage, inflammation, and chondrocyte hypertrophy in relation to energy metabolism-related gene expression in osteoarthritic (OA) articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with exogenous DFO (1-50 μM). Type II collagen cleavage and phospho-adenosine monophosphate-activated protein kinase (pAMPK) concentrations were measured using ELISAs. Gene expression studies employed real-time PCR and included AMPK analyses in PBMCs. In OA explants collagen cleavage was frequently downregulated by 10-50 μM DFO. PCR analysis of 7 OA patient cartilages revealed that 10 μM DFO suppressed expression of MMP-1, MMP-13, IL-1β, and TNFα and a marker of chondrocyte hypertrophy, COL10A1. No changes were observed in the expression of glycolysis-related genes. In contrast, expressions of genes associated with the mitochondrial Krebs cycle (TCA), AMPK, HIF1α, and COL2A1 were upregulated. AMPK gene expression was reduced in OA cartilage and increased in PBMCs from the same patients compared to healthy controls. Our studies demonstrate that DFO is capable of suppressing excessive collagenase-mediated type II collagen cleavage in OA cartilage and reversing phenotypic changes. The concomitant upregulation of proanabolic TCA-related gene expressions points to a potential for availability of energy generating substrates required for matrix repair by end-stage OA chondrocytes. This might normally be prevented by high whole-body energy requirements indicated by elevated AMPK expression in PBMCs of OA patients.

  9. Deferoxamine Suppresses Collagen Cleavage and Protease, Cytokine, and COL10A1 Expression and Upregulates AMPK and Krebs Cycle Genes in Human Osteoarthritic Cartilage

    Directory of Open Access Journals (Sweden)

    Elena V. Tchetina

    2016-01-01

    Full Text Available This study reports the effects of the iron chelator deferoxamine (DFO on collagen cleavage, inflammation, and chondrocyte hypertrophy in relation to energy metabolism-related gene expression in osteoarthritic (OA articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with exogenous DFO (1–50 μM. Type II collagen cleavage and phospho-adenosine monophosphate-activated protein kinase (pAMPK concentrations were measured using ELISAs. Gene expression studies employed real-time PCR and included AMPK analyses in PBMCs. In OA explants collagen cleavage was frequently downregulated by 10–50 μM DFO. PCR analysis of 7 OA patient cartilages revealed that 10 μM DFO suppressed expression of MMP-1, MMP-13, IL-1β, and TNFα and a marker of chondrocyte hypertrophy, COL10A1. No changes were observed in the expression of glycolysis-related genes. In contrast, expressions of genes associated with the mitochondrial Krebs cycle (TCA, AMPK, HIF1α, and COL2A1 were upregulated. AMPK gene expression was reduced in OA cartilage and increased in PBMCs from the same patients compared to healthy controls. Our studies demonstrate that DFO is capable of suppressing excessive collagenase-mediated type II collagen cleavage in OA cartilage and reversing phenotypic changes. The concomitant upregulation of proanabolic TCA-related gene expressions points to a potential for availability of energy generating substrates required for matrix repair by end-stage OA chondrocytes. This might normally be prevented by high whole-body energy requirements indicated by elevated AMPK expression in PBMCs of OA patients.

  10. Crosslinking by advanced glycation end products increases the stiffness of the collagen network in human articular cartilage: A possible mechanism through which age is a risk factor for osteoarthritis

    NARCIS (Netherlands)

    Verzijl, N.; Groot, J. de; Zaken, C.B.; Braun-Benjamin, O.; Maroudas, A.; Bank, R.A.; Mizrahi, J.; Schalkwijk, C.G.; Thorpe, S.R.; Baynes, J.W.; Bijlsma, J.W.J.; Lafeber, F.P.J.G.; TeKoppele, J.M.

    2002-01-01

    Objective. Age is an important risk factor for osteoarthritis (OA). During aging, nonenzymatic glycation results in the accumulation of advanced glycation end products (AGEs) in cartilage collagen. We studied the effect of AGE crosslinking on the stiffness of the collagen network in human articular

  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

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    Hosseininia Shahrzad

    2013-01-01

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

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

    Science.gov (United States)

    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

  13. Engineering Cartilage

    Science.gov (United States)

    ... Research Matters NIH Research Matters March 3, 2014 Engineering Cartilage Artistic rendering of human stem cells on ... situations has been a major goal in tissue engineering. Cartilage contains water, collagen, proteoglycans, and chondrocytes. Collagens ...

  14. A Novel Approach to Stimulate Cartilage Repair: Targeting Collagen Turnover

    NARCIS (Netherlands)

    Y.M. Bastiaansen-Jenniskens (Yvonne)

    2009-01-01

    textabstractOA is a complex disease of which the ethiopathology is not completely known and therapies to repair cartilage are still under investigation. The increase of collagen type II expression in osteoarthritic cartilage suggests an activated repair mechanism that is however ineffective in

  15. Combined role of type IX collagen and cartilage oligomeric matrix protein in cartilage matrix assembly: Cartilage oligomeric matrix protein counteracts type IX collagen-induced limitation of cartilage collagen fibril growth in mouse chondrocyte cultures

    NARCIS (Netherlands)

    Blumbach, K.; Bastiaansen-Jenniskens, Y.M.; Groot, J. de; Paulsson, M.; Osch, G.J.V.M. van; Zaucke, F.

    2009-01-01

    Objective. Defects in the assembly and composition of cartilage extracellular matrix are likely to result in impaired matrix integrity and increased susceptibility to cartilage degeneration. The aim of this study was to determine the functional interaction of the collagen fibril-associated proteins

  16. Induced collagen cross-links enhance cartilage integration.

    Directory of Open Access Journals (Sweden)

    Aristos A Athens

    Full Text Available Articular cartilage does not integrate due primarily to a scarcity of cross-links and viable cells at the interface. The objective of this study was to test the hypothesis that lysyl-oxidase, a metalloenzyme that forms collagen cross-links, would be effective in improving integration between native-to-native, as well as tissue engineered-to-native cartilage surfaces. To examine these hypotheses, engineered cartilage constructs, synthesized via the self-assembling process, as well as native cartilage, were implanted into native cartilage rings and treated with lysyl-oxidase for varying amounts of time. For both groups, lysyl-oxidase application resulted in greater apparent stiffness across the cartilage interface 2-2.2 times greater than control. The construct-to-native lysyl-oxidase group also exhibited a statistically significant increase in the apparent strength, here defined as the highest observed peak stress during tensile testing. Histology indicated a narrowing gap at the cartilage interface in lysyl-oxidase treated groups, though this alone is not sufficient to indicate annealing. However, when the morphological and mechanical data are taken together, the longer the duration of lysyl-oxidase treatment, the more integrated the interface appeared. Though further data are needed to confirm the mechanism of action, the enhancement of integration may be due to lysyl-oxidase-induced pyridinoline cross-links. This study demonstrates that lysyl-oxidase is a potent agent for enhancing integration between both native-to-native and native-to-engineered cartilages. The fact that interfacial strength increased manifold suggests that cross-linking agents should play a significant role in solving the difficult problem of cartilage integration. Future studies must examine dose, dosing regimen, and cellular responses to lysyl-oxidase to optimize its application.

  17. Contribution of collagen network features to functional properties of engineered cartilage

    NARCIS (Netherlands)

    Bastiaansen-Jenniskens, Y.M.; Koevoet, W.; Bart, A.C.W. de; Linden, J.C. van der; Zuurmond, A.M.; Weinans, H.; Verhaar, J.A.N.; Osch, G.J.V.M. van; Groot, J. de

    2008-01-01

    Background: Damage to articular cartilage is one of the features of osteoarthritis (OA). Cartilage damage is characterised by a net loss of collagen and proteoglycans. The collagen network is considered highly important for cartilage function but little is known about processes that control

  18. Cartilage turnover reflected by metabolic processing of type II collagen

    DEFF Research Database (Denmark)

    Gudmann, Karoline Natasja Stæhr; Wang, Jianxia; Hoielt, Sabine

    2014-01-01

    The aim of this study was to enable measurement of cartilage formation by a novel biomarker of type II collagen formation. The competitive enzyme-linked immunosorbent assay (ELISA) Pro-C2 was developed and characterized for assessment of the beta splice variant of type II procollagen (PIIBNP....... To our knowledge this is the first assay, which is able to specifically evaluate PIIBNP excretion. The Pro-C2 assay seems to provide a promising and novel marker of type II collagen formation....

  19. Quantification of collagen distributions in rat hyaline and fibro cartilages based on second harmonic generation imaging

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    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 wound repair following cartilage injury.

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

    NARCIS (Netherlands)

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

    2002-01-01

    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

  1. Collagen XII and XIV, New Partners of Cartilage Oligomeric Matrix Protein in the Skin Extracellular Matrix Suprastructure*

    Science.gov (United States)

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R.; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-01-01

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone. PMID:22573329

  2. Effect of epigallocatechin-3-gallate on the increase in type II collagen accumulation in cartilage-like MSC sheets.

    Science.gov (United States)

    Sato, Keigo; Mera, Hisashi; Wakitani, Shigeyuki; Takagi, Mutsumi

    2017-06-01

    With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10 μM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation.

  3. Human elastic cartilage engineering from cartilage progenitor cells using rotating wall vessel bioreactor.

    Science.gov (United States)

    Takebe, T; Kobayashi, S; Kan, H; Suzuki, H; Yabuki, Y; Mizuno, M; Adegawa, T; Yoshioka, T; Tanaka, J; Maegawa, J; Taniguchi, H

    2012-05-01

    Transplantation of bioengineered elastic cartilage is considered to be a promising approach for patients with craniofacial defects. We have previously shown that human ear perichondrium harbors a population of cartilage progenitor cells (CPCs). The aim of this study was to examine the use of a rotating wall vessel (RWV) bioreactor for CPCs to engineer 3-D elastic cartilage in vitro. Human CPCs isolated from ear perichondrium were expanded and differentiated into chondrocytes under 2-D culture conditions. Fully differentiated CPCs were seeded into recently developed pC-HAp/ChS (porous material consisted of collagen, hydroxyapatite, and chondroitinsulfate) scaffolds and 3-D cultivated utilizing a RWV bioreactor. 3-D engineered constructs appeared shiny with a yellowish, cartilage-like morphology. The shape of the molded scaffold was maintained after RWV cultivation. Hematoxylin and eosin staining showed engraftment of CPCs inside pC-HAp/ChS. Alcian blue and Elastica Van Gieson staining showed of proteoglycan and elastic fibers, which are unique extracellular matrices of elastic cartilage. Thus, human CPCs formed elastic cartilage-like tissue after 3-D cultivation in a RWV bioreactor. These techniques may assist future efforts to reconstruct complicate structures composed of elastic cartilage in vitro. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. An immunohistochemical study of matrix proteins in the craniofacial cartilage in midterm human fetuses

    Directory of Open Access Journals (Sweden)

    S. Shibata

    2013-12-01

    Full Text Available Immunohistochemical localization of collagen types I, II, and X, aggrecan, versican, dentin matrix protein (DMP-1, martix extracellular phosphoprotein (MEPE were performed for Meckel’s cartilage, cranial base cartilage, and mandibular condylar cartilage in human midterm fetuses; staining patterns within the condylar cartilage were compared to those within other cartilaginous structures. Mandibular condylar cartilage contained aggrecan; it also had more type I collagen and a thicker hypertrophic cell layer than the other two types of cartilage; these three characteristics are similar to those of the secondary cartilage of rodents. MEPE immunoreactivity was first evident in the cartilage matrix of all types of cartilage in the human fetuses and in Meckel’s cartilage of mice and rats. MEPE immunoreactivity was enhanced in the deep layer of the hypertrophic cell layer and in the cartilaginous core of the bone trabeculae in the primary spongiosa. These results indicated that MEPE is a component of cartilage matrix and may be involved in cartilage mineralization. DMP-1 immunoreactivity first became evident in human bone lacunae walls and canaliculi; this pattern of expression was comparable to the pattern seen in rodents. In addition, chondroid bone was evident in the mandibular (glenoid fossa of the temporal bone, and it had aggrecan, collagen types I and X, MEPE, and DMP-1 immunoreactivity; these findings indicated that chondroid bone in this region has phenotypic expression indicative of both hypertrophic chondrocytes and osteocytes.

  5. The increased swelling and instantaneous deformation of osteoarthritic cartilage is highly correlated with collagen degradation.

    Science.gov (United States)

    Bank, R A; Soudry, M; Maroudas, A; Mizrahi, J; TeKoppele, J M

    2000-10-01

    To provide evidence for the hypothesis that the loss of tensile strength of osteoarthritic (OA) cartilage (resulting in swelling-the hallmark of OA) is due to an impaired collagen network and not to loss or degradation of proteoglycans. The amount of degraded collagen molecules, the fixed charge density (FCD) on a dry-weight basis, the degree of swelling in saline, and the instantaneous deformation (ID; a test reflecting the tensile stiffness of the collagen network) were measured in full-depth OA femoral condyle samples. In addition, levels of the crosslink hydroxylysylpyridinoline (HP), the amount of degraded collagen molecules, and the degree of swelling were determined in the 3 zones (surface, middle, and deep) of OA cartilage. We also compared the ID of normal and OA cartilage. In full-depth OA cartilage, a close relationship was found between swelling and ID. Swelling and ID correlated strongly with the amount of degraded collagen molecules, and were not related to FCD. OA cartilage showed the same zonal pattern in HP levels as normal cartilage (i.e., an increase with depth). No relationship was found between collagen crosslinking and swelling of the surface, middle, and deep zones. In all 3 zones, swelling was proportional to the amount of degraded collagen molecules. Compared with that of normal cartilage, the change in ID of OA cartilage was most pronounced at the surface in a direction parallel to the direction of the collagen fibrils. The decreased stiffness of the OA collagen network (as measured by swelling and ID) is strongly related to the amount of degraded collagen molecules. The anisotropy in ID parallel and perpendicular to the direction of the fibrils revealed that the impairment of strength resides mainly in, and not between, the fibrils. Proteoglycans play only a minor role in the degeneration of the tensile stiffness of OA cartilage.

  6. A CARTILAGE GROWTH MIXTURE MODEL WITH COLLAGEN REMODELING: VALIDATION PROTOCOLS

    Science.gov (United States)

    Klisch, Stephen M.; Asanbaeva, Anna; Oungoulian, Sevan R.; Masuda, Koichi; Thonar, Eugene J-MA; Davol, Andrew; Sah, Robert L.

    2009-01-01

    A cartilage growth mixture (CGM) model is proposed to address limitations of a model used in a previous study. New stress constitutive equations for the solid matrix are derived and collagen (COL) remodeling is incorporated into the CGM model by allowing the intrinsic COL material constants to evolve during growth. An analytical validation protocol based on experimental data from a recent in vitro growth study is developed. Available data included measurements of tissue volume, biochemical composition, and tensile modulus for bovine calf articular cartilage (AC) explants harvested at three depths and incubated for 13 days in 20% FBS and 20% FBS+β-aminopropionitrile. The proposed CGM model can match tissue biochemical content and volume exactly while predicting theoretical values of tensile moduli that do not significantly differ from experimental values. Also, theoretical values of a scalar COL remodeling factor are positively correlated with COL crosslink content, and mass growth functions are positively correlated with cell density. The results suggest that the CGM model may help to guide in vitro growth protocols for AC tissue via the a priori prediction of geometric and biomechanical properties. PMID:18532855

  7. Type I collagen-based fibrous capsule enhances integration of tissue-engineered cartilage with native articular cartilage.

    Science.gov (United States)

    Yang, Yueh-Hsun; Ard, Mary B; Halper, Jaroslava T; Barabino, Gilda A

    2014-04-01

    Successful integration of engineered constructs with host tissues is crucial for cartilage repair, yet achieving it remains challenging. A collagen I-based fibrous capsule characterized by increased cell density and decreased glycosaminoglycan deposition usually forms at the periphery of tissue-engineered cartilage. The current study aimed to evaluate the effects of a solid fibrous capsule on construct integration with native articular cartilage. To this end, capsule-containing (CC) and capsule-free (CF) constructs were grown by culturing chondrocyte-seeded scaffolds with insulin-like growth factor-1 and transforming growth factor-β1, respectively, in a wavy-walled bioreactor that imparts hydrodynamic forces for 4 weeks. The ability of harvested constructs to integrate with native cartilage was determined using a cartilage explant model. Our results revealed that adhesive stress between native cartilage and the CC constructs was 57% higher than that in the CF group, potentially due to the absence of glycosaminoglycans and increased cell density in the capsule region and deposition of denser and thicker collagen fibrils at the integration site. The present work demonstrates that the fibrous capsule can effectively enhance early integration of engineered and native cartilage tissues and thus suggests the need to include the capsule as a variable in the development of cartilage tissue engineering strategies.

  8. Collagen/silk fibroin composite scaffold incorporated with PLGA microsphere for cartilage repair

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jianhua; Yang, Qiu; Cheng, Niangmei [Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002 (China); Tao, Xiaojun [Department of Pharmacy, School of Medicine, Hunan Normal University, Changsha, 410013, Hunan (China); Zhang, Zhihua; Sun, Xiaomin [Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002 (China); Zhang, Qiqing, E-mail: zhangqiq@126.com [Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002 (China); Key Laboratory of Biomedical Materials of Tianjin, Institute of Biomedical Engineering, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin 300192 (China)

    2016-04-01

    For cartilage repair, ideal scaffolds should mimic natural extracellular matrix (ECM) exhibiting excellent characteristics, such as biocompatibility, suitable porosity, and good cell affinity. This study aimed to prepare a collagen/silk fibroin composite scaffold incorporated with poly-lactic-co-glycolic acid (PLGA) microsphere that can be applied in repairing cartilage. To obtain optimum conditions for manufacturing a composite scaffold, a scaffold composed of different collagen-to-silk fibroin ratios was evaluated by determining porosity, water absorption, loss rate in hot water, and cell proliferation. Results suggested that the optimal ratio of collagen and silk fibroin composite scaffold was 7:3. The microstructure and morphological characteristics of the obtained scaffold were also examined through scanning electron microscopy and Fourier transform infrared spectroscopy. The results of in vitro fluorescence staining of bone marrow stromal cells revealed that collagen/silk fibroin composite scaffold enhanced cell proliferation without eliciting side effects. The prepared composite scaffold incorporated with PLGA microsphere was implanted in fully thick articular cartilage defects in rabbits. Collagen/silk fibroin composite scaffold with PLGA microspheres could enhance articular cartilage regeneration and integration between the repaired cartilage and the surrounding cartilage. Therefore, this composite will be a promising material for cartilage repair and regeneration. - Highlights: • Collagen/silk fibroin composite scaffold incorporated with PLGA microsphere proposed for cartilage repair was created. • In vivo, scaffold could enhance cartilage regeneration and integration between the repaired and surrounding cartilage. • In vitro, scaffold exhibits excellent characteristics, such as, improved porosity water absorption and good cell affinity.

  9. Caprine articular, meniscus and intervertebral disc cartilage : An integral analysis of collagen network and chondrocytes

    NARCIS (Netherlands)

    Vonk, Lucienne A.; Kroeze, Robert Jan; Doulabi, Behrouz Zandieh; Hoogendoorn, Roel J.; Huang, ChunLing; Helder, Marco N.; Everts, Vincent; Bank, Ruud A.

    Cartilage is a tissue with only limited reparative capacities. A small part of its volume is composed of cells, the remaining part being the hydrated extracellular matrix (ECM) with collagens and proteoglycans as its main constituents. The functioning of cartilage depends heavily on its ECM.

  10. Caprine articular, meniscus and intervertebral disc cartilage: an integral analysis of collagen network and chondrocytes

    NARCIS (Netherlands)

    Vonk, L.A.; Kroeze, R.J.; Doulabi, B.Z.; Hoogendoorn, R.J.; Huang, C.; Helder, M.N.; Everts, V.; Bank, R.A.

    2010-01-01

    Cartilage is a tissue with only limited reparative capacities. A small part of its volume is composed of cells, the remaining part being the hydrated extracellular matrix (ECM) with collagens and proteoglycans as its main constituents. The functioning of cartilage depends heavily on its ECM.

  11. Similar hyaline-like cartilage repair of osteochondral defects in rabbits using isotropic and anisotropic collagen scaffolds

    NARCIS (Netherlands)

    Mulder, E.L.W. de; Hannink, G.J.; Kuppevelt, T.H. van; Daamen, W.F.; Buma, P.

    2014-01-01

    Lesions in knee joint articular cartilage (AC) have limited repair capacity. Many clinically available treatments induce a fibrous-like cartilage repair instead of hyaline cartilage. To induce hyaline cartilage repair, we hypothesized that type I collagen scaffolds with fibers aligned perpendicular

  12. Proteolytic degradation of the collagen network results in cartilage with inferior biomechanical properties

    NARCIS (Netherlands)

    Bank, R.A.; Koppele, J.M. te

    1999-01-01

    Swelling of cartilage, one of the early signs of osteoarthritis (OA), is considered to be the result of a collagen network that has lost its integrity. So far, no quantitative data directly support this assertion: combined measurements of the state of the collagen network per se and the degree of

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

    Science.gov (United States)

    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.

  14. Prostaglandin E2 role in inhibition of joint cartilage collagen destruction in patients with osteoarthritis

    Directory of Open Access Journals (Sweden)

    E V Chetina

    2009-01-01

    Full Text Available Prostaglandin E2 role in inhibition of articular cartilage collagen degradation in patients with osteoarthritis. Objective. To assess prostaglandin E2 (PGE2 role in inhibition of type II collagen digestion in explants of articular cartilage of pts with osteoarthritis (OA. Material and methods. Explants of articular cartilage of pts with OA were cultured with PGE2 1pg to 10 ng/ml. Type II collagen digestion was assessed with immuno-enzyme assay. Gene expression was evaluated with PCR in real time. Results. PGE2 10 pg/ml as well as transforming growth factor β2 (TGFβ2 suppressed type II collagen digestion in explants of articular cartilage of pts with OA. This concentration of PGE2 did not suppress proteoglycan (aggrecan degradation. Gene expression analysis in 5 OA pts showed that PGE2 10 pg/ml suppressed metallomonooxigenase (MMP-13, MMP-1 and marker of chondrocyte hypertrophy type X collagen (COL10A1 as well as proinflammatory cytokines interleukine (IL-1β and tumor necrosis factor (TNFα. Naproxen, nonselective cyclooxygenase(COX-2 and 1 inhibitor concentration from 5 to 30 mcg/ml blocked TGFβ2 induced collagen digestion inhibition proving that PGE2 mediate influence of this growth factor. Naproxen concentration 5 mcg/ml increased collagen degradation. Conclusion. The study showed that PGE2 is a chondroprotector because it is able to suppress selectively OA pts cartilage collagen degradation. Beside that cartilage chondrocyte hypertrophy in OA connected functionally with increased collagen digestion is also regulated by low concentrations of PGE2

  15. Alteration of cartilage surface collagen fibers differs locally after immobilization of knee joints in rats

    Science.gov (United States)

    Nagai, Momoko; Aoyama, Tomoki; Ito, Akira; Tajino, Junichi; Iijima, Hirotaka; Yamaguchi, Shoki; Zhang, Xiangkai; Kuroki, Hiroshi

    2015-01-01

    The purpose of this study was to examine the ultrastructural changes of surface cartilage collagen fibers, which differ by region and the length of the experimental period in an immobilization model of rat. Male Wistar rats were randomly divided into histological or macroscopic and ultrastructural assessment groups. The left knees of all the animals were surgically immobilized by external fixation for 1, 2, 4, 8 or 16 weeks (n = 5/time point). Sagittal histological sections of the medial mid-condylar region of the knee were obtained and assessed in four specific regions (contact and peripheral regions of the femur and tibia) and two zones (superficial and deep). To semi-quantify the staining intensity of the collagen fibers in the cartilage, picrosirius red staining was used. The cartilage surface changes of all the assessed regions were investigated by scanning electron microscopy (SEM). From histological and SEM observations, the fibrillation and irregular changes of the cartilage surface were more severe in the peripheral region than in the contact region. Interestingly, at 16 weeks post-immobilization, we observed non-fibrous structures at both the contact and peripheral regions. The collagen fiber staining intensity decreased in the contact region compared with the peripheral region. In conclusion, the alteration of surface collagen fiber ultrastructure and collagen staining intensity differed by the specific cartilage regions after immobilization. These results demonstrate that the progressive degeneration of cartilage is region specific, and depends on the length of the immobilization period. PMID:25939458

  16. Chondroinduction Is the Main Cartilage Repair Response to Microfracture and Microfracture With BST-CarGel: Results as Shown by ICRS-II Histological Scoring and a Novel Zonal Collagen Type Scoring Method of Human Clinical Biopsy Specimens.

    Science.gov (United States)

    Hoemann, Caroline D; Tran-Khanh, Nicolas; Chevrier, Anik; Chen, Gaoping; Lascau-Coman, Viorica; Mathieu, Colleen; Changoor, Adele; Yaroshinsky, Alex; McCormack, Robert G; Stanish, William D; Buschmann, Michael D

    2015-10-01

    Current cartilage repair histological scoring systems are unable to explain the relationship between collagen type II deposition and overall repair quality. The purpose of this study was to develop a novel zonal collagen type (ZCT) 5-point scoring system to measure chondroinduction in human clinical biopsy specimens collected after marrow stimulation. The hypothesis was that the ZCT scores would correlate with the International Cartilage Repair Society-II (ICRS-II) overall histological repair assessment score and glycosaminoglycan (GAG) content. Descriptive laboratory study. After optimizing safranin O staining for GAG and immunostaining for human collagen type II and type I (Col2 and Col1, respectively), serial sections from clinical osteochondral repair biopsy specimens (13 months after microfracture or microfracture with BST-CarGel; n = 39 patients) were stained and 3 blinded readers performed histomorphometry for percentage of staining, ICRS-II histological scoring, polarized light microscopy (PLM) scoring, and 5-point ZCT scoring based on tidemark morphology, zonal distribution of Col2 and Col1, and Col1 percentage stain. Because 1 biopsy specimen was missing bone, 38 biopsy specimens were evaluated for ICRS-II, PLM, and ZCT scores. Chondroinduction was identified in 21 biopsy specimens as a Col2 matrix fused to bone that spanned the deep-middle-superficial zones ("full-thickness hyaline repair"), deep-middle zones, or deep zone ("stalled hyaline") that was covered with a variable-thickness Col1-positive matrix, and was scored, respectively, as ZCT = 1 (n = 4 biopsy specimens), ZCT = 2 (n = 6) and ZCT = 3 (n = 11). Other biopsy specimens (n = 17) were fibrocartilage (n = 9; ZCT = 4), fibrous tissue (n = 4, ZCT = 5), or non-marrow derived (n = 4; ZCT = 0). Non-marrow derived tissue had a mean mature tidemark score of 84 out of 100 versus a regenerating tidemark score of 24 for all other biopsy specimens (P = .005). Both "stalled hyaline" repair and

  17. 1991 Volvo Award in basic sciences. Collagen types around the cells of the intervertebral disc and cartilage end plate: an immunolocalization study.

    Science.gov (United States)

    Roberts, S; Menage, J; Duance, V; Wotton, S; Ayad, S

    1991-09-01

    Several types of collagen are known to exist in the intervertebral disc in addition to the fibrillar collagens, Types I and II. Although they constitute only a small percentage of the total collagen content, these minor collagens may have important functions. This study was designed to investigate the presence of Types I, II, III, IV, VI, and IX collagens in the intervertebral disc and cartilage end plate by immunohistochemistry, thereby establishing their location within the tissues. Types III and VI collagen have a pericellular distribution in animal and human tissue. No staining for Type IX collagen was present in normal human disc, but in rat and bovine intervertebral disc, it was also located pericellularly. These results show that cells of the intervertebral disc and cartilage end plate sit in fibrous capsules, forming chondrons similar to those described in articular cartilage. In pathologic tissue the amount and distribution of the collagen types, and the organization of the pericellular capsule, differ from that seen in control material.

  18. Collagens and collagen-related matrix components in the human and mouse eye.

    Science.gov (United States)

    Ihanamäki, Tapio; Pelliniemi, Lauri J; Vuorio, Eero

    2004-07-01

    The three-dimensional structure of the eye plays an important role in providing a correct optical environment for vision. Much of this function is dependent on the unique structural features of ocular connective tissue, especially of the collagen types and their supramolecular structures. For example, the organization of collagen fibrils is largely responsible for transparency and refraction of cornea, lens and vitreous body, and collagens present in the sclera are largely responsible for the structural strength of the eye. Phylogenetically, most of the collagens are highly conserved between different species, which suggests that collagens also share similar functions in mice and men. Despite considerable differences between the mouse and the human eye, particularly in the proportion of the different tissue components, the difficulty of performing systematic histologic and molecular studies on the human eye has made mouse an appealing alternative to studies addressing the role of individual genes and their mutations in ocular diseases. From a genetic standpoint, the mouse has major advantages over other experimental animals as its genome is better known than that of other species and it can be manipulated by the modern techniques of genetic engineering. Furthermore, it is easy, quick and relatively cheap to produce large quantities of mice for systematic studies. Thus, transgenic techniques have made it possible to study consequences of specific mutations in genes coding for structural components of ocular connective tissues in mice. As these changes in mice have been shown to resemble those in human diseases, mouse models are likely to provide efficient tools for pathogenetic studies on human disorders affecting the extracellular matrix. This review is aimed to clarify the role of collagenous components in the mouse and human eye with a closer look at the new findings of the collagens in the cartilage and the eye, the so-called "cartilage collagens".

  19. Influence of site and age on biochemical characteristics of the collagen network of equine articular cartilage.

    Science.gov (United States)

    Brama, P A; TeKoppele, J M; Bank, R A; van Weeren, P R; Barneveld, A

    1999-03-01

    To determine variations in biochemical characteristics of equine articular cartilage in relation to age and the degree of predisposition for osteochondral disease at a specific site. Articular cartilage specimens from 53 horses 4 to 30 years old. Healthy specimens were obtained from 2 locations on the proximal articular surface of the first phalanx that had different disease prevalences (site 1 at the mediodorsal margin and site 2 at the center of the medial cavity). Water, total collagen, and hydroxylysine contents and enzymatic (hydroxylysylpyridinoline [HP]) and nonenzymatic (pentosidine) crosslinking were determined at both sites. Differences between sites were analyzed by ANOVA (factors, site, and age), and age correlation was tested by Pearson's product-moment correlation analysis. Significance was set at Pcollagen, hydroxylysine contents, and enzymatic cross-linking. Nonenzymatic crosslinking was higher in older horses and was linearly related to age (r = 0.94). Water and collagen contents and HP and pentosidine crosslinks were significantly higher at site 1. Hydroxylysine content was significantly lower at site 1. Except for nonenzymatic glycation, the composition of articular cartilage collagen does not change significantly in adult horses. A significant topographic variation exists in biochemical characteristics of the articular cartilage collagen network in equine metacarpophalangeal joints. These differences may influence local biomechanical properties and, hence, susceptibility to osteochondral disease, as will greater pentosidine crosslinks in older horses that are likely to cause stiffer and more brittle cartilage.

  20. Helium ion microscopy for high-resolution visualization of the articular cartilage collagen network.

    Science.gov (United States)

    Vanden Berg-Foels, W S; Scipioni, L; Huynh, C; Wen, X

    2012-05-01

    The articular cartilage collagen network is an important research focus because network disruption results in cartilage degeneration and patient disability. The recently introduced helium ion microscope (HIM), with its smaller probe size, longer depth of field and charge neutralization, has the potential to overcome the inherent limitations of electron microscopy for visualization of collagen network features, particularly at the nanoscale. In this study, we evaluated the capabilities of the helium ion microscope for high-resolution visualization of the articular cartilage collagen network. Images of rabbit knee cartilage were acquired with a helium ion microscope; comparison images were acquired with a field emission scanning electron microscope (FE-SEM) and a transmission electron microscope (TEM). Sharpness of example high-resolution helium ion microscope and field emission scanning electron microscope images was quantified using the 25-75% rise distance metric. The helium ion microscope was able to acquire high-resolution images with unprecedented clarity, with greater sharpness and three-dimensional-like detail of nanoscale fibril morphologies and fibril connections, in samples without conductive coatings. These nanoscale features could not be resolved by field emission scanning electron microscopy, and three-dimensional network structure could not be visualized with transmission electron microscopy. The nanoscale three-dimensional-like visualization capabilities of the helium ion microscope will enable new avenues of investigation in cartilage collagen network research. © 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.

  1. The effect of collagen degradation on chondrocyte volume and morphology in bovine articular cartilage following a hypotonic challenge.

    Science.gov (United States)

    Turunen, S M; Lammi, M J; Saarakkala, S; Han, S-K; Herzog, W; Tanska, P; Korhonen, R K

    2013-06-01

    Collagen degradation is one of the early signs of osteoarthritis. It is not known how collagen degradation affects chondrocyte volume and morphology. Thus, the aim of this study was to investigate the effect of enzymatically induced collagen degradation on cell volume and shape changes in articular cartilage after a hypotonic challenge. Confocal laser scanning microscopy was used for imaging superficial zone chondrocytes in intact and degraded cartilage exposed to a hypotonic challenge. Fourier transform infrared microspectroscopy, polarized light microscopy, and mechanical testing were used to quantify differences in proteoglycan and collagen content, collagen orientation, and biomechanical properties, respectively, between the intact and degraded cartilage. Collagen content decreased and collagen orientation angle increased significantly (p < 0.05) in the superficial zone cartilage after collagenase treatment, and the instantaneous modulus of the samples was reduced significantly (p < 0.05). Normalized cell volume and height 20 min after the osmotic challenge (with respect to the original volume and height) were significantly (p < 0.001 and p < 0.01, respectively) larger in the intact compared to the degraded cartilage. These findings suggest that the mechanical environment of chondrocytes, specifically collagen content and orientation, affects cell volume and shape changes in the superficial zone articular cartilage when exposed to osmotic loading. This emphasizes the role of collagen in modulating cartilage mechanobiology in diseased tissue.

  2. Human acellular cartilage matrix powders as a biological scaffold for cartilage tissue engineering with synovium-derived mesenchymal stem cells.

    Science.gov (United States)

    Chang, Chih-Hung; Chen, Chia-Chun; Liao, Cheng-Hao; Lin, Feng-Huei; Hsu, Yuan-Ming; Fang, Hsu-Wei

    2014-07-01

    In our previous study, we found that cartilage fragments from osteoarthritic knee promoted chondrogenesis of mesenchymal stem cells. In this study, we further transformed the cartilage tissues into acellular cartilage matrix (ACM) and explored the feasibility of using ACM as a biological scaffold. Nonworn parts of cartilage tissues were obtained during total knee arthroplasty (TKA) surgery and were successfully fabricated into ACM powders. The ACM powders and human synovium-derived mesenchymal stem cells (SMSCs) were mixed into collagen gel for in vitro culture. Histological results showed a synergistic effect of ACM powders and chondrogenic growth factors in the formation of engineered cartilage. The findings of real-time polymerase chain reaction (PCR) suggested that ACM powders had the potential of promoting type II collagen gene expression in the growth factors-absent environment. Moreover, with growth factors induction, the ACM powders could reduce the hypertrophy in chondrogenesis of SMSCs. In summary, ACM powders could serve as a functional scaffold that benefited the chondrogenesis of SMSCs for cartilage tissue engineering. © 2013 Wiley Periodicals, Inc.

  3. Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy.

    Science.gov (United States)

    Fields, Mark; Spencer, Nicholas; Dudhia, Jayesh; McMillan, Paul F

    2017-06-01

    Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple-helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ∼60-80°C for hydrated samples. We report high temperature Raman spectra for freeze-dried cartilage samples that show an increase in laser-excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H2 O is retained within the freeze-dried tissue. This is confirmed by thermogravimetric analysis that show 5-7 wt% H2 O remaining within freeze-dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re-hydration following recovery indicate that the capacity of the denatured collagen to re-absorb water is reduced. Our results are important for revealing the presence of bound H2 O within the collagen component of connective tissue even after freeze-drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure. © 2017 Wiley Periodicals, Inc.

  4. Formation of Hyaline Cartilage Tissue by Passaged Human Osteoarthritic Chondrocytes.

    Science.gov (United States)

    Bianchi, Vanessa J; Weber, Joanna F; Waldman, Stephen D; Backstein, David; Kandel, Rita A

    2017-02-01

    When serially passaged in standard monolayer culture to expand cell number, articular chondrocytes lose their phenotype. This results in the formation of fibrocartilage when they are used clinically, thus limiting their use for cartilage repair therapies. Identifying a way to redifferentiate these cells in vitro is critical if they are to be used successfully. Transforming growth factor beta (TGFβ) family members are known to be crucial for regulating differentiation of fetal limb mesenchymal cells and mesenchymal stromal cells to chondrocytes. As passaged chondrocytes acquire a progenitor-like phenotype, the hypothesis of this study was that TGFβ supplementation will stimulate chondrocyte redifferentiation in vitro in serum-free three-dimensional (3D) culture. Human articular chondrocytes were serially passaged twice (P2) in monolayer culture. P2 cells were then placed in high-density (3D) culture on top of membranes (Millipore) and cultured for up to 6 weeks in chemically defined serum-free redifferentiation media (SFRM) in the presence or absence of TGFβ. The tissues were evaluated histologically, biochemically, by immunohistochemical staining, and biomechanically. Passaged human chondrocytes cultured in SFRM supplemented with 10 ng/mL TGFβ3 consistently formed a continuous layer of articular-like cartilage tissue rich in collagen type 2 and aggrecan and lacking collagen type 1 and X in the absence of a scaffold. The tissue developed a superficial zone characterized by expression of lubricin and clusterin with horizontally aligned collagen fibers. This study suggests that passaged human chondrocytes can be used to bioengineer a continuous layer of articular cartilage-like tissue in vitro scaffold free. Further study is required to evaluate their ability to repair cartilage defects in vivo.

  5. Tissue engineering of human cartilage and osteochondral composites using recirculation bioreactors.

    Science.gov (United States)

    Mahmoudifar, Nastaran; Doran, Pauline M

    2005-12-01

    Chondrocytes isolated from human foetal epiphyseal cartilage were seeded dynamically into polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to produce tissue-engineered cartilage. Several culture techniques with the potential to provide endogenous growth factors and other conditions beneficial for de novo cartilage synthesis were investigated. Osteochondral composite constructs were generated by seeding separate PGA scaffolds with either foetal chondrocytes or foetal osteoblasts then suturing the scaffolds together before bioreactor cultivation. This type of co-culture system provided direct contact between the tissue-engineered cartilage and developing tissue-engineered bone and yielded significant improvements in cartilage quality. In the cartilage section of the composites, the concentrations of glycosaminoglycan (GAG) and total collagen were increased by 55% and 2.5-fold, respectively, compared with control cartilage cultures, while levels of collagen type II were similar to those in the controls. The osteochondral composites were harvested from the bioreactors as single units with good integration between the cartilage and bone tissues. Only the cartilage layer contained GAG while only the bone layer was mineralised. In other experiments, co-culture of tissue-engineered cartilage with pieces of ex-vivo cartilage or ex-vivo bone did not improve the quality of the cartilage relative to control cultures. Addition of 10(-6) M diacerein to the culture medium also had no effect on the properties of engineered cartilage. This work demonstrates the beneficial effects of generating cartilage tissues in contact with developing bone. It also demonstrates the feasibility of producing composite osteochondral constructs for clinical application using recirculation column bioreactors.

  6. Site-specific immunostaining for type X collagen in noncalcified articular cartilage of canine stifle knee joint.

    Science.gov (United States)

    Lammi, P E; Lammi, M J; Hyttinen, M M; Panula, H; Kiviranta, I; Helminen, H J

    2002-12-01

    Type X collagen is a short-chain collagen that is strongly expressed in hypertrophic chondrocytes. In this study, we used an immunohistochemical technique exploiting a prolonged hyaluronidase unmasking of type X collagen epitopes to show that type X collagen is not restricted to calcified cartilage, but is also present in normal canine noncalcified articular cartilage. A 30 degrees valgus angulation procedure of the right tibia was performed in 15 dogs at the age of 3 months, whereas their nonoperated sister dogs served as controls. Samples were collected 7 and 18 months after the surgery and immunostained for type X collagen. The deposition of type X collagen increased during maturation from age 43 weeks to 91 weeks. In the patella, most of the noncalcified cartilage stained for type X collagen, whereas, in the patellar surface of the femur, it was present mainly in the femoral groove close to cartilage surface. In femoral condyles, the staining localized mostly in the superficial cartilage on the lateral and medial sides, but not in the central weight-bearing area. In tibial condyles, type X collagen was often observed close to the cartilage surface in medial parts of the condyles, although staining could also be seen in the deep zone of the cartilage. Staining for type X collagen appeared strongest at sites where the birefringence of polarized light was lowest, suggesting a colocalization of type X collagen with the collagen fibril arcades in the intermediate zone. No significant difference in type X collagen immunostaining was observed in lesion-free articular cartilage between controls and dogs that underwent a 30 degrees valgus osteotomy. In osteoarthritic lesions, however, there was strong immunostaining for both type X collagen and collagenase-induced collagen cleavage products. The presence of type X collagen in the transitional zone of cartilage in the patella, femoropatellar groove, and in tibial cartilage uncovered by menisci suggests that it may

  7. Polysulfated glycosaminoglycan accelerates net synthesis of collagen and glycosaminoglycans by arthritic equine cartilage tissues and chondrocytes.

    Science.gov (United States)

    Glade, M J

    1990-05-01

    Low molecular weight polysulfated glycosaminoglycan (PSGAG) stimulated net collagen and glycosaminoglycan synthesis by normal and arthritic equine fetlock cartilage tissues in organ culture. Arthritic tissues were more sensitive to PSGAG stimulation. The rates of cartilage-specific type-II collagen and chondroitin sulfate-rich glycosaminoglycan synthesis by confluent chondrocyte cell cultures obtained from normal and arthritic equine cartilage tissues were increased by 25 and 50 mg of PSGAG/ml. Cells from arthritic cartilage were also more sensitive to the presence of PSGAG. In addition, concentrations of PSGAG (25 and 50 mg/ml) approximate to those in synovial fluid after intra-articular injection of 250 mg of PSGAG inhibited the rate of collagen and glycosaminoglycan degradation in cell culture. These findings suggest that PSGAG may have a role in the healing of mild cartilage degeneration by encouraging the production of replacement hyaline matrix materials, while delaying their subsequent degradation. In contrast, growth of cell cultures was inhibited by PSGAG, suggesting that these compounds may fail to stimulate chondrocyte replication, a prerequisite for tissue regeneration. Nonetheless, these observations provide direct evidence of a truly chondroprotective role for low molecular weight PSGAG in the treatment of equine degenerative joint disease.

  8. Analysis of human collagen sequences.

    Science.gov (United States)

    Nassa, Manisha; Anand, Pracheta; Jain, Aditi; Chhabra, Aastha; Jaiswal, Astha; Malhotra, Umang; Rani, Vibha

    2012-01-01

    The extracellular matrix is fast emerging as important component mediating cell-cell interactions, along with its established role as a scaffold for cell support. Collagen, being the principal component of extracellular matrix, has been implicated in a number of pathological conditions. However, collagens are complex protein structures belonging to a large family consisting of 28 members in humans; hence, there exists a lack of in depth information about their structural features. Annotating and appreciating the functions of these proteins is possible with the help of the numerous biocomputational tools that are currently available. This study reports a comparative analysis and characterization of the alpha-1 chain of human collagen sequences. Physico-chemical, secondary structural, functional and phylogenetic classification was carried out, based on which, collagens 12, 14 and 20, which belong to the FACIT collagen family, have been identified as potential players in diseased conditions, owing to certain atypical properties such as very high aliphatic index, low percentage of glycine and proline residues and their proximity in evolutionary history. These collagen molecules might be important candidates to be investigated further for their role in skeletal disorders.

  9. Collagen-mimetic peptide-modifiable hydrogels for articular cartilage regeneration

    Science.gov (United States)

    Parmar, Paresh A.; Chow, Lesley W.; St-Pierre, Jean-Philippe; Horejs, Christine-Maria; Peng, Yong Y.; Werkmeister, Jerome A.; Ramshaw, John A.M.; Stevens, Molly M.

    2015-01-01

    Regenerative medicine strategies for restoring articular cartilage face significant challenges to recreate the complex and dynamic biochemical and biomechanical functions of native tissues. As an approach to recapitulate the complexity of the extracellular matrix, collagen-mimetic proteins offer a modular template to incorporate bioactive and biodegradable moieties into a single construct. We modified a Streptococcal collagen-like 2 protein with hyaluronic acid (HA) or chondroitin sulfate (CS)-binding peptides and then cross-linked with a matrix metalloproteinase 7 (MMP7)-sensitive peptide to form biodegradable hydrogels. Human mesenchymal stem cells (hMSCs) encapsulated in these hydrogels exhibited improved viability and significantly enhanced chondrogenic differentiation compared to controls that were not functionalized with glycosaminoglycan-binding peptides. Hydrogels functionalized with CS-binding peptides also led to significantly higher MMP7 gene expression and activity while the HA-binding peptides significantly increased chondrogenic differentiation of the hMSCs. Our results highlight the potential of this novel biomaterial to modulate cell-mediated processes and create functional tissue engineered constructs for regenerative medicine applications. PMID:25907054

  10. The effect of collagen fibril orientation on the biphasic mechanics of articular cartilage.

    Science.gov (United States)

    Meng, Qingen; An, Shuqiang; Damion, Robin A; Jin, Zhongmin; Wilcox, Ruth; Fisher, John; Jones, Alison

    2017-01-01

    The highly inhomogeneous distribution of collagen fibrils may have important effects on the biphasic mechanics of articular cartilage. However, the effect of the inhomogeneity of collagen fibrils has mainly been investigated using simplified three-layered models, which may have underestimated the effect of collagen fibrils by neglecting their realistic orientation. The aim of this study was to investigate the effect of the realistic orientation of collagen fibrils on the biphasic mechanics of articular cartilage. Five biphasic material models, each of which included a different level of complexity of fibril reinforcement, were solved using two different finite element software packages (Abaqus and FEBio). Model 1 considered the realistic orientation of fibrils, which was derived from diffusion tensor magnetic resonance images. The simplified three-layered orientation was used for Model 2. Models 3-5 were three control models. The realistic collagen orientations obtained in this study were consistent with the literature. Results from the two finite element implementations were in agreement for each of the conditions modelled. The comparison between the control models confirmed some functions of collagen fibrils. The comparison between Models 1 and 2 showed that the widely-used three-layered inhomogeneous model can produce similar fluid load support to the model including the realistic fibril orientation; however, an accurate prediction of the other mechanical parameters requires the inclusion of the realistic orientation of collagen fibrils. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. CARTILAGE OLIGOMERIC MATRIX PROTEIN ENHANCES MATRIX ASSEMBLY DURING CHONDROGENESIS OF HUMAN MESENCHYMAL STEM CELLS

    Science.gov (United States)

    Haleem-Smith, Hana; Calderon, Raul; Song, Yingjie; Tuan, Rocky S.; Chen, Faye H.

    2011-01-01

    Cartilage oligomeric matrix protein/thrombospondin-5 (COMP/TSP5) is an abundant cartilage extracellular matrix (ECM) protein that interacts with major cartilage ECM components, including aggrecan and collagens. To test our hypothesis that COMP/TSP5 functions in the assembly of the ECM during cartilage morphogenesis, we have employed mesenchymal stem cell (MSC) chondrogenesis in vitro as a model to examine the effects of COMP over-expression on neo-cartilage formation. Human bone marrow-derived MSCs were transfected with either full-length COMP cDNA or control plasmid, followed by chondrogenic induction in three-dimensional pellet or alginate-hydrogel culture. MSC chondrogenesis and ECM production was estimated based on quantitation of sulfated glycosaminoglycan (sGAG) accumulation, immunohistochemistry of the presence and distribution of cartilage ECM proteins, and real-time RT-PCR analyis of mRNA expression of cartilage markers. Our results showed that COMP over-expression resulted in increased total sGAG content during the early phase of MSC chondrogenesis, and increased immuno-detectable levels of aggrecan and collagen type II in the ECM of COMP-transfected pellet and alginate cultures, indicating more abundant cartilaginous matrix. COMP transfection did not significantly increase the transcript levels of the early chondrogenic marker, Sox9, or aggrecan, suggesting that enhancement of MSC cartilage ECM was effected at post-transcriptional levels. These findings strongly suggest that COMP functions in mesenchymal chondrogenesis by enhancing cartilage ECM organization and assembly. The action of COMP is most likely mediated not via direct changes in cartilage matrix gene expression but via interactions of COMP with other cartilage ECM proteins, such as aggrecan and collagens, that result in enhanced assembly and retention. PMID:22095699

  12. Hydrogels of collagen/chondroitin sulfate/hyaluronan interpenetrating polymer network for cartilage tissue engineering.

    Science.gov (United States)

    Guo, Yan; Yuan, Tun; Xiao, Zhanwen; Tang, Pingping; Xiao, Yumei; Fan, Yujiang; Zhang, Xingdong

    2012-09-01

    The network structure of a three-dimensional hydrogel scaffold dominates its performance such as mechanical strength, mass transport capacity, degradation rate and subsequent cellular behavior. The hydrogels scaffolds with interpenetrating polymeric network (IPN) structure have an advantage over the individual component gels and could simulate partly the structure of native extracellular matrix of cartilage tissue. In this study, to develop perfect cartilage tissue engineering scaffolds, IPN hydrogels of collagen/chondroitin sulfate/hyaluronan were prepared via two simultaneous processes of collagen self-assembly and cross linking polymerization of chondroitin sulfate-methacrylate (CSMA) and hyaluronic acid-methacrylate. The degradation rate, swelling performance and compressive modulus of IPN hydrogels could be adjusted by varying the degree of methacrylation of CSMA. The results of proliferation and fluorescence staining of rabbit articular chondrocytes in vitro culture demonstrated that the IPN hydrogels possessed good cytocompatibility. Furthermore, the IPN hydrogels could upregulate cartilage-specific gene expression and promote the chondrocytes secreting glycosaminoglycan and collagen II. These results suggested that IPN hydrogels might serve as promising hydrogel scaffolds for cartilage tissue engineering.

  13. Infrared microspectroscopic determination of collagen cross-links in articular cartilage

    Science.gov (United States)

    Rieppo, Lassi; Kokkonen, Harri T.; Kulmala, Katariina A. M.; Kovanen, Vuokko; Lammi, Mikko J.; Töyräs, Juha; Saarakkala, Simo

    2017-03-01

    Collagen forms an organized network in articular cartilage to give tensile stiffness to the tissue. Due to its long half-life, collagen is susceptible to cross-links caused by advanced glycation end-products. The current standard method for determination of cross-link concentrations in tissues is the destructive high-performance liquid chromatography (HPLC). The aim of this study was to analyze the cross-link concentrations nondestructively from standard unstained histological articular cartilage sections by using Fourier transform infrared (FTIR) microspectroscopy. Half of the bovine articular cartilage samples (n=27) were treated with threose to increase the collagen cross-linking while the other half (n=27) served as a control group. Partial least squares (PLS) regression with variable selection algorithms was used to predict the cross-link concentrations from the measured average FTIR spectra of the samples, and HPLC was used as the reference method for cross-link concentrations. The correlation coefficients between the PLS regression models and the biochemical reference values were r=0.84 (p<0.001), r=0.87 (p<0.001) and r=0.92 (p<0.001) for hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP), and pentosidine (Pent) cross-links, respectively. The study demonstrated that FTIR microspectroscopy is a feasible method for investigating cross-link concentrations in articular cartilage.

  14. Cartilage Turnover Reflected by Metabolic Processing of Type II Collagen: A Novel Marker of Anabolic Function in Chondrocytes

    Directory of Open Access Journals (Sweden)

    Natasja Stæhr Gudmann

    2014-10-01

    Full Text Available The aim of this study was to enable measurement of cartilage formation by a novel biomarker of type II collagen formation. The competitive enzyme-linked immunosorbent assay (ELISA Pro-C2 was developed and characterized for assessment of the beta splice variant of type II procollagen (PIIBNP. This is expected to originate primarily from remodeling of hyaline cartilage. A mouse monoclonal antibody (Mab was raised in mouse, targeting specifically PIIBNP (QDVRQPG and used in development of the assay. The specificity, sensitivity, 4-parameter fit and stability of the assay were tested. Levels of PIIBNP were quantified in human serum (0.6–2.2 nM, human amniotic fluid (163–188 nM and sera from different animal species, e.g., fetal bovine serum (851–901 nM with general good linearity (100% (SD 7.6 recovery and good intra- and inter-assay variation (CV% < 10. Dose (0.1 to 100 ng/mL and time (7, 14 and 21 days dependent release of PIIBNP were evaluated in the conditioned medium from bovine cartilage explants (BEX and human cartilage explants (HEX upon stimulation with insulin-like growth factor (IGF-1, transforming growth factor (TGF-β1 and fibroblastic growth factor-2 (FGF-2. TGF-β1 and IGF-1 in concentrations of 10–100 ng/mL significantly (p < 0.05 induced release of PIIBNP in BEX compared to conditions without treatment (WO. In HEX, IGF-1 100 ng/mL was able to induce a significant increase of PIIBNP after one week compared to WO. FGF-2 did not induce a PIIBNP release in our models. To our knowledge this is the first assay, which is able to specifically evaluate PIIBNP excretion. The Pro-C2 assay seems to provide a promising and novel marker of type II collagen formation.

  15. Immunohistochemical detection of interstitial collagens in bone and cartilage tissue remnants in an infant Peruvian mummy.

    Science.gov (United States)

    Nerlich, A G; Parsche, F; Kirsch, T; Wiest, I; von der Mark, K

    1993-07-01

    We investigated the immunohistochemical presence of various collagen types in bone and cartilage tissue from an infant Peruvian mummy dating between 500 and 1000 A.D. which had been excavated at the necropolis of Las Trancas in the Nazca region in Peru. Following careful rehydration and decalcification of the tissue, the mummy tissue showed morphologically good preservation of the matrix, which could be shown to be composed of various collagen types in a typical pattern. Bone consisted of a collagen I matrix with a small rim of collagen III and V at the endosteal lining and a pericellular collagen V staining around osteocytic holes. In the hypertrophic cartilage of the epiphyseal growth plate, a typical pattern of collagen types II and X could be found. These observations provide evidence that in well-preserved mummy tissue the antigenic determinants of major matrix components are still adequately preserved for an immunohistochemical analysis. This technique may thus be a very helpful tool for the analysis of pathologic processes of historic bone tissue. It may also allow in certain circumstances a distinction between pseudopathologic tissue destruction and pathologic tissue alteration.

  16. The response to estrogen deprivation on cartilage collagen degradation markers; CTX-II is unique compared to other markers of collagen turnover

    DEFF Research Database (Denmark)

    Bay-Jensen, Anne-Christine; Tabassi, Nadine; Sondergaard, Lene

    2009-01-01

    ABSTRACT: INTRODUCTION: The urinary level of type II collagen degradation marker CTX-II is increased in postmenopausal women and in ovariectomized rats, suggesting that estrogen deprivation induces cartilage breakdown. Here we investigate whether this response to estrogen holds 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 body fluids of pre- and postmenopausal women and of ovariectomized...... rats receiving estrogen or not. Levels of PIIANP, a marker of type II collagen synthesis, were also measured in rats. Rat knee cartilage was analyzed for immunoreactivity of CTX-II and PIIANP and for type II collagen expression. RESULTS: As expected, urinary levels of CTX-II are significantly increased...

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

    DEFF Research Database (Denmark)

    Bay-Jensen, Anne-Christine; Tabassi, Nadine C B; Sondergaard, Lene V

    2009-01-01

    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...

  18. A new collagen from the extracellular matrix of Sepia officinalis cartilage.

    Science.gov (United States)

    Rigo, Cristina; Bairati, Aurelio

    2002-11-01

    Guanidinium chloride treatment of Sepia officinalis cartilage solubilized a component that contained hydroxyproline. Electron-microscopy observation of rotary-shadowed preparations of this component revealed it to consist of rod-like units themselves consisting of filaments. Dialysis of an acetic acid solution against ATP afforded polymeric aggregates consisting of a succession of two or three thick sections showing transverse electron-opaque banding, separated by thinner sections without banding. Electrophoresis produced a main band of about 140 kDa sensitive to bacterial collagenase. After reduction with mercaptoethanol, electrophoresis afforded a 40-kDa band. Pepsin digestion resulted in additional electrophoretic bands. These data suggest the presence of a collagen in Sepia cartilage with characteristics unlike those of any known collagen.

  19. Collagen-based porous scaffolds containing PLGA microspheres for controlled kartogenin release in cartilage tissue engineering.

    Science.gov (United States)

    Sun, Xiaomin; Wang, Jianhua; Wang, Yingying; Zhang, Qiqing

    2017-11-06

    A scaffold composed of different collagen (COL)/chitosan (CS)/hyaluronic acid sodium (HAS) salt ratios was evaluated by determining porosity, swelling, loss rate in hot water, mechanical property, and cell proliferation to obtain optimum conditions for manufacturing porous scaffolds. Results showed that the optimal ratio of COL/CS/HAS salt porous scaffold was 1:1:0.1. High swelling and loss rate of scaffolds/microspheres (MPs) could lead to high diffusion rate of MPs from the scaffolds, causing an increase in the kartogenin (KGN) release. The porous scaffolds at optimum conditions had a maximum amount of KGN release. Results of in vitro fluorescence staining and cell proliferation suggested that scaffolds/MPs had good biocompatibility and the capability to promote bone marrow stromal cell proliferation, cartilage tissue regeneration, and integration between the repaired and surrounding cartilages. Therefore, this composite could be a promising material for cartilage repair and regeneration, which could be effective in the knee osteoarthritis treatment.

  20. Cartilage regeneration using a porous scaffold, a collagen sponge incorporating a hydroxyapatite/chondroitinsulfate composite

    Energy Technology Data Exchange (ETDEWEB)

    Ohyabu, Yohimi, E-mail: ooyabu.yoshimi@aist.go.jp [Department of Metallurgy and Ceramics Science, Tokyo Institute of Technology, 2-12-1, S7-5 Ookayama, Meguro, Tokyo 152-8550 (Japan); Nanotechnology Research Institute (NRI), National Institute of Advanced Industrial Science and Technology (AIST), Central-4, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566 (Japan); Adegawa, Takuro; Yoshioka, Tomohiko [Department of Metallurgy and Ceramics Science, Tokyo Institute of Technology, 2-12-1, S7-5 Ookayama, Meguro, Tokyo 152-8550 (Japan); Ikoma, Toshiyuki [Biomaterials Center, National Institute for Materials Science, 1-1 Sengen, Tsukuba, Ibaraki, 305-0047 (Japan); Uemura, Toshimasa [Nanotechnology Research Institute (NRI), National Institute of Advanced Industrial Science and Technology (AIST), Central-4, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566 (Japan); Tanaka, Junzo [Department of Metallurgy and Ceramics Science, Tokyo Institute of Technology, 2-12-1, S7-5 Ookayama, Meguro, Tokyo 152-8550 (Japan)

    2010-10-15

    Because cartilage has limited potential for self-repair, tissue engineering is expected to replace the present therapies for damaged cartilage, such as total knee arthroplasty. However, scaffolds suitable for cartilage tissue engineering have not been established. We synthesized a novel porous scaffold, a collagen sponge incorporating a hydroxyapatite/chondroitinsulfate composite (pCol-HAp/ChS), containing materials which resemble extracellular matrices in bone and cartilage tissues, which needs high compressive strength for clinical use. HAp/ChS had smaller crystals and a larger total surface area than HAp. SEM images showed pCol-HAp/ChS to have the roughest surface compared with pCol and pCol-HAp. The mechanical properties suggest that pCol-HAp/ChS and pCol/HAp are similar, and superior to pCol. Seeding experiments showed a uniform distribution of mesenchymal stem cells (MSCs) in pCol-HAp/ChS and pCol/HAp. Safranin O, Toluidine blue and Alcian blue staining after 2 weeks of culture revealed pCol-HAp/ChS to be the most chondrogenic in each case. In addition, MSCs in pCol-HAp/ChS produced more glycosaminoglycans, a cartilage matrix, than those in pCol-HAp. Further, pCol-HAp/ChS regenerated 15 times more cartilaginous tissue than pCol. From these results, pCol-HAp/ChS is expected to be a candidate for a scaffold for cartilage tissue engineering in place of collagen sponge.

  1. Collagene order of articular cartilage by clinical magnetic resonance images and its age dependency

    Energy Technology Data Exchange (ETDEWEB)

    Seidel, P.; Gruender, W. [Inst. of Medical Physics and Biophysics, Univ. of Leipzig (Germany)

    2005-07-01

    The present papers describes a novel method to obtain information on the degree of order of the collagen network of the knee meniscal cartilage by means of a single clinical MRI. Images were obtained from 34 healthy volunteers aged between 6 and 76 years as well as from one patient with clinically-diagnosed arthrosis at the age of 32 and 37 years. A siemens vision (1.5 T) MRT with TR = 750 ms, TE = 50 ms, FoV = 160 mm, and Matrix 512 x 512 was used for this purpose. The MR signal intensities of the cartilage were read out along slices with constant height above the subchondral bone and plotted versus the actual angle to the external magnetic field. The obtained intensity curves were fitted by a model distribution, and the degree of order of the collagen fibers was calculated. For the knee meniscal cartilage, there was an age-dependency of the degree of order and a significant deviation of the volunteer with arthrosis from the normal curve. The results are discussed in view of the arcade model and of a possible use of non-invasive clinical MRT for the detection of early arthrotic changes of cartilage. (orig.)

  2. Tissue engineering of human cartilage in bioreactors using single and composite cell-seeded scaffolds.

    Science.gov (United States)

    Mahmoudifar, Nastaran; Doran, Pauline M

    2005-08-05

    Chondrocytes isolated from human fetal epiphyseal cartilage were seeded under mixed conditions into 15-mm-diameter polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to generate cartilage constructs. After seeding, the cell distributions in thick (4.75 mm) and thin (2.15 mm) PGA disks were nonuniform, with higher cell densities accumulating near the top surfaces. Composite scaffolds were developed by suturing together two thin PGA disks after seeding to manipulate the initial cell distribution before bioreactor culture. The effect of medium flow direction in the bioreactors, including periodic reversal of medium flow, was also investigated. The quality of the tissue-engineered cartilage was assessed after 5 weeks of culture in terms of the tissue wet weight, glycosaminoglycan (GAG), total collagen and collagen type II contents, histological analysis of cell, GAG and collagen distributions, and immunohistochemical analysis of collagen types I and II. Significant enhancement in construct quality was achieved using composite scaffolds compared with single PGA disks. Operation of the bioreactors with periodic medium flow reversal instead of unidirectional flow yielded further improvements in tissue weight and GAG and collagen contents with the composite scaffolds. At harvest, the constructs contained GAG concentrations similar to those measured in ex vivo human adult articular cartilage; however, total collagen and collagen type II levels were substantially lower than those in adult tissue. This study demonstrates that the location of regions of high cell density in the scaffold coupled with application of dynamic bioreactor operating conditions has a significant influence on the quality of tissue-engineered cartilage. Copyright 2005 Wiley Periodicals, Inc.

  3. C2K77 ELISA detects cleavage of type II collagen by cathepsin K in equine articular cartilage.

    Science.gov (United States)

    Noé, B; Poole, A R; Mort, J S; Richard, H; Beauchamp, G; Laverty, S

    2017-12-01

    Develop a species-specific ELISA for a neo-epitope generated by cathepsin K cleavage of equine type II collagen to: (1) measure cartilage type II collagen degradation by cathepsin K in vitro, (2) identify cytokines that upregulate cathepsin K expression and (3) compare cathepsin K with matrix metalloproteinase (MMP) collagenase activity in stimulated cartilage explants and freshly isolated normal and osteoarthritic (OA) articular cartilages. A new ELISA (C2K77) was developed and tested by measuring the activity of exogenous cathepsin K on equine articular cartilage explants. The ELISA was then employed to measure endogenous cathepsin K activity in cultured cartilage explants with or without stimulation by interleukin-1 beta (IL-1β), tumour necrosis-alpha (TNF-α), oncostatin M (OSM) and lipopolysaccharide (LPS). Cathepsin K activity in cartilage explants (control and osteoarthritic-OA) and freshly harvested cartilage (control and OA) was compared to that of MMPs employing C2K77 and C1,2C immunoassays. The addition of Cathepsin K to normal cartilage caused a significant increase (P K77 epitope release. Whereas the content of C1,2C, that reflects MMP collagenase activity, was increased in media by the addition to cartilage explants of TNF-α and OSM (P K77 which also unchanged in OA cartilages compared to normal. The ELISA C2K77 measured the activity of cathepsin K in equine cartilage which was unchanged in OA cartilage. Cytokines that upregulate MMP collagenase activity had no effect on endogenous cathepsin K activity, suggesting a different activation mechanism that requires further study. Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  4. The effects of proteoglycan and type II collagen on T1rho relaxation time of articular cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Won Seok; Yoo, Hye Jin; Hong, Sung Hwan; Choi, Ja Young [Dept. of Radiology and Institute of Radiation Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul (Korea, Republic of)

    2015-02-15

    To evaluate the effects of proteoglycan and type II collagen within articular cartilage on T1rho relaxation time of articular cartilage. This study was exempted by the institutional and animal review boards, and informed consent was not required. Twelve porcine patellae were assigned to three groups of control, trypsin-treated (proteoglycan-degraded), or collagenase-treated (collagen-degraded). The T1rho images were obtained with a 3 tesla magnetic resonance imaging scanner with a single loop coil. Statistical differences were detected by analysis of variance to evaluate the effects of the enzyme on T1rho relaxation time. Safranin-O was used to stain proteoglycan in the articular cartilage and immunohistochemical staining was performed for type II collagen. Mean T1rho values of the control, trypsin-treated, and collagenase-treated groups were 37.72 +/- 5.82, 57.53 +/- 8.24, and 45.08 +/- 5.31 msec, respectively (p < 0.001). Histology confirmed a loss of proteoglycan and type II collagen in the trypsin- and collagenase-treated groups. Degradation of proteoglycans and collagen fibers in the articular cartilage increased the articular cartilage T1rho value.

  5. Hydrogels derived from cartilage matrices promote induction of human mesenchymal stem cell chondrogenic differentiation.

    Science.gov (United States)

    Burnsed, Olivia A; Schwartz, Zvi; Marchand, Katherine O; Hyzy, Sharon L; Olivares-Navarrete, René; Boyan, Barbara D

    2016-10-01

    Limited supplies of healthy autologous or allogeneic cartilage sources have inspired a growing interest in xenogeneic cartilage matrices as biological scaffolds for cartilage tissue engineering. The objectives of this study were to determine if shark and pig cartilage extracellular matrix (ECM) hydrogels can stimulate chondrocytic differentiation of mesenchymal stem cells (MSCs) without exogenous growth factors and to determine if the soluble factors retained by these ECM hydrogels are responsible. Human MSCs cultured on hydrogels from shark skull cartilage, pig articular cartilage, and pig auricular cartilage ECM had increased expression of chondrocyte markers and decreased secretion of angiogenic factors VEGF-A and FGF2 in comparison to MSCs cultured on tissue culture polystyrene (TCPS) at one week. MSCs grown on shark ECM gels had decreased type-1 collagen mRNA as compared to all other groups. Degradation products of the cartilage ECM gels and soluble factors released by the matrices increased chondrogenic and decreased angiogenic mRNA levels, indicating that the processed ECM retains biochemically active proteins that can stimulate chondrogenic differentiation. In conclusion, this work supports the use of cartilage matrix-derived hydrogels for chondrogenic differentiation of MSCs and cartilage tissue engineering. Longer-term studies and positive controls will be needed to support these results to definitively demonstrate stimulation of chondrocyte differentiation, and particularly to verify that calcification without endochondral ossification does not occur as it does in shark cartilage. The objectives of this study were to determine if shark and pig cartilage extracellular matrix (ECM) hydrogels can stimulate chondrocytic differentiation of mesenchymal stem cells (MSCs) without exogenous growth factors and to determine if the soluble factors retained by these ECM hydrogels are responsible for this induction. Sharks are an especially interesting model for

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

    DEFF Research Database (Denmark)

    Szabova, Ludmila; Yamada, Susan S; Wimer, Helen F

    2009-01-01

    Skeletal formation is dependent on timely recruitment of skeletal stem cells and their ensuing synthesis and remodeling of the major fibrillar collagens, type I collagen and type II collagen, in bone and cartilage tissues during development and postnatal growth. Loss of the major collagenolytic...... in bone cells and skeletal stem/progenitor cells of wildtype mice. Moreover, bone marrow stromal cells isolated from mice expressing MT1-MMP under the control of the type II collagen promoter in an MT1-MMP-deficient background showed enhanced bone formation in vitro and in vivo compared with cells derived...... from nontransgenic MT1-MMP-deficient littermates. These observations show that type II collagen is not stringently confined to the chondrocyte but is expressed in skeletal stem/progenitor cells (able to regenerate bone, cartilage, myelosupportive stroma, marrow adipocytes) and in the chondrogenic...

  7. Collagens in the aged human macular sclera.

    Science.gov (United States)

    Marshall, G E; Konstas, A G; Lee, W R

    1993-02-01

    Scleral tissue from the region of the human macula was studied by the immunogold labeling technique (cryoultramicrotomy and LR white resin embedding) in an attempt to identify the fine structural distribution of collagen types I-VI. Labeling of the striated collagen fibrils suggested colocalisation of collagen types I, III and V with type V occurring at the fibril surface. Both types V and VI collagen were localised to filamentous strands in the interfibrillar matrix. Collagen types II and IV were absent from the scleral stroma.

  8. The influence of collagen network integrity on the accumulation of gadolinium-based MR contrast agents in articular cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Wiener, Edzard; Schmidt, C.; Diederichs, G. [Charite - Universitaetsmedizin Berlin (Germany). Inst. fuer Radiologie; Settles, M. [Klinikum rechts der Isar, Muenchen (Germany). Inst. fuer Roentgendiagnostik; Weirich, G. [Klinikum Rechts der Isar, Muenchen (Germany). Inst. fuer Pathologie und Pathologische Anatomie

    2011-03-15

    Delayed gadolinium-enhanced MR imaging of cartilage is used to quantify the proteoglycan loss in early osteoarthritis. It is assumed that T 1 after Gd-DTPA administration in the near equilibrium state reflects selective proteoglycan loss from cartilage. To investigate the influence of the collagen network integrity on contrast accumulation, the relaxation rates {delta}R1 and {delta}R2 were compared after Gd-DTPA administration in a well established model of osteoarthritis. Collagen or proteoglycan depletion was induced by the proteolytic enzymes papain and collagenase in healthy bovine patellar cartilage. Using a dedicated MRI sequence, T{sub 1} and T{sub 2} maps were simultaneously acquired before and 11 h after Gd-DTPA administration. Depth-dependent profiles of {delta}R1 and {delta}R2 were calculated in healthy, proteoglycan and collagen-depleted articular cartilage and the mean values of different cartilage layers were compared using the Mann-Whitney-U test. In superficial layers (1 mm) there was no significant difference (p > 0.05) in either {delta}R1 or {delta}R2 between proteoglycan-depleted (16.6 {+-} 1.2 s{sup -1}, 15.9 {+-} 1.0 s{sup -1}) and collagen-depleted articular cartilage (15.3 {+-} 0.9 s{sup -1}, 15.5 {+-} 0.9 s{sup -1}). In deep layers (3 mm) both parameters were significantly higher (p = 0.005, 0.03) in proteoglycan-depleted articular cartilage (12.3 {+-} 1.1 s{sup -1}, 9.8 {+-} 0.8 s{sup -1}) than in collagen-depleted articular cartilage (9.1 {+-} 1.1 s{sup -1}, 8.7 {+-} 0.7 s{sup -1}). Both proteoglycan loss and alterations in the collagen network influence the accumulation of Gd-DTPA in articular cartilage with significant differences between superficial and deep cartilage layers. (orig.)

  9. Mesenchymal Stromal/stem Cell-derived Extracellular Vesicles Promote Human Cartilage RegenerationIn Vitro.

    Science.gov (United States)

    Vonk, Lucienne A; van Dooremalen, Sanne F J; Liv, Nalan; Klumperman, Judith; Coffer, Paul J; Saris, Daniël B F; Lorenowicz, Magdalena J

    2018-01-01

    Osteoarthritis (OA) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. Recently, allogeneic human mesenchymal stromal/stem cells (MSC) entered clinical trials as a novel therapy for OA. Increasing evidence suggests that therapeutic efficacy of MSC depends on paracrine signalling. Here we investigated the role of extracellular vesicles (EVs) secreted by human bone marrow derived MSC (BMMSC) in human OA cartilage repair. To test the effect of BMMSC-EVs on OA cartilage inflammation, TNF-alpha-stimulated OA chondrocyte monolayer cultures were treated with BMMSC-EVs and pro-inflammatory gene expression was measured by qRT-PCR after 48 h. To assess the impact of BMMSC-EVs on cartilage regeneration, BMMSC-EVs were added to the regeneration cultures of human OA chondrocytes, which were analyzed after 4 weeks for glycosaminoglycan content by 1,9-dimethylmethylene blue (DMMB) assay. Furthermore, paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-O) and type II collagen (immunostaining). We show that BMMSC-EVs inhibit the adverse effects of inflammatory mediators on cartilage homeostasis. When co-cultured with OA chondrocytes, BMMSC-EVs abrogated the TNF-alpha-mediated upregulation of COX2 and pro-inflammatory interleukins and inhibited TNF-alpha-induced collagenase activity. BMMSC-EVs also promoted cartilage regeneration in vitro . Addition of BMMSC-EVs to cultures of chondrocytes isolated from OA patients stimulated production of proteoglycans and type II collagen by these cells. Our data demonstrate that BMMSC-EVs can be important mediators of cartilage repair and hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis.

  10. High-resolution diffusion tensor imaging of human patellar cartilage: feasibility and preliminary findings.

    Science.gov (United States)

    Filidoro, L; Dietrich, O; Weber, J; Rauch, E; Oerther, T; Wick, M; Reiser, M F; Glaser, C

    2005-05-01

    MR diffusion tensor imaging (DTI) was used to analyze the microstructural properties of articular cartilage. Human patellar cartilage-on-bone samples were imaged at 9.4T using a diffusion-weighted SE sequence (12 gradient directions, resolution = 39 x 78 x 1500 microm(3)). Voxel-based maps of the mean diffusivity, fractional anisotropy (FA), and eigenvectors were calculated. The mean diffusivity decreased from the surface (1.45 x 10(-3) mm(2)/s) to the tide mark (0.68 x 10(-3) mm(2)/s). The FA was low (0.04-0.28) and had local maxima near the surface and in the portion of the cartilage corresponding to the radial layer. The eigenvector corresponding to the largest eigenvalue showed a distinct zonal pattern, being oriented tangentially and radially in the upper and lower portions of the cartilage, respectively. The findings correspond to current scanning electron microscopy (SEM) data on the zonal architecture of cartilage. The eigenvector maps appear to reflect the alignment of the collagenous fibers in cartilage. In view of current efforts to develop and evaluate structure-modifying therapeutic approaches in osteoarthritis (OA), DTI may offer a tool to assess the structural properties of cartilage. Copyright 2005 Wiley-Liss, Inc.

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

    Directory of Open Access Journals (Sweden)

    Zheng Ming H

    2008-07-01

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

  12. Genipin-cross-linked collagen/chitosan biomimetic scaffolds for articular cartilage tissue engineering applications.

    Science.gov (United States)

    Yan, Le-Ping; Wang, Ying-Jun; Ren, Li; Wu, Gang; Caridade, Sofia G; Fan, Jia-Bing; Wang, Ling-Yun; Ji, Pei-Hong; Oliveira, Joaquim M; Oliveira, João T; Mano, João F; Reis, Rui L

    2010-11-01

    In this study, genipin-cross-linked collagen/chitosan biodegradable porous scaffolds were prepared for articular cartilage regeneration. The influence of chitosan amount and genipin concentration on the scaffolds physicochemical properties was evaluated. The morphologies of the scaffolds were characterized by scanning electron microscope (SEM) and cross-linking degree was investigated by ninhydrin assay. Additionally, the mechanical properties of the scaffolds were assessed under dynamic compression. To study the swelling ratio and the biostability of the collagen/chitosan scaffold, in vitro tests were also carried out by immersion of the scaffolds in PBS solution or digestion in collagenase, respectively. The results showed that the morphologies of the scaffolds underwent a fiber-like to a sheet-like structural transition by increasing chitosan amount. Genipin cross-linking remarkably changed the morphologies and pore sizes of the scaffolds when chitosan amount was less than 25%. Either by increasing the chitosan ratio or performing cross-linking treatment, the swelling ratio of the scaffolds can be tailored. The ninhydrin assay demonstrated that the addition of chitosan could obviously increase the cross-linking efficiency. The degradation studies indicated that genipin cross-linking can effectively enhance the biostability of the scaffolds. The biocompatibility of the scaffolds was evaluated by culturing rabbit chondrocytes in vitro. This study demonstrated that a good viability of the chondrocytes seeded on the scaffold was achieved. The SEM analysis has revealed that the chondrocytes adhered well to the surface of the scaffolds and contacted each other. These results suggest that the genipin-cross-linked collagen/chitosan matrix may be a promising formulation for articular cartilage scaffolding.

  13. Tissue-engineered cartilaginous constructs for the treatment of caprine cartilage defects, including distribution of laminin and type IV collagen.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Elango Jeevithan

    2014-06-01

    Full Text Available 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.

  15. Collagen Scaffolds with Controlled Insulin Release and Controlled Pore Structure for Cartilage Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Himansu Sekhar Nanda

    2014-01-01

    Full Text Available Controlled and local release of growth factors and nutrients from porous scaffolds is important for maintenance of cell survival, proliferation, and promotion of tissue regeneration. The purpose of the present research was to design a controlled release porous collagen-microbead hybrid scaffold with controlled pore structure capable of releasing insulin for application to cartilage tissue regeneration. Collagen-microbead hybrid scaffold was prepared by hybridization of insulin loaded PLGA microbeads with collagen using a freeze-drying technique. The pore structure of the hybrid scaffold was controlled by using preprepared ice particulates having a diameter range of 150–250 μm. Hybrid scaffold had a controlled pore structure with pore size equivalent to ice particulates and good interconnection. The microbeads showed an even spatial distribution throughout the pore walls. In vitro insulin release profile from the hybrid scaffold exhibited a zero order release kinetics up to a period of 4 weeks without initial burst release. Culture of bovine articular chondrocytes in the hybrid scaffold demonstrated high bioactivity of the released insulin. The hybrid scaffold facilitated cell seeding and spatial cell distribution and promoted cell proliferation.

  16. Streamlined bioreactor-based production of human cartilage tissues.

    Science.gov (United States)

    Tonnarelli, B; Santoro, R; Adelaide Asnaghi, M; Wendt, D

    2016-05-27

    Engineered tissue grafts have been manufactured using methods based predominantly on traditional labour-intensive manual benchtop techniques. These methods impart significant regulatory and economic challenges, hindering the successful translation of engineered tissue products to the clinic. Alternatively, bioreactor-based production systems have the potential to overcome such limitations. In this work, we present an innovative manufacturing approach to engineer cartilage tissue within a single bioreactor system, starting from freshly isolated human primary chondrocytes, through the generation of cartilaginous tissue grafts. The limited number of primary chondrocytes that can be isolated from a small clinically-sized cartilage biopsy could be seeded and extensively expanded directly within a 3D scaffold in our perfusion bioreactor (5.4 ± 0.9 doublings in 2 weeks), bypassing conventional 2D expansion in flasks. Chondrocytes expanded in 3D scaffolds better maintained a chondrogenic phenotype than chondrocytes expanded on plastic flasks (collagen type II mRNA, 18-fold; Sox-9, 11-fold). After this "3D expansion" phase, bioreactor culture conditions were changed to subsequently support chondrogenic differentiation for two weeks. Engineered tissues based on 3D-expanded chondrocytes were more cartilaginous than tissues generated from chondrocytes previously expanded in flasks. We then demonstrated that this streamlined bioreactor-based process could be adapted to effectively generate up-scaled cartilage grafts in a size with clinical relevance (50 mm diameter). Streamlined and robust tissue engineering processes, as the one described here, may be key for the future manufacturing of grafts for clinical applications, as they facilitate the establishment of compact and closed bioreactor-based production systems, with minimal automation requirements, lower operating costs, and increased compliance to regulatory guidelines.

  17. Concentration profiles of collagen and proteoglycan in articular cartilage by Fourier transform infrared imaging and principal component regression

    Science.gov (United States)

    Yin, Jianhua; Xia, Yang; Lu, Mei

    2012-03-01

    Fourier-transform infrared imaging (FT-IRI) technique with the principal component regression (PCR) method was used to quantitatively determine the 2D images and the depth-dependent concentration profiles of two principal macromolecular components (collagen and proteoglycan) in articular cartilage. Ten 6 μm thick sections of canine humeral cartilage were imaged at a pixel size of 6.25 μm in FT-IRI. The infrared spectra extracted from FT-IRI experiments were imported into a PCR program to calculate the quantitative distributions of both collagen and proteoglycan in dry cartilage, which were subsequently converted into the wet-weight based concentration profiles. The proteoglycan profiles by FT-IRI and PCR significantly correlated in linear regression with the proteoglycan profiles by the non-destructive μMRI (the goodness-of-fit 0.96 and the Pearson coefficient 0.98). Based on these concentration relationships, the concentration images of collagen and proteoglycan in both healthy and lesioned articular cartilage were successfully constructed two dimensionally. The simultaneous construction of both collagen and proteoglycan concentration images demonstrates that this combined imaging and chemometrics approach could be used as a sensitive tool to accurately resolve and visualize the concentration distributions of macromolecules in biological tissues.

  18. The monoclonal antibody GRC1 produced against human cornea recognizes a common determinant of collagen.

    Science.gov (United States)

    Lopez Nevot, M A; Cardona, L; Doblaré, E; Muñoz, C; Ruiz-Cabello, F; Garrido, F

    1990-02-01

    The monoclonal antibody GRC1 was obtained by immunizing BALB/c mice with human cornea. Screening was performed by indirect immunofluorescence in cryostatic sections of several tissues: cornea, skin, placenta, hyaline cartilage, blood vessels, and nerves. GRC1 was seen to recognize fibrillar structures in all of these tissues. The pattern of reaction was interstitial and membranous. On cornea, GRC1 reacts definitely with Bowman's membrane and diffusely with the stroma, while on skin it shows strongly positive reactivity with the papillary dermis and with the basement membrane. It also reacts on hyaline cartilage at the periphery of the condrocytic lacunae. These immunohistologic results suggest that GRC1 recognized human collagen. In order to investigate further the subtype of collagen defined by GRC1, an ELISA was performed with purified collagens of several types: I, II, III, IV, and V. The monoclonal antibody GRC1 defines a common determinant in types III, IV, and V.

  19. A novel computational modelling to describe the anisotropic, remodelling and reorientation behaviour of collagen fibrres in articular cartilage

    CERN Document Server

    Cortez, S; Alves, J L

    2016-01-01

    In articular cartilage the orientation of collagen fibres is not uniform, varying mostly with the depth on the tissue. Besides, the biomechanical response of each layer of the articular cartilage differs from the neighbouring ones, evolving through thickness as a function of the distribution, density and orientation of the collagen fibres. Based on a finite element implementation, a new continuum formulation is proposed to describe the remodelling and reorientation of the collagen fibres under arbitrary mechanical loads: the cartilaginous tissue is modelled based on a hyperelastic formulation, being the ground isotropic matrix described by a neo-Hookean law and the fibrillar anisotropic part modelled by a new anisotropic formulation introduced for the first time in the present work, in which both reorientation and remodelling are taken into account. To characterize the orientation of fibres, a structure tensor is defined to represent the expected distribution and orientation of fibres around a reference direc...

  20. Cartilage collagen type II seromarker patterns in axial spondyloarthritis and psoriatic arthritis

    DEFF Research Database (Denmark)

    Munk, Heidi Lausten; Gudmann, Natasja Staehr; Christensen, Anne Friesgaard

    2016-01-01

    The aim of the study was to assess the possible association between type II collagen turnover seromarkers and disease profile in patients with axial spondyloarthritis (SpA) and psoriatic arthritis (PsA). Outpatients with axial SpA (n = 110) or PsA (n = 101) underwent clinical examination including......-smokers, 0.43 ng/ml (p = 0.02), while PIIANP was higher in HLA-B27 positive, 2312 ng/ml versus negative patients, 2021 ng/ml (p = 0.03). In PsA, PIIANP and C2M did not differ between patients and controls, but PIIANP was elevated in patients not receiving DMARDs, 2726 ng/ml. In PsA, PIIANP and C2M did...... not differ according to smoking and HLA-B27. Cartilage degradation assessed by C2M is increased in SpA irrespective of treatment but not in PsA. Cartilage synthesis reflected by PIIANP is increased in untreated SpA and PsA. PIIANP correlates with CRP in SpA while not in PsA. In DMARD-naïve SpA but not in PsA...

  1. Microstructural analysis of collagen and elastin fibres in the kangaroo articular cartilage reveals a structural divergence depending on its local mechanical environment.

    Science.gov (United States)

    He, B; Wu, J P; Chim, S M; Xu, J; Kirk, T B

    2013-01-01

    To assess the microstructure of the collagen and elastin fibres in articular cartilage under different natural mechanical loading conditions and determine the relationship between the microstructure of collagen and its mechanical environment. Articular cartilage specimens were collected from the load bearing regions of the medial femoral condyle and the medial distal humerus of adult kangaroos. The microstructure of collagen and elastin fibres of these specimens was studied using laser scanning confocal microscopy (LSCM) and the orientation and texture features of the collagen were analysed using ImageJ. A zonal arrangement of collagen was found in kangaroo articular cartilage: the collagen fibres aligned parallel to the surface in the superficial zone and ran perpendicular in the deep zone. Compared with the distal humerus, the collagen in the femoral condyle was less isotropic and more clearly oriented, especially in the superficial and deep zones. The collagen in the femoral condyle was highly heterogeneous, less linear and more complex. Elastin fibres were found mainly in the superficial zone of the articular cartilage of both femoral condyle and distal humerus. The present study demonstrates that the collagen structure and texture of kangaroo articular cartilage is joint-dependent. This finding emphasizes the effects of loading on collagen development and suggests that articular cartilage with high biochemical and biomechanical qualities could be achieved by optimizing joint loading, which may benefit cartilage tissue engineering and prevention of joint injury. The existence of elastin fibres in articular cartilage could have important functional implications. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  2. Interstitial Perfusion Culture with Specific Soluble Factors Inhibits Type I Collagen Production from Human Osteoarthritic Chondrocytes in Clinical-Grade Collagen Sponges.

    Science.gov (United States)

    Mayer, Nathalie; Lopa, Silvia; Talò, Giuseppe; Lovati, Arianna B; Pasdeloup, Marielle; Riboldi, Stefania A; Moretti, Matteo; Mallein-Gerin, Frédéric

    2016-01-01

    Articular cartilage has poor healing ability and cartilage injuries often evolve to osteoarthritis. Cell-based strategies aiming to engineer cartilaginous tissue through the combination of biocompatible scaffolds and articular chondrocytes represent an alternative to standard surgical techniques. In this context, perfusion bioreactors have been introduced to enhance cellular access to oxygen and nutrients, hence overcoming the limitations of static culture and improving matrix deposition. Here, we combined an optimized cocktail of soluble factors, the BIT (BMP-2, Insulin, Thyroxin), and clinical-grade collagen sponges with a bidirectional perfusion bioreactor, namely the oscillating perfusion bioreactor (OPB), to engineer in vitro articular cartilage by human articular chondrocytes (HACs) obtained from osteoarthritic patients. After amplification, HACs were seeded and cultivated in collagen sponges either in static or dynamic conditions. Chondrocyte phenotype and the nature of the matrix synthesized by HACs were assessed using western blotting and immunohistochemistry analyses. Finally, the stability of the cartilaginous tissue produced by HACs was evaluated in vivo by subcutaneous implantation in nude mice. Our results showed that perfusion improved the distribution and quality of cartilaginous matrix deposited within the sponges, compared to static conditions. Specifically, dynamic culture in the OPB, in combination with the BIT cocktail, resulted in the homogeneous production of extracellular matrix rich in type II collagen. Remarkably, the production of type I collagen, a marker of fibrous tissues, was also inhibited, indicating that the association of the OPB with the BIT cocktail limits fibrocartilage formation, favoring the reconstruction of hyaline cartilage.

  3. Human conchal cartilage and temporal fascia: an evidence-based roadmap from rhinoplasty to an in vivo study and beyond.

    Science.gov (United States)

    Cimpean, Anca Maria; Crăiniceanu, Zorin; Mihailovici, Dorina; Bratu, Tiberiu; Raica, Marius

    2014-01-01

    Conchal cartilage or cartilage/ temporal fascia composite grafting (DC-F) used for rhinoplasty is applied by plastic surgeons for reconstructive purposes. Previous studies on experimental models such as mice or rabbits have elucidated on the late events following grafting, with tissue specimens being harvested two months after implantation. Early microscopic and molecular events following DC-F grafting are completely unknown. We designed a chick embryo chorioallantoic membrane model for human grafts study, regarding the dynamic observation of graft survival and its mutual interrelation with the chick embryo chorioallantoic membrane microenvironment. The DC-F graft preserved its cartilage component in a normal state compared to cartilage graft-only because of protective factors provided by temporal fascia. Its strong adherence to the cartilage, lack of angiogenic factors and high content of collagen IV-derived fragments with anti-angiogenic effects make the temporal fascia a good protective tissue to prevent implanted cartilage degeneration. The cartilage graft produced high inflammation, stromal fibrosis and activated angiogenic cascade through VEGF-mediated pathways followed by cartilage degeneration. Also, high content of podoplanin from conchal cartilage chondrocytes exerted a major role in inflammation accompanying cartilage graft. The presently employed experimental model allowed us to characterize the early histological and molecular events triggered by temporal fascia, cartilage or composite graft DC-F implanted on chick embryo chorioallantoic membrane. Our microscopic and molecular observations may help explain some post-surgical complications generated after using cartilage alone as biomaterial for nasal augmentation, supporting the use of DC-F composite graft, with the aim to reduce unwanted post-surgical events. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    activity associated with the membrane-type 1 matrix metalloproteinase (MT1-MMP) results in disrupted skeletal development and growth in both cartilage and bone, where MT1-MMP is required for pericellular collagen dissolution. We show here that reconstitution of MT1-MMP activity in the type II collagen......-expressing cells of the skeleton rescues not only diminished chondrocyte proliferation, but surprisingly, also results in amelioration of the severe skeletal dysplasia associated with MT1-MMP deficiency through enhanced bone formation. Consistent with this increased bone formation, type II collagen was identified...... in bone cells and skeletal stem/progenitor cells of wildtype mice. Moreover, bone marrow stromal cells isolated from mice expressing MT1-MMP under the control of the type II collagen promoter in an MT1-MMP-deficient background showed enhanced bone formation in vitro and in vivo compared with cells derived...

  5. Projection Stereolithographic Fabrication of Human Adipose Stem Cell-incorporated Biodegradable Scaffolds for Cartilage Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Aaron X Sun

    2015-08-01

    Full Text Available Poor self-healing ability of cartilage necessitates the development of methods for cartilage regeneration. Scaffold construction with live stem cell incorporation and subsequent differentiation presents a promising route. Projection stereolithography (PSL offers high resolution and processing speed as well as the ability to fabricate scaffolds that precisely fit the anatomy of cartilage defects using medical imaging as the design template. We report here the use of a visible-light based PSL (VL-PSL system to encapsulate human adipose-derived stem cells (hASCs into a biodegradable polymer (poly-D,L-lactic acid/polyethylene glycol/ poly-D,L-lactic acid (PDLLA-PEG/hyaluronic acid (HA matrix to produce live cell constructs with customized architectures. After fabrication, hASCs showed high viability (84% and were uniformly distributed throughout the constructs, which possessed high mechanical property with a compressive modulus of 780 kPa. The hASC-seeded constructs were then cultured in Control or TGF-β3-containing chondrogenic medium for up to 28 days. In chondrogenic medium treated group (TGF-β3 group hASCs maintained 77% viability and expressed chondrogenic genes Sox9, collagen type II, and aggrecan at 11, 232, and 2.29 x 10(5 fold increases, respectively, compared to levels at day 0 in non-chondrogenic medium. The TGF-β3 group also produced a collagen type II and glycosaminoglycan (GAG-rich extracellular matrix, detected by immunohistochemistry, and Alcian blue and Safranin O staining suggesting robust chondrogenesis within the scaffold. Without chondroinductive addition (Control group, cell viability decreased with time (65% at 28 days and showed poor cartilage matrix deposition. After 28 days, mechanical strength of the TGF-β3 group remained high at 240 kPa. Thus, the PSL- and PLLA-PEG/HA based fabrication method using adult stem cells is a promising approach in producing mechanically competent engineered cartilage for joint cartilage

  6. Mold-shaped, nanofiber scaffold-based cartilage engineering using human mesenchymal stem cells and bioreactor.

    Science.gov (United States)

    Janjanin, Sasa; Li, Wan-Ju; Morgan, Meredith T; Shanti, Rabie M; Tuan, Rocky S

    2008-09-01

    Mesenchymal stem cell (MSC)-based tissue engineering is a promising future alternative to autologous cartilage grafting. This study evaluates the potential of using MSCs, seeded into electrospun, biodegradable polymeric nanofibrous scaffolds, to engineer cartilage with defined dimensions and shape, similar to grafts used for subcutaneous implantation in plastic and reconstructive surgery. Human bone marrow derived MSCs seeded onto nanofibrous scaffolds and placed in custom-designed molds were cultured for up to 42 days in bioreactors. Chondrogenesis was induced with either transforming growth factor-beta1 (TGF-beta1) alone or in combination with insulin-like growth factor-I (IGF-I). Constructs exhibited hyaline cartilage histology with desired thickness and shape as well as favorable tissue integrity and shape retention, suggesting the presence of elastic tissue. Time-dependent increase in cartilage matrix gene expression was seen in both types of culture: at Day 42, TGF-beta1/IGF-I treated cultures showed higher collagen Type 2 and aggrecan expression. Both culture conditions showed significant time-dependent increase in sulfated glycosaminoglycan and hydroxyproline contents. TGF-beta1/IGF-I-treated samples were significantly stiffer; with equilibrium compressive Young's modulus values reaching 17 kPa by Day 42. The successful ex vivo development of geometrically defined cartilaginous construct using customized molding suggests the potential of cell-based cartilage tissue for reconstructive surgery.

  7. Application of tissue engineering technology for formation of human articular cartilage in perfusion bioreactor

    Directory of Open Access Journals (Sweden)

    V. I. Sevastianov

    2017-01-01

    Full Text Available Formation of tissue-engineered construct was performed in a specially developed bioreactor. At first, a cellengineered construct of human cartilage tissue consisting of biopolymer microstructured collagen-containing hydrogel, mesenchymal stromal cells of human adipose tissue (hADMSCs and induction chondrogenic culture medium was prepared and placed in a perfusion bioreactor. As a result, on the 16th day of the study hADMSCs obtain a flattened shape typical for chondroblasts and demonstrate high proliferative activity with the formation of their own extracellular matrix. Histological analysis of the cultured system indicates the beginning of the formation of a tissue-engineered construct of human cartilage tissue.

  8. Immunogenicity of unprocessed and photooxidized bovine and human osteochondral grafts in collagen-sensitive mice

    Directory of Open Access Journals (Sweden)

    Lehmann Paul V

    2006-03-01

    Full Text Available Abstract Background Autologous and allogeneic osteochondral grafts have been used to repair damaged or diseased cartilage. There are drawbacks to both of these methods, however. Another possible source for osteochondral grafting is photooxidized xenograft scaffolds. The purpose of this study was to evaluate the adaptive immune response to unprocessed and photooxidized xenogeneic osteochondral grafts in a collagen-sensitive mouse model. Methods Unprocessed and photooxidized bovine and human osteochondral grafts were used. The grafts were implanted subcutaneously in collagen-sensitive DBA/1LacJ mice for four or twelve weeks. ELISPOT assays were conducted with spleen cells to evaluate the number of collagen-specific T cells that produce IL-2, IL-4, IL-5 or IFN-γ. Serum was collected and ELISA assays were performed to determine the titers of collagen-specific and total IgG, IgG1, IgG2a, or IgM antibodies. Histology was conducted on the retrieved osteochondral grafts. Results Results indicated that, with respect to adaptive T cell immunity, the photooxidized bovine grafts, unprocessed human grafts and photooxidized human grafts did not induce a significant response to collagen. The unprocessed bovine grafts, however, were slightly more immunogenic, inducing a weak immune response. With respect to antibody production, the bovine grafts were less immunogenic than the human grafts. Bovine collagen-specific IgG antibodies were not induced by these grafts, but production of IgM after twelve weeks was observed with both the unprocessed and photooxidized bovine grafts. In contrast, photooxidized human osteochondral grafts induced IgG1 and IgG2a antibodies, while the unprocessed human grafts did not. Pre-existing human collagen-specific IgM antibodies were present in all mice, including sham-operated negative controls that did not receive an implant. Histological analysis revealed some degree of fibrous encapsulation and inflammatory infiltrations in both

  9. Cartilage.

    Science.gov (United States)

    Caplan, Arnold I.

    1984-01-01

    Cartilage is a fundamental biological material that helps to shape the body and then helps to support it. Its fundamental properties of strength and resilience are explained in terms of the tissue's molecular structure. (JN)

  10. Inhibition of glycosaminoglycan incorporation influences collagen network formation during cartilage matrix production

    NARCIS (Netherlands)

    Bastiaansen-Jenniskens, Y.M.; Koevoet, W.; Jansen, K.M.B.; Verhaar, J.A.N.; Groot, J. de; Vanosch, G.J.V.M.

    2009-01-01

    To understand cartilage degenerative diseases and improve repair procedures, we investigate the influence of glycosaminoglycans (GAGs) on cartilage matrix biochemistry and functionality. Bovine articular chondrocytes were cultured in alginate beads with(out) para-nitrophenyl-beta-d-xyloside (PNPX)

  11. Engineering of human tracheal tissue with collagen-enforced poly-lactic-glycolic acid non-woven mesh: a preliminary study in nude mice.

    Science.gov (United States)

    Wu, Wei; Feng, Xue; Mao, Tianqiu; Feng, Xinghua; Ouyang, Hong-Wei; Zhao, Guifang; Chen, Fulin

    2007-06-01

    The purpose of the current study is to fabricate tissue engineered trachea with poly-lactic-glycolic acid (PLGA) non-woven mesh enforced by collagen type I. PLGA fibres coated with collagen solution were put together and fabricated into the shape of a human trachea, after drying and cross-linking treatment, a non-woven mesh with "C" shape formed. Chondrocytes from sheep nasal septum cartilage were expanded in vitro and seeded into PLGA/collagen non-woven mesh in the density of 5.0 x 10(7)mL(-1). After 5 days of in vitro incubation, six Cell-PLGA/collagen composites were implanted subcutaneously into the back of 6 nude mice to prefabricate a tissue engineering trachea. Eight weeks later, the cartilage formation was observed by gross inspection and histological examination. Cartilage-like tissue in the shape of the initial PLGA/collagen scaffold had been regenerated successfully without obvious inflammatory response. The tissue engineered trachea cartilage consisted of evenly spaced lacunae embedded in matrix stained red with safranin-O staining. The amount of GAGs in tissue engineered trachea cartilage reached 71.42% of normal value in native cartilage. This study demonstrated that collagen-enforced PLGA non-woven mesh facilitated the adhesion and proliferation of chondrocytes, it also owned adequate mechanical strength to serve as an ideal scaffold for trachea tissue engineering without internal support.

  12. Cross-linked type I and type II collagenous matrices for the repair of full-thickness articular cartilage defects--a study in rabbits.

    NARCIS (Netherlands)

    Buma, P.; Pieper, J.S.; Tienen, Tony van; Susante, J.L.C. van; Kraan, P.M. van der; Veerkamp, J.H.; Berg, W.B. van den; Veth, R.P.H.; Kuppevelt, A.H.M.S.M. van

    2003-01-01

    The physico-chemical properties of collagenous matrices may determine the tissue response after insertion into full-thickness articular cartilage defects. In this study, cross-linked type I and type II collagen matrices, with and without attached chondroitin sulfate, were implanted into

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

    Science.gov (United States)

    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.

  14. Vitamin D prevents articular cartilage erosion by regulating collagen II turnover through TGF-β1 in ovariectomized rats.

    Science.gov (United States)

    Li, S; Niu, G; Wu, Y; Du, G; Huang, C; Yin, X; Liu, Z; Song, C; Leng, H

    2016-02-01

    To explore the effect of vitamin D on turnover of articular cartilage with ovariectomy (OVX) induced OA, and to investigate transforming growth factor-β1 (TGF-β1) as a possible underlying mechanism mediated by 1α,25(OH)2D3. Sixty-six rats were randomly allocated into seven groups: sham plus control diet (SHAM+CTL), OVX+CTL diet, sham plus vitamin D-deficient (VDD) diet, OVX+VDD diet, and three groups of ovariectomized rats treated with different doses of 1α,25(OH)2D3. The cartilage erosion and the levels of serum 17β-estradiol, 1α,25(OH)2D3 and C-telopeptide of type II collagen (CTX-II) were measured. TGF-β1, type II Collagen (CII), matrix metalloproteinases (MMP)-9,-13 in articular cartilage were assessed by immunohistochemistry. TGF-β1 and CTX-II expression were measured in articular cartilage chondrocytes treated with/without tumor necrosis factor (TNF-α), 1α,25(OH)2D3, and TGF-β receptor inhibitor (SB505124) in vitro. Cartilage erosion due to OVX was significantly reduced in a dose-dependent manner by 1α,25(OH)2D3 supplementation, and exacerbated by VDD. The expressions of TGF-β1 and CII in articular cartilage were suppressed by OVX and VDD, and rescued by 1α,25(OH)2D3 supplementation. The expression of MMP-9,-13 in articular cartilage increased with OVX and VDD, and decreased with 1α,25(OH)2D3 supplementation. In vitro experiments showed that 1α,25(OH)2D3 increased the TGF-β1 expression of TNF-α stimulated chondrocytes in a dose-dependent manner. 1α,25(OH)2D3 significantly counteracted the increased CTX-II release due to TNF-α stimulation, and this effect was significantly suppressed by SB505124. VDD aggravated cartilage erosion, and 1α,25(OH)2D3 supplementation showed protective effects in OVX-induced OA partly through the TGF-β1 pathway. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  15. Biochemical effects on long-term frozen human costal cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Santin, Stefany P.; Martinho Junior, Antonio C.; Yoshito, Daniele; Soares, Fernando A.N.; Mathor, Monica B., E-mail: mathor@ipen.b [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2011-07-01

    Currently, the progresses on treatment of musculoskeletal diseases with the evolving of artificial implants and the success of tissue transplantation between genetically different individuals have conducted to an increase in radiosterilization. Regarding to tissue transplantation, it is essential to have sterile tissue and many tissue banks use radiosterilization as an effective method to sterilize these tissues. However, high doses of ionizing radiation and the preservation method may induce structural modifications in the tissues, as degradation of structural scaffold, decreasing its mechanical properties. Particularly, cartilage have been preserved in high concentrations of glycerol or deep-frozen at -70 degree C for storage after radiosterilization. Therefore, it is important to study the modifications induced in cartilage by preservation methods and by radiosterilization to determine the appropriated parameters for high quality of human allografts. Costal cartilages were obtained from cadaveric donors and were frozen at -20 degree C for 2 years long in order to compare with previous studies for fresh, deep-frozen and glycerolised cartilages. The mechanical tests were carried out in a universal testing machine until sample failure. According our results, there is no significant statistical difference between stress at break of fresh, long-term - 20 degree C frozen cartilages and deep-frozen cartilage. This early result suggests, regarding to tensile property, that long-term - 20 degree C frozen cartilages corresponds to glycerolised costal cartilages irradiated with 25 kGy or deep-frozen cartilages irradiated with 25 and 50 kGy. Thus, this long-term frozen cartilages may be used for tissue banks, but more studies about effects of ionizing radiation are necessary. (author)

  16. Physical mechanisms underlying the strain-rate-dependent mechanical behavior of kangaroo shoulder cartilage

    Science.gov (United States)

    Thibbotuwawa, Namal; Oloyede, Adekunle; Li, Tong; Singh, Sanjleena; Senadeera, Wijitha; Gu, YuanTong

    2015-09-01

    Due to anatomical and biomechanical similarities to human shoulder, kangaroo was chosen as a model to study shoulder cartilage. Comprehensive enzymatic degradation and indentation tests were applied on kangaroo shoulder cartilage to study mechanisms underlying its strain-rate-dependent mechanical behavior. We report that superficial collagen plays a more significant role than proteoglycans in facilitating strain-rate-dependent behavior of the kangaroo shoulder cartilage. By comparing the mechanical properties of degraded and normal cartilages, it was noted that proteoglycan and collagen degradation significantly compromised strain-rate-dependent mechanical behavior of the cartilage. Superficial collagen contributed equally to the tissue behavior at all strain-rates. This is different to the studies reported on knee cartilage and confirms the importance of superficial collagen on shoulder cartilage mechanical behavior. A porohyperelastic numerical model also indicated that collagen disruption would lead to faster damage of the shoulder cartilage than when proteoglycans are depleted.

  17. Extracellular matrix components and culture regimen selectively regulate cartilage formation by self-assembling human mesenchymal stem cells in vitro and in vivo.

    Science.gov (United States)

    Ng, Johnathan; Wei, Yiyong; Zhou, Bin; Burapachaisri, Aonnicha; Guo, Edward; Vunjak-Novakovic, Gordana

    2016-12-09

    Cartilage formation from self-assembling mesenchymal stem cells (MSCs) in vitro recapitulate important cellular events during mesenchymal condensation that precedes native cartilage development. The goal of this study was to investigate the effects of cartilaginous extracellular matrix (ECM) components and culture regimen on cartilage formation by self-assembling human MSCs in vitro and in vivo. Human bone marrow-derived MSCs (hMSCs) were seeded and compacted in 6.5-mm-diameter transwell inserts with coated (type I, type II collagen) or uncoated (vehicle) membranes, at different densities (0.5 × 10(6), 1.0 × 10(6), 1.5 × 10(6) per insert). Pellets were formed by aggregating hMSCs (0.25 × 10(6)) in round-bottomed wells. All tissues were cultured for up to 6 weeks for in vitro analyses. Discs (cultured for 6, 8 or 10 weeks) and pellets (cultured for 10 weeks) were implanted subcutaneously in immunocompromised mice to evaluate the cartilage stability in vivo. Type I and type II collagen coatings enabled cartilage disc formation from self-assembling hMSCs. Without ECM coating, hMSCs formed dome-shaped tissues resembling the pellets. Type I collagen, expressed in the prechondrogenic mesenchyme, improved early chondrogenesis versus type II collagen. High seeding density improved cartilage tissue properties but resulted in a lower yield of disc formation. Discs and pellets exhibited compositional and organizational differences in vitro and in vivo. Prolonged chondrogenic induction of the discs in vitro expedited endochondral ossification in vivo. The outcomes of cartilage tissues formed from self-assembling MSCs in vitro and in vivo can be modulated by the control of culture parameters. These insights could motivate new directions for engineering cartilage and bone via a cartilage template from self-assembling MSCs.

  18. Latent Transforming Growth Factor-beta1 Functionalised Electrospun Scaffolds Promote Human Cartilage Differentiation: Towards an Engineered Cartilage Construct

    Directory of Open Access Journals (Sweden)

    Erh-Hsuin Lim

    2013-11-01

    Full Text Available BackgroundTo overcome the potential drawbacks of a short half-life and dose-related adverse effects of using active transforming growth factor-beta 1 for cartilage engineering, a cell-mediated latent growth factor activation strategy was developed incorporating latent transforming growth factor-β1 (LTGF into an electrospun poly(L-lactide scaffold.MethodsThe electrospun scaffold was surface modified with NH3 plasma and biofunctionalised with LTGF to produce both random and orientated biofunctionalised electrospun scaffolds. Scaffold surface chemical analysis and growth factor bioavailability assays were performed. In vitro biocompatibility and human nasal chondrocyte gene expression with these biofunctionalised electrospun scaffold templates were assessed. In vivo chondrogenic activity and chondrocyte gene expression were evaluated in athymic rats.ResultsChemical analysis demonstrated that LTGF anchored to the scaffolds was available for enzymatic, chemical and cell activation. The biofunctionalised scaffolds were non-toxic. Gene expression suggested chondrocyte re-differentiation after 14 days in culture. By 6 weeks, the implanted biofunctionalised scaffolds had induced highly passaged chondrocytes to re-express Col2A1 and produce type II collagen.ConclusionsWe have demonstrated a proof of concept for cell-mediated activation of anchored growth factors using a novel biofunctionalised scaffold in cartilage engineering. This presents a platform for development of protein delivery systems and for tissue engineering.

  19. Latent Transforming Growth Factor-beta1 Functionalised Electrospun Scaffolds Promote Human Cartilage Differentiation: Towards an Engineered Cartilage Construct.

    Science.gov (United States)

    Lim, Erh-Hsuin; Sardinha, Jose Paulo; Myers, Simon; Stevens, Molly

    2013-11-01

    To overcome the potential drawbacks of a short half-life and dose-related adverse effects of using active transforming growth factor-beta 1 for cartilage engineering, a cell-mediated latent growth factor activation strategy was developed incorporating latent transforming growth factor-β1 (LTGF) into an electrospun poly(L-lactide) scaffold. The electrospun scaffold was surface modified with NH3 plasma and biofunctionalised with LTGF to produce both random and orientated biofunctionalised electrospun scaffolds. Scaffold surface chemical analysis and growth factor bioavailability assays were performed. In vitro biocompatibility and human nasal chondrocyte gene expression with these biofunctionalised electrospun scaffold templates were assessed. In vivo chondrogenic activity and chondrocyte gene expression were evaluated in athymic rats. Chemical analysis demonstrated that LTGF anchored to the scaffolds was available for enzymatic, chemical and cell activation. The biofunctionalised scaffolds were non-toxic. Gene expression suggested chondrocyte re-differentiation after 14 days in culture. By 6 weeks, the implanted biofunctionalised scaffolds had induced highly passaged chondrocytes to re-express Col2A1 and produce type II collagen. We have demonstrated a proof of concept for cell-mediated activation of anchored growth factors using a novel biofunctionalised scaffold in cartilage engineering. This presents a platform for development of protein delivery systems and for tissue engineering.

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

    Directory of Open Access Journals (Sweden)

    Schwartz SR

    2012-07-01

    Full Text Available Stephen R Schwartz,1 Joosang Park21International Research Services Inc, Port Chester, NY, USA; 2BioCell Technology, LLC, Newport Beach, CA, USAAbstract: Skin aging and its clinical manifestation is associated with altered molecular metabolism in the extracellular matrix of the dermis. In a pilot open-label study, we investigated the effect of a dietary supplement, BioCell Collagen® (BCC, which contains a naturally occurring matrix of hydrolyzed collagen type II and low-molecular-weight hyaluronic acid and chondroitin sulfate, in 26 healthy females who displayed visible signs of natural and photoaging in the face. Daily supplementation with 1 g of BCC for 12 weeks led to a significant reduction of skin dryness/scaling (76%, P = 0.002 and global lines/wrinkles (13.2%, P = 0.028 as measured by visual/tactile score. Additionally, a significant increase in the content of hemoglobin (17.7%, P = 0.018 and collagen (6.3%, P = 0.002 in the skin dermis was observed after 6 weeks of supplementation. At the end of the study, the increase in hemoglobin remained significant (15%, P = 0.008, while the increase in collagen content was maintained, but the difference from baseline was not significant (3.5%, P = 0.134. This study provides preliminary data suggesting that dietary supplementation with BCC elicits several physiological events which can be harnessed to counteract natural photoaging processes to reduce visible aging signs in the human face. A controlled study is necessary to verify these observations.Keywords: BioCell Collagen, chicken sternal cartilage extract, hydrolyzed collagen type II, low-molecular-weight hyaluronic acid, skin aging

  1. The effects of glycosaminoglycan content on the compressive modulus of cartilage engineered in type II collagen scaffolds.

    Science.gov (United States)

    Pfeiffer, E; Vickers, S M; Frank, E; Grodzinsky, A J; Spector, M

    2008-10-01

    The current study determined the unconfined compressive modulus of tissue-engineered constructs with varying sulfated glycosaminoglycan (GAG) density produced by goat articular chondrocytes in type II collagen scaffolds prepared with a range of cross-link densities and various times in culture. The purpose of this work is to establish a basis for future studies employing constructs of selected maturity (e.g., 25%, 50%, or 75% normal GAG content) for cartilage repair in vivo. Porous scaffolds (8 mm diameter by 2 mm thick) were fabricated from porcine type II collagen by freeze-drying, followed by dehydrothermal treatment and carbodiimide cross-linking. In a pilot study, passage 3 adult caprine articular chondrocytes isolated from one goat were grown in scaffolds with six cross-link densities for 2, 3, 4, and 6 weeks (n=3). The goal was to select scaffold cross-link densities and times in culture that would produce constructs with approximately 25%, 50% and 75% the GAG density of native articular cartilage. Based on the results of the pilot study, chondrocytes from three goats were grown in scaffolds with two cross-link densities for three time periods: 3, 5, and 9 weeks (n=6; one of the cross-link groups was run in quadruplicate). The equilibrium modulus from unconfined compression testing of these samples was correlated with GAG content. There was a notable increase in GAG density with decreasing cross-link density. Histological analysis verified a chondrogenic phenotype and revealed various amounts of GAG and type II collagen-containing cartilage. The correlation between modulus and GAG density had a linear coefficient of determination of 0.60. One group with a mean GAG density of 22 microg/mm(3), which was 140% the GAG density of normal caprine articular cartilage, averaged a compressive modulus of 31.5 kPa, which was 10% of caprine articular cartilage tested in this study. The GAG density and modulus of tissue-engineered constructs can be controlled by the

  2. Effect of seeding and bioreactor culture conditions on the development of human tissue-engineered cartilage.

    Science.gov (United States)

    Mahmoudifar, Nastaran; Doran, Pauline M

    2006-06-01

    Human cartilage was produced using fetal chondrocytes seeded into polyglycolic acid (PGA) mesh scaffolds and cultured in recirculation bioreactors. The effect of scaffold thickness, seeding cell density, and bioreactor operating conditions on the quality of the engineered cartilage was investigated. Thin (2.15-mm-thick) PGA scaffolds lost their structural integrity during bioreactor culture and the resulting constructs were small and misshapen compared with tissues generated using 4.75-mm-thick scaffolds. Increasing the seeding cell number from 1.2 x 10(7) to 2.2 x 10(7 )per 4.75-mm-thick scaffold resulted in a doubling of the construct wet weight, a 4.4-fold increase in glycosaminoglycan (GAG) concentration, and a 2.9-fold increase in total collagen concentration in the tissues. Levels of GAG and total collagen were also improved significantly when 100 mL or 50% v/v of the culture medium was replaced periodically during operation of the bioreactors compared with 50, 25, or 5 mL. The proportion of GAG lost from the tissues into the medium was reduced by increasing the seeding cell number and replaced medium volume. This work demonstrates that the quality of tissue-engineered cartilage can be manipulated substantially depending on the cell seeding and bioreactor culture conditions employed.

  3. Repair of articular cartilage defects one year after treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2).

    Science.gov (United States)

    Sellers, R S; Zhang, R; Glasson, S S; Kim, H D; Peluso, D; D'Augusta, D A; Beckwith, K; Morris, E A

    2000-02-01

    Damaged articular cartilage has a limited ability to repair. Operative removal of damaged cartilage and penetration into the subchondral bone to allow population of the defect with progenitor cells can result in filling of the defect with repair tissue. However, this repair tissue often degenerates over time because of its inability to withstand the mechanical forces to which it is subjected. We previously reported that recombinant human bone morphogenetic protein-2 (rhBMP-2) improves the repair of full-thickness defects of cartilage as long as six months postoperatively. We have now extended that study to examine the quality of the repair tissue at one year. Full-thickness defects of cartilage were created in the trochlear groove of twenty-five adult New Zealand White rabbits. Eight defects were left empty, eight were filled with a collagen sponge, and nine were filled with a collagen sponge impregnated with five micrograms of rhBMP-2. The animals were killed at fifty-two weeks postoperatively, and the gross appearance of the healed defect was assessed. The repair tissue was examined histologically and was evaluated, according to a grading scale, by four individuals who were blinded with respect to the treatment. The tissue sections were immunostained with antibodies against type-I collagen, type-II collagen, aggrecan, and link protein. The residence time of the rhBMP-2 in the cartilage defect was evaluated in vivo with use of scintigraphic imaging of radiolabeled protein. One year after a single implantation of a collagen sponge containing five micrograms of rhBMP-2, the defects had a significantly better histological appearance than the untreated defects (those left empty or filled with a collagen sponge). The histological features that showed improvement were integration at the margin, cellular morphology, architecture within the defect, and reformation of the tidemark. The total scores were also better for the defects treated with rhBMP-2 than for the

  4. Concentration determination of collagen and proteoglycan in bovine nasal cartilage by Fourier transform infrared imaging and PLS

    Science.gov (United States)

    Zhang, Xuexi; Xiao, Zhi-Yan; Yin, Jianhua; Xia, Yang

    2014-09-01

    Fourier transform infrared imaging (FTIRI) combined with chemometrics can be used to detect the structure of bio-macromolecule, measure the concentrations of some components, and so on. In this study, FTIRI with Partial Least-Squares (PLS) regression was applied to study the concentration of two main components in bovine nasal cartilage (BNC), collagen and proteoglycan. An infrared spectrum library was built by mixing the collagen and chondroitin 6-sulfate (main of proteoglycan) at different ratios. Some pretreatments are needed for building PLS model. FTIR images were collected from BNC sections at 6.25μm and 25μm pixel size. The spectra extracted from BNC-FTIR images were imported into the PLS regression program to predict the concentrations of collagen and proteoglycan. These PLS-determined concentrations are agreed with the result in our previous work and biochemical analytical results. The prediction shows that the concentrations of collagen and proteoglycan in BNC are comparative on the whole. However, the concentration of proteoglycan is a litter higher than that of collagen, to some extent.

  5. Age-associated glycopeptide pigment in human costal cartilage.

    Science.gov (United States)

    van der Korst, J K; Willekens, F L; Lansink, A G; Henrichs, A M

    1977-12-01

    Age-associated pigmentation of human costal cartilage is caused by the accumulation of a brown water-soluble substance which can be only be extracted after proteolytic disruption of the cartilage. After isolation by gel filtration and ion exchange chromatography, the compound was identified as an acid glycopeptide. In contrast to ochronotic pigment and an artificial pigment derived by oxidation of homogentistic acid in alkaline solution, the age-associated cartilage pigment was strongly fluorescent and did not form insoluble complexes with cetylpyridinium chloride. Moreover, age-associated cartilage pigment is alkali resistant, in contrast to the ochronotic pigment. The pigment differs from lipofuscin in being strongly hydrophilic and having no affinity for fat stains. The unidentified chromophore could not be separated from the glycopeptide molecule.

  6. The composition of engineered cartilage at the time of implantation determines the likelihood of regenerating tissue with a normal collagen architecture.

    Science.gov (United States)

    Nagel, Thomas; Kelly, Daniel J

    2013-04-01

    The biomechanical functionality of articular cartilage is derived from both its biochemical composition and the architecture of the collagen network. Failure to replicate this normal Benninghoff architecture in regenerating articular cartilage may in turn predispose the tissue to failure. In this article, the influence of the maturity (or functionality) of a tissue-engineered construct at the time of implantation into a tibial chondral defect on the likelihood of recapitulating a normal Benninghoff architecture was investigated using a computational model featuring a collagen remodeling algorithm. Such a normal tissue architecture was predicted to form in the intact tibial plateau due to the interplay between the depth-dependent extracellular matrix properties, foremost swelling pressures, and external mechanical loading. In the presence of even small empty defects in the articular surface, the collagen architecture in the surrounding cartilage was predicted to deviate significantly from the native state, indicating a possible predisposition for osteoarthritic changes. These negative alterations were alleviated by the implantation of tissue-engineered cartilage, where a mature implant was predicted to result in the formation of a more native-like collagen architecture than immature implants. The results of this study highlight the importance of cartilage graft functionality to maintain and/or re-establish joint function and suggest that engineering a tissue with a native depth-dependent composition may facilitate the establishment of a normal Benninghoff collagen architecture after implantation into load-bearing defects.

  7. Comparative digital cartilage histology for human and common osteoarthritis models

    Directory of Open Access Journals (Sweden)

    Pedersen DR

    2013-02-01

    Full Text Available Douglas R Pedersen, Jessica E Goetz, Gail L Kurriger, James A MartinDepartment of Orthopaedics and Rehabilitation, University of Iowa, Iowa City, IA, USAPurpose: This study addresses the species-specific and site-specific details of weight-bearing articular cartilage zone depths and chondrocyte distributions among humans and common osteoarthritis (OA animal models using contemporary digital imaging tools. Histological analysis is the gold-standard research tool for evaluating cartilage health, OA severity, and treatment efficacy. Historically, evaluations were made by expert analysts. However, state-of-the-art tools have been developed that allow for digitization of entire histological sections for computer-aided analysis. Large volumes of common digital cartilage metrics directly complement elucidation of trends in OA inducement and concomitant potential treatments.Materials and methods: Sixteen fresh human knees, 26 adult New Zealand rabbit stifles, and 104 bovine lateral plateaus were measured for four cartilage zones and the cell densities within each zone. Each knee was divided into four weight-bearing sites: the medial and lateral plateaus and femoral condyles.Results: One-way analysis of variance followed by pairwise multiple comparisons (Holm–Sidak method at a significance of 0.05 clearly confirmed the variability between cartilage depths at each site, between sites in the same species, and between weight-bearing articular cartilage definitions in different species.Conclusion: The present study clearly demonstrates multisite, multispecies differences in normal weight-bearing articular cartilage, which can be objectively quantified by a common digital histology imaging technique. The clear site-specific differences in normal cartilage must be taken into consideration when characterizing the pathoetiology of OA models. Together, these provide a path to consistently analyze the volume and variety of histologic slides necessarily generated

  8. Influence of site and age on biochemical characteristics of the collagen network of equine articular cartilage

    NARCIS (Netherlands)

    Brama, P.A.J.; TeKoppele, J.M.; Bank, R.A.; Weeren, P.R. van; Barneveld, A.

    1999-01-01

    Objective - To determine variations in biochemical characteristics of equine articular cartilage in relation to age and the degree of predisposition for osteochondral disease at a specific site. Sample Population - Articular cartilage specimens from 53 horses 4 to 30 years old. Procedure - Healthy

  9. Oriented Collagen Scaffolds for Tissue Engineering

    OpenAIRE

    Shohta Kodama; Taro Saku; Hiroshi Mikami; Go Kuwahara; Toru Kosaka; Yoshihiro Isobe

    2012-01-01

    Oriented collagen scaffolds were developed in the form of sheet, mesh and tube by arraying flow-oriented collagen string gels and dehydrating the arrayed gels. The developed collagen scaffolds can be any practical size with any direction of orientation for tissue engineering applications. The birefringence of the collagen scaffolds was quantitatively analyzed by parallel Nicols method. Since native collagen in the human body has orientations such as bone, cartilage, tendon and cornea, and the...

  10. In vivo cartilage formation using chondrogenic-differentiated human adipose-derived mesenchymal stem cells mixed with fibrin glue.

    Science.gov (United States)

    Jung, Sung-No; Rhie, Jong Won; Kwon, Ho; Jun, Young Joon; Seo, Je-Won; Yoo, Gyeol; Oh, Deuk Young; Ahn, Sang Tae; Woo, Jihyoun; Oh, Jieun

    2010-03-01

    Human adipose-derived mesenchymal stem cells (MSCs) were differentiated into chondrogenic MSCs, and fibrin glue was used together to explore the feasibility of whether cartilages can be generated in vivo by injecting the differentiated cells. Mesenchymal stem cells extracted from human adipose were differentiated into chondrogenic MSCs, and such differentiated cells mixed with fibrin glue were injected subcutaneously into the back of the nude mouse. In addition to visual evaluation of the tissues formed after 4, 8, and 12 weeks, hematoxylin-eosin staining, Masson trichrome staining, measurement of glycosaminoglycan concentration using dimethylmethylene blue, agreecan through reverse transcriptase-polymerase chain reaction, type II collagen, and expression of SOX-9 were verified. Moreover, the results were compared with 2 groups of controls: 1 control group that received only injection of chondrogenic-differentiated MSC and the supporting control group that received only fibrin glue injection. For the experimental group, cartilage-like tissues were formed after 4, 8, and 12 weeks. Formation of cartilage tissues was not observed in any of 4, 8, and 12 weeks of the control group. The supporting control group had only a small structure formation after 4 weeks, but the formed structure was completely decomposed by the 8th and 12th weeks. The range of staining dramatically increased with time at 4, 8, and 12 weeks in Masson trichrome staining. The concentration of glycosaminoglycan also increased with time. The increased level was statistically significant with more than 3 times more after 8 weeks compared with 4 weeks and more than 2 times more after 12 weeks compared with 8 weeks. Also, in reverse transcriptase-polymerase chain reaction at 4, 8, and 12 weeks, all results expressed a cartilage-specific gene called aggrecan, type II collagen, and SOX-9. The study verified that the chondrogenic-differentiated MSCs derived from human adipose tissues with fibrin glue can

  11. Moderate loading of the human osteoarthritic knee joint leads to lowering of intraarticular cartilage oligomeric matrix protein

    DEFF Research Database (Denmark)

    Helmark, Ida C; Petersen, Marie C H; Christensen, Helle E

    2012-01-01

    osteoarthritic joint determined by biochemical markers of cartilage turnover and inflammation in the synovial fluid (SF), serum and urine. Eleven subjects with OA of the knee(s), but with no other joint- or inflammatory disorders, volunteered for the study and had samples of blood, urine and synovial fluid drawn...... both at baseline and following 30-min one-legged knee-extension exercise. Workload: 60% of 1 RM (Repetition Maximum). Determination of cartilage oligomeric matrix protein (COMP), aggrecan, C-terminal collagen II peptide (CTX-II) and interleukin (IL)-6 were performed in synovial fluid (SF), serum......The non-pharmacological treatment of osteoarthritis (OA) includes exercise therapy; however, little is known about the specific effect of exercise on the joint per se. The purpose of the present study was to investigate the direct effects of a load-bearing exercise upon cartilage in a single, human...

  12. Organization of fibrillar collagen in the human and bovine cornea: collagen types V and III.

    Science.gov (United States)

    White, J; Werkmeister, J A; Ramshaw, J A; Birk, D E

    1997-01-01

    The localization and fibrillar organization of collagen types V and III in the human and bovine corneal stromas were studied. In the chicken cornea, type V co-assembles with type I collagen as heterotypic fibrils and this interaction is involved in the regulation of fibril diameter necessary for corneal transparency. To determine whether this is a regulatory mechanism common to the corneas of different species the human and bovine corneal stroma were studied. Collagen type V was found in the epithelium and Bowman's membrane in the untreated adult human and bovine cornea using immunofluorescence microscopy. In the absence of any treatment, there was no type V reactivity within the stroma. However, type V collagen was detected homogeneously throughout the corneal stroma after treatments that partially disrupt fibril structure. The reactivity was strongest in the cornea, weaker in the limbus and weakest in the sclera. Fetal corneas showed similar reactivity for type V collagen, but unlike the adult, the stroma was slightly reactive. Immunoelectron microscopy demonstrated that type V collagen was associated with disrupted, but not with intact, fibrils in both human and bovine corneal stroma. Type III collagen reactivity was not detected in the cornea, but was present subepithelially in the limbus and in the scleral stroma. These data indicate that type V collagen is a component of striated collagen fibrils throughout the human and bovine corneal stromas. The interaction of type I and V collagen as heterotypic fibrils masks the helical epitope recognized by the monoclonal antibody against type V collagen. The heterotypic interactions of collagen type V indicate a role in the regulation of fibril diameter analogous to that described in the avian cornea.

  13. Anisotropy of collagen fibre alignment in bovine cartilage: comparison of polarised light microscopy and spatially resolved diffusion-tensor measurements.

    Science.gov (United States)

    de Visser, S K; Bowden, J C; Wentrup-Byrne, E; Rintoul, L; Bostrom, T; Pope, J M; Momot, K I

    2008-06-01

    To compare collagen fibre alignment angles obtained from polarised light microscopy (PLM) and diffusion-tensor imaging (DTI) in bovine articular cartilage. Five samples of bovine articular cartilage from five different animals were studied using magnetic resonance imaging and PLM techniques. T(2)-weighted, diffusion-tensor (DT), and PLM images were acquired for each sample and average depth profiles of the PLM and DTI angles, as well as the banding patterns observed in T(2)-weighted magnetic resonance (MR) images, were compared. Statistical properties of the distributions of the DTI and PLM angles were examined. The samples exhibited a range of alignment morphologies. In the samples with the "conventional" three-zone alignment pattern, a correlation between the PLM and DTI alignment zones and the banding in T(2)-weighted MR images was observed. The shapes of the depth profiles of the PLM and DTI alignment angles were qualitatively similar for each sample. Three samples showed good quantitative correlation between the DT and PLM alignment angles. The correlation between the diffusion and PLM alignment angles was best in the regions of low degree of disorder of fibre alignment. This study provides the first quantitative comparison of DTI of cartilage with the more established PLM techniques. The correlation between alignment angles derived from PLM and DTI data was evident across a wide range of alignment morphologies. The results support the use of DTI for the quantitative measurement of collagen fibre alignment. The microscopic-scale (~10 microm) dispersion of fibre alignment angles appears to be an important factor for understanding the extent of quantitative correlation between PLM and DTI results.

  14. Is T1ρ Mapping an Alternative to Delayed Gadolinium-enhanced MR Imaging of Cartilage in the Assessment of Sulphated Glycosaminoglycan Content in Human Osteoarthritic Knees? An in Vivo Validation Study.

    Science.gov (United States)

    van Tiel, Jasper; Kotek, Gyula; Reijman, Max; Bos, Pieter K; Bron, Esther E; Klein, Stefan; Nasserinejad, Kazem; van Osch, Gerjo J V M; Verhaar, Jan A N; Krestin, Gabriel P; Weinans, Harrie; Oei, Edwin H G

    2016-05-01

    To determine if T1ρ mapping can be used as an alternative to delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) in the quantification of cartilage biochemical composition in vivo in human knees with osteoarthritis. This study was approved by the institutional review board. Written informed consent was obtained from all participants. Twelve patients with knee osteoarthritis underwent dGEMRIC and T1ρ mapping at 3.0 T before undergoing total knee replacement. Outcomes of dGEMRIC and T1ρ mapping were calculated in six cartilage regions of interest. Femoral and tibial cartilages were harvested during total knee replacement. Cartilage sulphated glycosaminoglycan (sGAG) and collagen content were assessed with dimethylmethylene blue and hydroxyproline assays, respectively. A four-dimensional multivariate mixed-effects model was used to simultaneously assess the correlation between outcomes of dGEMRIC and T1ρ mapping and the sGAG and collagen content of the articular cartilage. T1 relaxation times at dGEMRIC showed strong correlation with cartilage sGAG content (r = 0.73; 95% credibility interval [CI] = 0.60, 0.83) and weak correlation with cartilage collagen content (r = 0.40; 95% CI: 0.18, 0.58). T1ρ relaxation times did not correlate with cartilage sGAG content (r = 0.04; 95% CI: -0.21, 0.28) or collagen content (r = -0.05; 95% CI = -0.31, 0.20). dGEMRIC can help accurately measure cartilage sGAG content in vivo in patients with knee osteoarthritis, whereas T1ρ mapping does not appear suitable for this purpose. Although the technique is not completely sGAG specific and requires a contrast agent, dGEMRIC is a validated and robust method for quantifying cartilage sGAG content in human osteoarthritis subjects in clinical research. (©) RSNA, 2015.

  15. Troponin I is present in human cartilage and inhibits angiogenesis

    Science.gov (United States)

    Moses, Marsha A.; Wiederschain, Dmitri; Wu, Inmin; Fernandez, Cecilia A.; Ghazizadeh, Vahid; Lane, William S.; Flynn, Evelyn; Sytkowski, Arthur; Tao, Terence; Langer, Robert

    1999-01-01

    Cartilage is an avascular and relatively tumor-resistant tissue. Work from a number of laboratories, including our own, has demonstrated that cartilage is an enriched source of endogenous inhibitors of angiogenesis. In the course of a study designed to identify novel cartilage-derived inhibitors of new capillary growth, we have purified an inhibitory protein that was identified by peptide microsequencing and protein database analysis as troponin I (TnI). TnI is a subunit of the troponin complex (troponin-C and troponin-T being the other two), which, along with tropomyosin, is responsible for the calcium-dependent regulation of striated muscle contraction; independently, TnI is capable of inhibiting actomyosin ATPase. Because troponin has never previously been reported to be present in cartilage, we have cloned and expressed the cDNA of human cartilage TnI, purified this protein to apparent homogeneity, and demonstrated that it is a potent and specific inhibitor of angiogenesis in vivo and in vitro, as well as of tumor metastasis in vivo. PMID:10077564

  16. Histochemical characterization of human osteochondral tissue: comparison between healthy cartilage, arthrotic tissues, and cartilage defect treated with MACI technique

    Directory of Open Access Journals (Sweden)

    F. Tessarolo

    2011-01-01

    Full Text Available Matrix-induced sutologous chondrocytes implantation (MACI is a promising technique for the treatment of articular cartilage lesions, but long time outcome have to be established. We developed and optimized specific techniques of histochemical staining to characterize healthy and pathologic osteochondral tissue. Seven different staining protocols were applied to assess tissue architecture, cells morphology, proteoglycan content, and collagen fibers distribution. Potentialities of histochemical staining and histomorphology of biopsies from second look arthroscopy will be presented.

  17. Successful Low-Cost Scaffold-Free Cartilage Tissue Engineering Using Human Cartilage Progenitor Cell Spheroids Formed by Micromolded Nonadhesive Hydrogel

    Directory of Open Access Journals (Sweden)

    Mellannie P. Stuart

    2017-01-01

    Full Text Available The scaffold-free tissue engineering using spheroids is pointed out as an approach for optimizing the delivery system of cartilage construct. In this study, we aimed to evaluate the micromolded nonadhesive hydrogel (MicroTissues® for spheroid compaction (2-day culture and spontaneous chondrogenesis (21-day culture using cartilage progenitors cells (CPCs from human nasal septum without chondrogenic stimulus. CPC spheroids showed diameter stability (486 μm ± 65, high percentage of viable cells (88.1 ± 2.1, and low percentage of apoptotic cells (2.3%. After spheroid compaction, the synthesis of TGF-β1, TGF-β2, and TGF-β3 was significantly higher compared to monolayer (p<0.005. Biomechanical assay revealed that the maximum forces applied to spheroids after chondrogenesis were 2.6 times higher than for those cultured for 2 days. After spontaneous chondrogenesis, CPC spheroids were entirely positive for N-cadherin, collagen type II and type VI, and aggrecan and chondroitin sulfate. Comparing to monolayer, the expression of SOX5 and SOX6 genes analyzed by qPCR was significantly upregulated (p<0.01. Finally, we observed the capacity of CPC spheroids starting to fuse. To the best of our knowledge, this is the first time in the scientific literature that human CPC spheroids were formed by micromolded nonadhesive hydrogel, achieving a successful scaffold-free cartilage engineering without chondrogenic stimulus (low cost.

  18. Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilage.

    Science.gov (United States)

    Sakai, Shinsuke; Mishima, Hajime; Ishii, Tomoo; Akaogi, Hiroshi; Yoshioka, Tomokazu; Ohyabu, Yoshimi; Chang, Fei; Ochiai, Naoyuki; Uemura, Toshimasa

    2009-04-01

    The method of constructing cartilage tissue from bone marrow-derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow-derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per microg DNA of the tissues were 10.01 +/- 3.49 microg/microg DNA in the case of the RWV bioreactor and 6.27 +/- 3.41 microg/microg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three-dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow-derived cells.

  19. Intermittent hydrostatic compressive force stimulates exclusively the proteoglycan synthesis of osteoarthritic human cartilage

    NARCIS (Netherlands)

    Lafeber, F.; Veldhuijzen, J. P.; Vanroy, J. L.; Huber-Bruning, O.; Bijlsma, J. W.

    1992-01-01

    In paired observations the in vitro proteoglycan turnover was studied of human normal and osteoarthritic cartilage in the absence and presence of intermittent hydrostatic compressive force. Shortly after collection, osteoarthritic cartilage showed a higher proteoglycan synthesis rate than normal

  20. Special pattern of endochondral ossification in human laryngeal cartilages: X-ray and light-microscopic studies on thyroid cartilage.

    Science.gov (United States)

    Claassen, Horst; Schicht, Martin; Sel, Saadettin; Paulsen, Friedrich

    2014-04-01

    Endochondral ossification is a process that also occurs in the skeleton of the larynx. Differences in the ossification mechanism in comparison to growth plates are not understood until now. To get deeper insights into this process, human thyroid cartilage was investigated by the use of X-rays and a series of light-microscopic stainings. A statistical analysis of mineralization was done by scanning areas of mineralized cartilage and of ossification. We detected a special mode of endochondral ossification which differs from the processes in growth plates. Thyroid cartilage ossifies very slowly and in a gender-specific manner. Compared with age-matched women, bone formation in thyroid cartilage of men is significantly higher in the age group 41-60 years. Endochondral ossification is prepared by internal changes of extracellular matrix leading to areas of asbestoid fibers with ingrowing cartilage canals. In contrast to growth plates, bone is deposited on large areas of mineralized cartilage, which appear at the rims of cartilage canals. Furthermore, primary parallel fibered bone was observed which was deposited on woven bone. The predominant bone type is cancellous bone with trabeculae, whereas compact bone with Haversian systems was seldom found. Trabeculae contain a great number of reversal and arresting lines meaning that the former were often reconstructed and that bone formation was arrested and resumed again with advancing age. It is hypothesized that throughout life trabeculae of ossified thyroid cartilage undergo adaptation to different loads due to the use of voice. Copyright © 2014 Wiley Periodicals, Inc.

  1. Human Endogenous Retrovirus W Activity in Cartilage of Osteoarthritis Patients

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    Signy Bendiksen

    2014-01-01

    Full Text Available The etiology of viruses in osteoarthritis remains controversial because the prevalence of viral nucleic acid sequences in peripheral blood or synovial fluid from osteoarthritis patients and that in healthy control subjects are similar. Until now the presence of virus has not been analyzed in cartilage. We screened cartilage and chondrocytes from advanced and non-/early osteoarthritis patients for parvovirus B19, herpes simplex virus-1, Epstein Barr virus, cytomegalovirus, human herpes virus-6, hepatitis C virus, and human endogenous retroviruses transcripts. Endogenous retroviruses transcripts, but none of the other viruses, were detected in 15 out the 17 patients. Sequencing identified the virus as HERV-WE1 and E2. HERV-W activity was confirmed by high expression levels of syncytin, dsRNA, virus budding, and the presence of virus-like particles in all advanced osteoarthritis cartilages examined. Low levels of HERV-WE1, but not E2 envelope RNA, were observed in 3 out of 8 non-/early osteoarthritis patients, while only 3 out of 7 chondrocytes cultures displayed low levels of syncytin, and just one was positive for virus-like particles. This study demonstrates for the first time activation of HERV-W in cartilage of osteoarthritis patients; however, a causative role for HERV-W in development or deterioration of the disease remains to be proven.

  2. The distribution of YKL-40 in osteoarthritic and normal human articular cartilage

    DEFF Research Database (Denmark)

    Volck, B; Ostergaard, K; Johansen, J S

    1999-01-01

    YKL-40, also called human cartilage glycoprotein-39, is a major secretory protein of human chondrocytes in cell culture. YKL-40 mRNA is expressed by cartilage from patients with rheumatoid arthritis, but is not detectable in normal human cartilage. The aim was to investigate the distribution of YKL......-40 in osteoarthritic (n=9) and macroscopically normal (n=5) human articular cartilage, collected from 12 pre-selected areas of the femoral head, to discover a potential role for YKL-40 in cartilage remodelling in osteoarthritis. Immunohistochemical analysis showed that YKL-40 staining was found...

  3. In Vivo Articular Cartilage Regeneration Using Human Dental Pulp Stem Cells Cultured in an Alginate Scaffold: A Preliminary Study

    Directory of Open Access Journals (Sweden)

    Manuel Mata

    2017-01-01

    Full Text Available Osteoarthritis is an inflammatory disease in which all joint-related elements, articular cartilage in particular, are affected. The poor regeneration capacity of this tissue together with the lack of pharmacological treatment has led to the development of regenerative medicine methodologies including microfracture and autologous chondrocyte implantation (ACI. The effectiveness of ACI has been shown in vitro and in vivo, but the use of other cell types, including bone marrow and adipose-derived mesenchymal stem cells, is necessary because of the poor proliferation rate of isolated articular chondrocytes. In this investigation, we assessed the chondrogenic ability of human dental pulp stem cells (hDPSCs to regenerate cartilage in vitro and in vivo. hDPSCs and primary isolated rabbit chondrocytes were cultured in chondrogenic culture medium and found to express collagen II and aggrecan. Both cell types were cultured in 3% alginate hydrogels and implanted in a rabbit model of cartilage damage. Three months after surgery, significant cartilage regeneration was observed, particularly in the animals implanted with hDPSCs. Although the results presented here are preliminary, they suggest that hDPSCs may be useful for regeneration of articular cartilage.

  4. Dietary extra-virgin olive oil prevents inflammatory response and cartilage matrix degradation in murine collagen-induced arthritis.

    Science.gov (United States)

    Rosillo, María Angeles; Sánchez-Hidalgo, Marina; Sánchez-Fidalgo, Susana; Aparicio-Soto, Marina; Villegas, Isabel; Alarcón-de-la-Lastra, Catalina

    2016-02-01

    Current experimental studies support a beneficial role of extra-virgin olive oil (EVOO) in several inflammatory diseases. The present study was designed to evaluate the effects of dietary EVOO on type II collagen-induced arthritis (CIA) in mice. DBA-1/J mice were randomized in four experimental groups (10 or 15 animals per group): (1) Sham sunflower diet (SO-Sham), (2) CIA sunflower diet (SO-CIA), (3) Sham EVOO diet (EVOO-Sham) and (4) CIA EVOO diet (EVOO-CIA) group. After 6 weeks, arthritis was induced by type II collagen. Mice were sacrified 42 days after first immunization. In addition to macroscopic and histological analyses, serum levels of cartilage olimeric matrix protein (COMP), metalloproteinase-3 (MMP-3) and pro-inflammatory cytokines levels were evaluated by ELISA. The expressions of heme oxygenase-1 (HO-1), nuclear factor E2-related factor 2 (Nrf2), mitogen-activated protein kinases (MAPKs), Janus kinase-signal transducer and activator of transcription (JAK/STAT) and nuclear transcription factor-kappa B (NF-κB) pathways were studied by western blotting. EVOO diet significantly reduced joint edema and cartilage destruction, preventing the arthritis development. Dietary EVOO significantly decreased serum COMP and MMP-3 levels, as well as, the pro-inflammatory cytokines levels (TNF-α, IL-1β and IL-17). Moreover, the activation of JAK/STAT, MAPKs and NF-κB pathways was drastically ameliorated. According to Nrf2 and HO-1, the protein expressions were up-regulated in those mice fed with EVOO. These results support the interest of EVOO as a beneficial functional food to prevent the development of the rheumatoid arthritis (RA).

  5. Enzymatic Breakdown of Type II Collagen in the Human Vitreous

    NARCIS (Netherlands)

    van Deemter, Marielle; Pas, Hendri H.; Kuijer, Roel; van der Worp, Roelofje J.; Hooymans, Johanna M. M.; Los, Leonoor I.

    2009-01-01

    PURPOSE. To investigate whether enzymatic collagen breakdown is an active process in the human vitreous. METHODS. Human donor eyes were used for immunohistochemistry to detect the possible presence of the matrix metalloproteinase (MMP)-induced type II collagen breakdown product col2-3/4C-short in

  6. Mechanical properties of the normal human cartilage-bone complex in relation to age

    DEFF Research Database (Denmark)

    Ding, Ming; Dalstra, M; Linde, F

    1998-01-01

    OBJECTIVE: This study investigates the age-related variations in the mechanical properties of the normal human tibial cartilage-bone complex and the relationships between cartilage and bone. DESIGN: A novel technique was applied to assess the mechanical properties of the cartilage and bone by means...... of testing the cartilage-bone complex. BACKGROUND: Up to now, mechanical testing of cartilage and bone has been reported separately, and little is known about the mechanical behaviour of both tissues when examined as a unit. METHODS: Cylindrical human proximal tibial cartilage-bone complex specimens from 31...... demonstrates that similar age-related trends were seen in cartilage and bone, as if they behaved as a single mechanical unit. RELEVANCE: The basic information presented here on the mechanical properties of cartilage and bone and the correlations between them reveals the unit function of both tissues...

  7. Scaffold-free cartilage tissue engineering with a small population of human nasoseptal chondrocytes.

    Science.gov (United States)

    Chiu, Loraine L Y; To, William T H; Lee, John M; Waldman, Stephen D

    2017-03-01

    Cartilage tissue engineering is a promising approach to provide suitable materials for nasal reconstruction; however, it typically requires large numbers of cells. We have previously shown that a small number of chondrocytes cultivated within a continuous flow bioreactor can elicit substantial tissue growth, but translation to human chondrocytes is not trivial. Here, we aimed to demonstrate the application of the bioreactor to generate large-sized tissues from a small population of primary human nasoseptal chondrocytes. Experimental study. Chondrocytes were cultured in the bioreactor using different medium compositions, with varying amounts of serum and with or without growth factors. Resulting engineered tissues were analyzed for physical properties, biochemical composition, tissue microstructure, and protein localization. Bioreactor-cultivated constructs grown with serum and growth factors (basic fibroblast growth factor and transforming growth factor beta 2) had greater thickness, as well as DNA and glycosaminoglycan (GAG) contents, compared to low serum and no growth factor controls. These constructs also showed the most intense proteoglycan and collagen II staining. The combination of bioreactor conditions, serum, and growth factors allowed the generation of large, thick scaffold-free human cartilaginous tissues that resembled the native nasoseptal cartilage. There also may be implications for patient selection in future clinical applications of these engineered tissues because their GAG content decreased with donor age. NA. Laryngoscope, 127:E91-E99, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

  8. Oriented Collagen Scaffolds for Tissue Engineering

    Science.gov (United States)

    Isobe, Yoshihiro; Kosaka, Toru; Kuwahara, Go; Mikami, Hiroshi; Saku, Taro; Kodama, Shohta

    2012-01-01

    Oriented collagen scaffolds were developed in the form of sheet, mesh and tube by arraying flow-oriented collagen string gels and dehydrating the arrayed gels. The developed collagen scaffolds can be any practical size with any direction of orientation for tissue engineering applications. The birefringence of the collagen scaffolds was quantitatively analyzed by parallel Nicols method. Since native collagen in the human body has orientations such as bone, cartilage, tendon and cornea, and the orientation has a special role for the function of human organs, the developed various types of three-dimensional oriented collagen scaffolds are expected to be useful biomaterials for tissue engineering and regenerative medicines. PMID:28817059

  9. Oriented Collagen Scaffolds for Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Shohta Kodama

    2012-03-01

    Full Text Available Oriented collagen scaffolds were developed in the form of sheet, mesh and tube by arraying flow-oriented collagen string gels and dehydrating the arrayed gels. The developed collagen scaffolds can be any practical size with any direction of orientation for tissue engineering applications. The birefringence of the collagen scaffolds was quantitatively analyzed by parallel Nicols method. Since native collagen in the human body has orientations such as bone, cartilage, tendon and cornea, and the orientation has a special role for the function of human organs, the developed various types of three-dimensional oriented collagen scaffolds are expected to be useful biomaterials for tissue engineering and regenerative medicines.

  10. Oriented Collagen Scaffolds for Tissue Engineering.

    Science.gov (United States)

    Isobe, Yoshihiro; Kosaka, Toru; Kuwahara, Go; Mikami, Hiroshi; Saku, Taro; Kodama, Shohta

    2012-03-16

    Oriented collagen scaffolds were developed in the form of sheet, mesh and tube by arraying flow-oriented collagen string gels and dehydrating the arrayed gels. The developed collagen scaffolds can be any practical size with any direction of orientation for tissue engineering applications. The birefringence of the collagen scaffolds was quantitatively analyzed by parallel Nicols method. Since native collagen in the human body has orientations such as bone, cartilage, tendon and cornea, and the orientation has a special role for the function of human organs, the developed various types of three-dimensional oriented collagen scaffolds are expected to be useful biomaterials for tissue engineering and regenerative medicines.

  11. Collagen synthesis in human musculoskeletal tissues and skin

    DEFF Research Database (Denmark)

    Babraj, J A; Cuthbertson, D J R; Smith, K

    2005-01-01

    We have developed a direct method for the measurement of human musculoskeletal collagen synthesis on the basis of the incorporation of stable isotope-labeled proline or leucine into protein and have used it to measure the rate of synthesis of collagen in tendon, ligament, muscle, and skin....... In postabsorptive, healthy young men (28 +/- 6 yr) synthetic rates for tendon, ligament, muscle, and skin collagen were 0.046 +/- 0.005, 0.040 +/- 0.006, 0.016 +/- 0.002, and 0.037 +/- 0.003%/h, respectively (means +/- SD). In postabsorptive, healthy elderly men (70 +/- 6 yr) the rate of skeletal muscle collagen...... synthesis is greater than in the young (0.023 +/- 0.002%/h, P collagen are similar to those of mixed skeletal muscle protein in the postabsorptive state, whereas the rate for muscle collagen synthesis is much lower in both young and elderly men...

  12. Computational biomechanics of articular cartilage of human knee joint: effect of osteochondral defects.

    Science.gov (United States)

    Shirazi, R; Shirazi-Adl, A

    2009-11-13

    Articular cartilage and its supporting bone functional conditions are tightly coupled as injuries of either adversely affects joint mechanical environment. The objective of this study was set to quantitatively investigate the extent of alterations in the mechanical environment of cartilage and knee joint in presence of commonly observed osteochondral defects. An existing validated finite element model of a knee joint was used to construct a refined model of the tibial lateral compartment including proximal tibial bony structures. The response was computed under compression forces up to 2000 N while simulating localized bone damage, cartilage-bone horizontal split, bone overgrowth and absence of deep vertical collagen fibrils. Localized tibial bone damage increased overall joint compliance and substantially altered pattern and magnitude of contact pressures and cartilage strains in both tibia and femur. These alterations were further exacerbated when bone damage was combined with base cartilage split and absence of deep vertical collagen fibrils. Local bone boss markedly changed contact pressures and strain patterns in neighbouring cartilage. Bone bruise/fracture and overgrowth adversely perturbed the homeostatic balance in the mechanical environment of articulate cartilage surrounding and opposing the lesion as well as the joint compliance. As such, they potentially contribute to the initiation and development of post-traumatic osteoarthritis.

  13. Effects of mechanical loading on human mesenchymal stem cells for cartilage tissue engineering.

    Science.gov (United States)

    Choi, Jane Ru; Yong, Kar Wey; Choi, Jean Yu

    2018-03-01

    Today, articular cartilage damage is a major health problem, affecting people of all ages. The existing conventional articular cartilage repair techniques, such as autologous chondrocyte implantation (ACI), microfracture, and mosaicplasty, have many shortcomings which negatively affect their clinical outcomes. Therefore, it is essential to develop an alternative and efficient articular repair technique that can address those shortcomings. Cartilage tissue engineering, which aims to create a tissue-engineered cartilage derived from human mesenchymal stem cells (MSCs), shows great promise for improving articular cartilage defect therapy. However, the use of tissue-engineered cartilage for the clinical therapy of articular cartilage defect still remains challenging. Despite the importance of mechanical loading to create a functional cartilage has been well demonstrated, the specific type of mechanical loading and its optimal loading regime is still under investigation. This review summarizes the most recent advances in the effects of mechanical loading on human MSCs. First, the existing conventional articular repair techniques and their shortcomings are highlighted. The important parameters for the evaluation of the tissue-engineered cartilage, including chondrogenic and hypertrophic differentiation of human MSCs are briefly discussed. The influence of mechanical loading on human MSCs is subsequently reviewed and the possible mechanotransduction signaling is highlighted. The development of non-hypertrophic chondrogenesis in response to the changing mechanical microenvironment will aid in the establishment of a tissue-engineered cartilage for efficient articular cartilage repair. © 2017 Wiley Periodicals, Inc.

  14. Human cornea modeling using artificial collagen

    OpenAIRE

    Bukanina, V.; Khokhlov, A.; Lovetskiy, K.

    2011-01-01

    This article focuses on spectrophotometric analysis of thin films of synthetic collagen with subsequent use of the received data to recover the optical properties of collagen and modeling a multilayer optical structure similar to the properties of human cornea.

  15. Hyaline Cartilage Tissue Is Formed through the Co-culture of Passaged Human Chondrocytes and Primary Bovine Chondrocytes

    Science.gov (United States)

    Taylor, Drew W.; Ahmed, Nazish; Hayes, Anthony J.; Ferguson, Peter; Gross, Allan E.; Caterson, Bruce

    2012-01-01

    To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks. PMID:22610463

  16. Osteoarthritic human cartilage is more sensitive to transforming growth factor beta than is normal cartilage

    NARCIS (Netherlands)

    Lafeber, F. P.; Vander Kraan, P. M.; Huber-Bruning, O.; Vanden Berg, W. B.; Bijlsma, J. W.

    1993-01-01

    Osteoarthritis is a degenerative joint disease, characterized by the destruction of the articular cartilage. One of the first changes in the osteoarthritic articular cartilage is a reduction in proteoglycan content. In this study we demonstrate that transforming growth factor beta (TGF beta), a

  17. Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation

    Directory of Open Access Journals (Sweden)

    Adila A Hamid

    2012-01-01

    Full Text Available OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adiposederived stem cells was most prominent after one week of chondrogenic induction.

  18. The immunomodulatory effects of shark cartilage on the mouse and human immune system

    Directory of Open Access Journals (Sweden)

    ali Sheikhian

    2007-01-01

    Materials and methods: In an experimental study, the effects of different doses of shark cartilage on humoral (antibody titer immune response against sheep red blood cells (SRBC, were measured in mouse. In addition, we evaluated the modulatory effects of the shark cartilage on the natural killer (NK activity of the peritoneal cells of mouse against a tumor cell line called K562, according to the standard methods. The proliferative response of the human peripheral blood mononuclear cells was measured under the influence of shark cartilage. Results: Pure shark cartilage enhanced antibody response against SRBC in vivo. The hemagglutination titer which was 1/147 in the control group (injected with hen cartilage, increased to 1/1355 in the test group. The optimal dose was 100 mg/ml. both type of cartilage had blastogenic effect on peripheral blood mononuclear cells (the blastogenic index was 6.7 and 4.9 for impure shark cartilage and hen cartilage, respectively. NK activity was inhibited completely by pure shark cartilage (the amount of the killing activity of the effector peritoneal cells for the control and test groups against target cells was 25.9% and 5.5% respectively. Conclusion: Shark cartilage has a potent immunomodulatory effect on the specific immune mechanisms and some inhibitory effects on the innate immune mechanisms such as NC activity. Since the specific immunity has a more pivotal role against tumor formation, shark cartilage can be used as a cancer immunotherapeutic.

  19. Arrangement of collagen fibers in human placental stem villi.

    Science.gov (United States)

    Sati, Leyla; Demir, Ayse Yasemin; Sarikcioglu, Levent; Demir, Ramazan

    2008-01-01

    The aim of the study was to investigate the arrangements and related localization patterns of different collagen types in the stroma of placental stem villi by immunohistochemistry and electron microscopy. A total of 14 normal human term placental tissue samples were studied. Immunohistochemistry was performed in order to localize collagen types I, III, IV, V and cytokeratin 7 on tissue sections. Parallel tissue samples were examined by transmission electron microscopy. Semi-quantitative analysis of immunolabeling intensities was also performed to determine the distribution of fibers in stem villi stroma. All collagen types, especially collagen type V, were strongly immunopositive in the triangular areas of the stem villi stroma. However, there was no collagen type I or type III immunolabeling in the sub-trophoblastic regions. Membrane collagen type IV immunolabeling was also observed in the stroma of stem villi. Ultrastructurally, collagen fibers showed different configurations in cross, longitudinal, circular, oblique and parallel directions compared to the villous axis. We conclude that the organization of collagen fiber bundles in stem villi shows a very specific arrangement: a compact coat formed by fibrillar bundles between the vascular wall and extravascular stroma of stem villi correlated with the functional activity.

  20. The search for negative amplitude components in quasi-continuous distributions of relaxation times: the example of 1H magnetization exchange in articular cartilage and hydrated collagen

    Science.gov (United States)

    Fantazzini, Paola; Galassi, Francesca; Bortolotti, Villiam; Brown, Robert J. S.; Vittur, Franco

    2011-06-01

    When inverting nuclear magnetic resonance relaxation data in order to obtain quasi-continuous distributions of relaxation times for fluids in porous media, it is common practice to impose a non-negative (NN) constraint on the distributions. While this approach can be useful in reducing the effects of data distortion and/or preventing wild oscillations in the distributions, it may give misleading results in the presence of real negative amplitude components. Here, some examples of valid negative components for articular cartilage and hydrated collagen are given. Articular cartilage is a connective tissue, consisting mainly of collagen, proteoglycans and water, which can be considered, in many aspects, as a porous medium. Separate T1 relaxation data are obtained for low-mobility ('solid') macromolecular 1H and for higher-mobility ('liquid') 1H by the separation of these components in free induction decays, with α denoting the solid/liquid 1H ratio. When quasi-continuous distributions of relaxation times (T1) of the solid and liquid signal components of cartilage or collagen are computed from experimental relaxation data without imposing the usual NN constraint, valid negative peaks may appear. The features of the distributions, in particular negative peaks, and the fact that peaks at longer times for macromolecular and water protons are at essentially the same T1, are interpreted as the result of a magnetization exchange between these two spin pools. For the only-slightly-hydrated collagen samples, with α>1, the exchange leads to small negative peaks at short T1 times for the macromolecular component. However, for the cartilage, with substantial hydration or for a strongly hydrated collagen sample, both with αLt1, the behavior is reversed, with a negative peak for water at short times. The validity of a negative peak may be accepted (dismissed) by a high (low) cost of NN in error of fit. Computed distributions for simulated data using observed signal

  1. Role of Fibulin 3 in Aging-Related Joint Changes and Osteoarthritis Pathogenesis in Human and Mouse Knee Cartilage.

    Science.gov (United States)

    Hasegawa, Akihiko; Yonezawa, Tomo; Taniguchi, Noboru; Otabe, Koji; Akasaki, Yukio; Matsukawa, Tetsuya; Saito, Masahiko; Neo, Masashi; Marmorstein, Lihua Y; Lotz, Martin K

    2017-03-01

    The EFEMP1 gene encoding fibulin 3 is specifically expressed in the superficial zone (SZ) of articular cartilage. The aims of this study were to examine the expression patterns of fibulin 3 in the knee joints during aging and during osteoarthritis (OA) and to determine the role of fibulin 3 in the pathogenesis of OA. Immunohistochemical analysis was performed on normal and OA knee cartilage samples from humans and mice. Experimental OA was induced in wild-type and fibulin 3 -/- mice, and the severity of OA was evaluated by histologic scoring. To examine fibulin 3 function, human chondrocyte monolayer cultures were transfected with small interfering RNA (siRNA), followed by quantitative polymerase chain reaction and Western blot analyses. Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transduced with an EFEMP1 lentivirus and analyzed for markers of chondrogenesis. Fibulin 3 was specifically expressed in the SZ of normal knee joint cartilage from humans and mice, and the expression levels declined with aging. Both aging-related OA and experimental OA were significantly more severe in fibulin 3 -/- mice compared with wild-type mice. Fibulin 3 expression was high in undifferentiated human BM-MSCs and decreased during chondrogenesis. Suppression of fibulin 3 by siRNA significantly increased the expression of SOX9, type II collagen, and aggrecan in human articular chondrocytes, while overexpression of fibulin 3 inhibited chondrogenesis in BM-MSCs. Fibulin 3 is specifically expressed in the SZ of articular cartilage and its expression is reduced in aging and OA. Fibulin 3 regulates differentiation of adult progenitor cells, and its aging-related decline is an early event in the pathogenesis of OA. Preventing aging-associated loss of fibulin 3 or restoring it to normal levels in SZ chondrocytes has the potential to delay or prevent the onset of OA. © 2016, American College of Rheumatology.

  2. Cartilage-selective genes identified in genome-scale analysis of non-cartilage and cartilage gene expression

    Directory of Open Access Journals (Sweden)

    Cohn Zachary A

    2007-06-01

    Full Text Available Abstract Background Cartilage plays a fundamental role in the development of the human skeleton. Early in embryogenesis, mesenchymal cells condense and differentiate into chondrocytes to shape the early skeleton. Subsequently, the cartilage anlagen differentiate to form the growth plates, which are responsible for linear bone growth, and the articular chondrocytes, which facilitate joint function. However, despite the multiplicity of roles of cartilage during human fetal life, surprisingly little is known about its transcriptome. To address this, a whole genome microarray expression profile was generated using RNA isolated from 18–22 week human distal femur fetal cartilage and compared with a database of control normal human tissues aggregated at UCLA, termed Celsius. Results 161 cartilage-selective genes were identified, defined as genes significantly expressed in cartilage with low expression and little variation across a panel of 34 non-cartilage tissues. Among these 161 genes were cartilage-specific genes such as cartilage collagen genes and 25 genes which have been associated with skeletal phenotypes in humans and/or mice. Many of the other cartilage-selective genes do not have established roles in cartilage or are novel, unannotated genes. Quantitative RT-PCR confirmed the unique pattern of gene expression observed by microarray analysis. Conclusion Defining the gene expression pattern for cartilage has identified new genes that may contribute to human skeletogenesis as well as provided further candidate genes for skeletal dysplasias. The data suggest that fetal cartilage is a complex and transcriptionally active tissue and demonstrate that the set of genes selectively expressed in the tissue has been greatly underestimated.

  3. Ultrasound can detect macroscopically undetectable changes in osteoarthritis reflecting the superficial histological and biochemical degeneration: ex vivo study of rabbit and human cartilage.

    Directory of Open Access Journals (Sweden)

    Kohei Nishitani

    Full Text Available Recognizing subtle cartilage changes in the preclinical stage of osteoarthritis (OA is essential for early diagnosis. To this end, the ability of the ultrasound signal intensity to detect macroscopically undetectable cartilage change was investigated. In this study, cartilage of rabbit OA model and human OA samples was examined by macroscopic evaluation, ultrasound signal intensity, histology with Mankin scores, and Fourier transform infrared imaging (FTIRI analysis. Rabbit OA was induced by anterior cruciate ligament transection and evaluated at 1, 2, 4 and 12 weeks. Twenty human samples were harvested during total knee arthroplasty from OA patients who had macroscopically normal human cartilage (ICRS grade 0 on the lateral femoral condyle. In the animal study, there was no macroscopic OA change at 2 weeks, but histology detected degenerative changes at this time point. Ultrasound signal intensity also detected degeneration at 2 weeks. In human samples, all samples were obtained from macroscopically intact site, however nearly normal (0 ≤ Mankin score <2, early OA (2 ≤ Mankin score <6, and moderate OA (6 ≤ Mankin score <10 samples were actually intermixed. Ultrasound signal intensity was significantly different among these 3 stages and was well correlated with Mankin scores (R = -0.80 and FTIR parameters related to collagen and proteoglycan content in superficial zone. In conclusion, ultrasound can detect microscopic cartilage deterioration when such changes do not exist macroscopically, reflecting superficial histological and biochemical changes.

  4. Proteomic analysis of human articular cartilage: identification of differentially expressed proteins in knee osteoarthritis.

    Science.gov (United States)

    Guo, Dunming; Tan, Wenfeng; Wang, Fang; Lv, Zheng; Hu, Jun; Lv, Tianrun; Chen, Qun; Gu, Xiaoyuan; Wan, Bing; Zhang, Zhongnan

    2008-07-01

    The mechanisms underlying the development of age related osteoarthritis (OA) remain unclear. To better understand the pathogenesis of OA and the molecular basis of progressive destruction of articular cartilage in OA, we compared the proteome of OA cartilage with that of normal cartilage. After removal of proteoglycans and collagens, proteins extracted from either normal or OA knee joint cartilage were separated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins in OA cartilage were chosen to be further identified by linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry (LTQ-FT/MS). A total of 1436+/-49 or 1472+/-7 protein spots were resolved by 2-DE of normal or OA cartilage extractions, respectively. Sixteen spots from OA cartilage samples were found to have statistically significant changes in the amount of protein compared with normal samples. Of 16 spots, the identities of 14 proteins were unambiguously determined by LTQ-FT/MS. These OA associated proteins fell into five groups, including glycolysis and energy production (ADH, ADK, ENOA, KPYM and FR), signaling (ANNX-I, PEBP and TUB), Redox (PRDX3 and SODM), and cartilage matrix (COLL-I and COLL-VI). Interestingly, two novel RING (Really Interesting New Gene) domain-containing proteins, RF, Zn-RF, were identified, suggesting novel pathways of cartilage protein regulation. This study shows that 2-DE followed by LTQ-FT/MS can be successfully used to characterize the proteome of cartilage without in vitro culturing which could obfuscate physiological differences. The definition of unique OA-associated proteins described here provides significant mechanistic insights into OA by corroborating previously suggested mechanisms and by defining unique players with roles yet to be defined in disease pathogenesis.

  5. Effects of cross-linking type II collagen-GAG scaffolds on chondrogenesis in vitro: dynamic pore reduction promotes cartilage formation.

    Science.gov (United States)

    Vickers, Scott M; Squitieri, Lee S; Spector, Myron

    2006-05-01

    Articular cartilage tissue-engineering investigations often implement bioassays for chondrogenesis in vitro using articular chondrocytes or mesenchymal stem cells in cell pellets that contract with time in culture, suggesting an association between the processes of contraction of the cell pellet and cartilage formation. The objective of the present study was to investigate this relationship further using adult canine articular chondrocyte-seeded type II collagen-GAG scaffolds. The collagen-GAG scaffolds were chemically cross-linked to achieve a range of cross-link densities. Chondrocyte-seeded scaffolds of varying cross-link densities were then cultured for 2 weeks to evaluate the effect of crosslink density on scaffold contraction and chondrogenesis. Scaffolds with low cross-link densities experienced cell-mediated contraction, increased cell number densities, a greater degree of chondrogenesis (viz., chondrocytic morphology of cells, synthesis of type II collagen), and an apparent increase in the rate of degradation of the scaffold compared to more highly cross-linked scaffolds that resisted cellular contraction. The results of this study suggest the promise of "dynamic pore reduction" for scaffolds for articular cartilage tissue engineering. In this approach, scaffolds would have an initial pore diameter large enough to facilitate cell seeding and a mechanical stiffness low enough to allow for cell-mediated contraction to yield a reduced pore volume to favor chondrogenesis. This approach may provide a useful alternative to traditional means of increasing cell number density and retention of synthesized molecules that promote cartilage formation in tissue-engineered constructs.

  6. Cartilage damage as a result of hemarthrosis in a human in vitro model

    NARCIS (Netherlands)

    Roosendaal, G.; Vianen, M. E.; van den Berg, H. M.; Lafeber, F. P.; Bijlsma, J. W.

    1997-01-01

    To investigate the direct effect of blood and blood components on human cartilage in vitro. Healthy human articular cartilage tissue was obtained post mortem and cultured according to standard procedures. The harmful effects of whole blood and various isolated blood components as well as the

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

    Science.gov (United States)

    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.

  8. Effect of homologous synovial membrane on adult human articular cartilage in organ culture, and failure to influence it with D-penicillamine.

    OpenAIRE

    Jacoby, R K

    1980-01-01

    Adult human articular cartilage has been maintained in organ culture for 8 days, and the culture medium, which was changed on alternate days, was pooled. Normal and rheumatoid cartilage was obtained from patients and 4 types of culture were prepared: (1) cartilage alone; (2) cartilage + D-penicillamine; (3) cartilage + homologous synovium; (4) cartilage, synovium, and D-penicillamine. The hexosamines and hexuronic acid were measured in the cartilage explants and in the medium. The quantity re...

  9. Characterization of a migrative subpopulation of adult human nasoseptal chondrocytes with progenitor cell features and their potential for in vivo cartilage regeneration strategies.

    Science.gov (United States)

    Elsaesser, A F; Schwarz, S; Joos, H; Koerber, L; Brenner, R E; Rotter, N

    2016-01-01

    Progenitor cells display interesting features for tissue repair and reconstruction. In the last years, such cells have been identified in different cartilage types. In this study, we isolated a migrative subpopulation of adult human nasoseptal chondrocytes with progenitor cell features by outgrowth from human nasal septum cartilage. These putative progenitor cells were comparatively characterized with mesenchymal stem cells (MSC) and human nasal septum chondrocytes with respect to their cellular characteristics as well as surface marker profile using flow cytometric analyses. Differentiation capacity was evaluated on protein and gene expression levels. The migrative subpopulation differentiated into osteogenic and chondrogenic lineages with distinct differences to chondrocytes and MSC. Cells of the migrative subpopulation showed an intermediate surface marker profile positioned between MSC and chondrocytes. Significant differences were found for CD9, CD29, CD44, CD90, CD105 and CD106. The cells possessed a high migratory ability in a Boyden chamber assay and responded to chemotactic stimulation. To evaluate their potential use in tissue engineering applications, a decellularized septal cartilage matrix was either seeded with cells from the migrative subpopulation or chondrocytes. Matrix production was demonstrated immunohistochemically and verified on gene expression level. Along with secretion of matrix metalloproteinases, cells of the migrative subpopulation migrated faster into the collagen matrix than chondrocytes, while synthesis of cartilage specific matrix was comparable. Cells of the migrative subpopulation, due to their migratory characteristics, are a potential cell source for in vivo regeneration of nasal cartilage. The in vivo mobilization of nasal cartilage progenitor cells is envisioned to be the basis for in situ tissue engineering procedures, aiming at the use of unseeded biomaterials which are able to recruit local progenitor cells for cartilage

  10. Adenovirus-mediated osteoprotegerin ameliorates cartilage destruction by inhibiting proteoglycan loss and chondrocyte apoptosis in rats with collagen-induced arthritis.

    Science.gov (United States)

    Feng, Zhi-yun; He, Zhen-nian; Zhang, Bin; Li, Yi-qiao; Guo, Jian; Xu, Yuan-lin; Han, Ming-yuan; Chen, Zhong

    2015-10-01

    Our aim is to elucidate the effects of osteoproteogerin (OPG) on cartilage destruction in rats as a model of collagen-induced arthritis (CIA). To establish the CIA model, Sprague Dawley rats were injected with bovine type II collagen solution subcutaneously via the tails. Adenovirus-mediated OPG (Ad-OPG) was then injected intra-articularly either at the beginning of CIA (early OPG treatment) or one week after CIA establishment (late OPG treatment); vehicle or Ad-green fluorescent protein were injected as controls. The rats were killed 4 weeks after treatment. Ankle-joint sections were obtained for histology. Serum samples were collected for enzyme-linked immunosorbent assay. Safranin O staining showed that proteoglycan loss was inhibited in the early and late Ad-OPG groups. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining revealed that both early and late Ad-OPG treatments significantly prevented chondrocyte apoptosis in CIA rats. Furthermore, disintegrin and metalloproteinase with thrombospondin motif-5 expression decreased remarkably in the early and late OPG treatment groups. However, the cartilage destruction score, cartilage oligomeric matrix protein level and caspase-3 expression were only decreased in the early Ad-OPG treatment group. Additionally, ankle-joint swelling and the interleukin-1β expression level in CIA rats were not notably altered by Ad-OPG treatment. Taken together, our results suggest that early Ad-OPG treatment has potent protective effects against cartilage destruction during rheumatoid arthritis progression, mainly by reducing proteoglycan loss and chondrocyte apoptosis.

  11. Xeno-free and shrinkage-free preparation of scaffold-free cartilage-like disc-shaped cell sheet using human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Sato, Yasushi; Wakitani, Shigeyuki; Takagi, Mutsumi

    2013-12-01

    Aiming for the clinical application of cartilage regeneration, the xeno-free cultivation method to obtain a scaffold-free cartilage-like disc-shaped cell sheet using mesenchymal stem cells (MSCs) derived from human bone marrow without the shrinkage of the sheet was investigated. MSCs were inoculated into Cell Culture Insert (0.3 cm(2), pore size; 0.4 μm, pore density; 1.0 × 10(8)/cm(2)) using serum-free chondrogenic differentiation medium containing TGF-β3, IGF-1 and dexamethasone or other modified media, and cultured at 37 °C in 5% CO2 for 3 weeks. Sheet thickness, cartilage specific genes expression, ECM accumulation were determined, and the sections of sheets were stained with alcian blue. A novel mixed medium consisting of a growth medium (10% FCS) with a serum-free chondrogenic differentiation medium could prevent the shrinkage of the sheet and produced a disc-shaped cell sheet. The depth of the sheet was approximately 0.7 mm and the gene expression levels were higher than those in cells in normal human cartilage. The use of human serum instead of FCS did not cause shrinkage and did not decrease the accumulation levels of sGAG and type 2 collagen in the sheet. The cultivation of MSCs grown with completely xeno-free materials using the mixed medium containing human serum in a cell culture insert showed a sheet depth of 1.0 mm and gene expression levels higher than those in normal cartilage. The scaffold-free and xeno-free cartilage-like cell sheet was successfully formed without shrinkage using human bone marrow MSCs and the chondrogenic differentiation medium containing human serum. Copyright © 2013. Published by Elsevier B.V.

  12. Collagen crosslinks in human lumbar intervertebral disc aging.

    Science.gov (United States)

    Pokharna, H K; Phillips, F M

    1998-08-01

    Human lumbar intervertebral discs from individuals of varying ages were obtained at autopsy and analyzed for collagen crosslinks. To analyze alterations in collagen crosslinks in human lumbar intervertebral discs with aging and disc degeneration. Crosslinks studied were pyridinoline, which is a collagen maturation crosslink, and pentosidine, a nonenzymatically initiated age-related crosslink. Crosslinking of collagen fibers within the matrix affects intervertebral disc biomechanics. In various connective tissues, alterations in pyridinoline and pentosidine crosslinks have been shown to predispose the tissue to mechanical failure. Little is known about the fate of intervertebral disc collagen crosslinks with advancing age and disc degeneration. Forty-two postmortem lumbar intervertebral discs were harvested from nine individuals whose ages were 24, 44, 47, 52, 67, 72, 75, 82, and 89 years. Degree of disc degeneration was graded macroscopically. Each lumbar disc was extracted with 4 mol/L guanidine hydrochloride, and the residual collagen was acid hydrolyzed and analyzed by reverse-phase high-performance liquid chromatography for pyridinoline and pentosidine crosslinks. The findings indicate a decrease in pyridinoline and an increase in pentosidine crosslink levels with disc aging. The decrease in pyridinoline crosslinks with disc aging is a novel finding and may have detrimental effects on matrix resilience. Increased pentosidine levels have been implicated in the age-related deterioration of connective tissue. With advancing degrees of macroscopic disc degeneration, pentosidine levels increase, and pyridinoline levels are diminished. Alterations in concentrations of pyridinoline and pentosidine collagen crosslinks occur with intervertebral disc aging and degeneration. These changes may contribute to the loss of disc integrity and play a role in the pathogenesis of the degenerative process.

  13. Effect of silver nanoparticles and hydroxyproline, administered in ovo, on the development of blood vessels and cartilage collagen structure in chicken embryos

    DEFF Research Database (Denmark)

    Beck, Iwona; Hotowy, Anna; Sawosz, Ewa

    2015-01-01

    . Experiments were performed on Ross 308 chicken embryos from 160 fertilised eggs. Experimental solutions of silver nanoparticles (Ag), hydroxyproline solution (Hyp) and a complex of silver nanoparticles with hydroxyproline (AgHyp) were injected into albumen, and embryos were incubated until day 20...... microscopy was performed. Our results clearly indicate that Hyp, Ag and AgHyp administered in ovo to chicken embryos did not harm embryos. Comparing to the control group, Hyp, Ag and the AgHyp complex significantly upregulated expression of the FGF-2 at the mRNA and protein levels. Moreover, Hyp, Ag and......, in particular, the complex of AgHyp significantly increased blood vessel size, cartilage collagen fibre lattice size and bundle thickness. The general conclusion from this study is that AgHyp treatment may help to build a stronger and longer lasting form of collagen fibres....

  14. Nonuniform distribution of collagen density in human knee ligaments

    NARCIS (Netherlands)

    Mommersteeg, T. J.; Blankevoort, L.; Kooloos, J. G.; Hendriks, J. C.; Kauer, J. M.; Huiskes, R.

    1994-01-01

    It is generally recognized that the mechanical properties of soft connective tissues are affected by their structural components. We documented collagen density distributions in human knee ligaments to quantify differences in density within and between these ligaments. In order to explain the

  15. Arrangement of collagen fibers in human placental stem villi

    NARCIS (Netherlands)

    Sati, Leyla; Demir, Ayse Yasemin; Sarikcioglu, Levent; Demir, Ramazan

    2008-01-01

    The aim of the study was to investigate the arrangements and related localization patterns of different collagen types in the stroma of placental stem villi by immunohistochemistry and electron microscopy. A total of 14 normal human term placental tissue samples were studied. Immunohistochemistry

  16. Applying Knowledge on Collagen of CLRI: In Human Health Care

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. Applying Knowledge on Collagen of CLRI: In Human Health Care. India's first natural immunogenic sterile biological skin cover for burns and wounds. An ideal skin substitute for the management of 1st & 2nd degree non-infected burns, 3rd degree burns. Kollagen ...

  17. Imaging of irradiated human costal cartilage birefringence by PS-OCT

    Energy Technology Data Exchange (ETDEWEB)

    Martinho Junior, Antonio C.; Freitas, Anderson Z.; Santin, Stefany P.; Soares, Fernando A.N.; Mosca, Rodrigo C.; Bringel, Fabiana A.; Mathor, Monica B., E-mail: freitas.az@ipen.b, E-mail: rmosca@usp.b, E-mail: mathor@ipen.b [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2011-07-01

    Sterilization by ionizing radiation is a technique used for tissue banks around the world to avoid transmission of infectious diseases by human allografts. However, high doses of ionizing radiation may cause undesirable changes in tissue structure, decreasing its mechanical properties, for example. Optical Coherence Tomography (OCT) is a non destructive, non ionizing and real time method to investigate biological tissues without promote any change in tissue structure. Polarization Sensitive Optical Coherence Tomography (PS-OCT) is an OCT technique that combines polarimetry with low coherence reflectometry to provide depth resolved measurements from birefringent structures as collagen. Costal cartilages from 15 cadaveric donors were preserved in high concentration glycerol and each individual sample was divided in 6 fragments. One of them was kept as a control group and the others were irradiated with gamma radiation from a Co-60 source with doses of 15, 25, 50, 75 and 100 kGy. OCT and PS-OCT images of the same region of the samples were obtained from a device OCS 1300 SS (Thorlabs, USA) with a coupling polarization module PSOCT 1300 (Thorlabs, USA). According with our results, birefringence may be visualized in all test groups as well in the control group, suggesting that sterilization by ionizing radiation does not affect the collagen structure significantly to cause total loss of birefringence, even if high doses as 75 and 100 kGy are used. The next step of our work is to develop a new method to quantify the birefringence using the optical properties of the tissue. (author)

  18. Cartilage integrity and proteoglycan turnover are comparable in canine experimentally induced and human joint degeneration

    Directory of Open Access Journals (Sweden)

    Femke Intema

    2010-10-01

    Full Text Available The value of experimental models of osteoarthritis (OA largely depends on the ability to translate observations to human OA. Surprisingly, direct comparison of characteristics of human and experimental OA is scarce. In the present study, cartilage integrity and matrix turnover in a canine model of joint degeneration were compared to human clinical OA. In 23 Beagle dogs, joint degeneration was induced in one knee, the contra-lateral knee served as a control. For comparison, human osteoarthritic and healthy knee cartilage were obtained at arthroplasty (n=14 and post-mortem (n=13. Cartilage was analyzed by histology and biochemistry. Values for cartilage integrity and proteoglycan (PG synthesis showed species specific differences; GAG content of healthy cartilage was 2-fold higher in canine cartilage and PG synthesis even 8-fold. However, the relative decrease in PG content between healthy and OA cartilage was similar for humans and canines (-17% vs. -15%, respectively, as was the histological damage (+7.0 vs. +6.1, respectively and the increase of PG synthesis (+100% vs. +70%, respectively. Remarkably, the percentage release of total and of newly formed PGs in human and canine controls was similar, as was the increase due to degeneration (+65% vs. +81% and +91% vs. +52%, respectively. Despite differences in control conditions, the observed changes in characteristics of cartilage integrity and matrix turnover are similar in a canine model of joint degeneration and human clinical OA. The canine Groove model shows that its characteristics reflect those of human OA which makes the model appropriate for studying human OA.

  19. Viscoelastic behavior of discrete human collagen fibrils

    DEFF Research Database (Denmark)

    Svensson, René; Hassenkam, Tue; Hansen, Philip

    2010-01-01

    Whole tendon and fibril bundles display viscoelastic behavior, but to the best of our knowledge this property has not been directly measured in single human tendon fibrils. In the present work an atomic force microscopy (AFM) approach was used for tensile testing of two human patellar tendon fibr...

  20. Mechanical characterization of articular cartilage by combining magnetic resonance imaging and finite-element analysis-a potential functional imaging technique

    Energy Technology Data Exchange (ETDEWEB)

    Julkunen, P; Korhonen, R K; Nissi, M J; Jurvelin, J S [Department of Physics, University of Kuopio, PO Box 1627, FI-70211 Kuopio (Finland)], E-mail: petro.julkunen@uku.fi

    2008-05-07

    Magnetic resonance imaging (MRI) provides a method for non-invasive characterization of cartilage composition and structure. We aimed to see whether T{sub 1} and T{sub 2} relaxation times are related to proteoglycan (PG) and collagen-specific mechanical properties of articular cartilage. Specifically, we analyzed whether variations in the depthwise collagen orientation, as assessed by the laminae obtained from T{sub 2} profiles, affect the mechanical characteristics of cartilage. After MRI and unconfined compression tests of human and bovine patellar cartilage samples, fibril-reinforced poroviscoelastic finite-element models (FEM), with depthwise collagen orientations implemented from quantitative T{sub 2} maps (3 laminae for human, 3-7 laminae for bovine), were constructed to analyze the non-fibrillar matrix modulus (PG specific), fibril modulus (collagen specific) and permeability of the samples. In bovine cartilage, the non-fibrillar matrix modulus (R = -0.64, p < 0.05) as well as the initial permeability (R = 0.70, p < 0.05) correlated with T{sub 1}. In bovine cartilage, T{sub 2} correlated positively with the initial fibril modulus (R = 0.62, p = 0.05). In human cartilage, the initial fibril modulus correlated negatively (R = -0.61, p < 0.05) with T{sub 2}. Based on the simulations, cartilage with a complex collagen architecture (5 or 7 laminae), leading to high bulk T{sub 2} due to magic angle effects, provided higher compressive stiffness than tissue with a simple collagen architecture (3 laminae). Our results suggest that T{sub 1} reflects PG-specific mechanical properties of cartilage. High T{sub 2} is characteristic to soft cartilage with a classical collagen architecture. Contradictorily, high bulk T{sub 2} can also be found in stiff cartilage with a multilaminar collagen fibril network. By emerging MRI and FEM, the present study establishes a step toward functional imaging of articular cartilage.

  1. Full-Length Recombinant Human Proteoglycan 4 Interacts with Hyaluronan to Provide Cartilage Boundary Lubrication.

    Science.gov (United States)

    Abubacker, Saleem; Dorosz, Samuel G; Ponjevic, Dragana; Jay, Gregory D; Matyas, John R; Schmidt, Tannin A

    2016-04-01

    Proteoglycan 4 (PRG4) is a mucin-like glycoprotein present in synovial fluid and at the surface of articular cartilage. The objectives of this study were to (1) assess the articular cartilage surface adsorption and in vitro cartilage boundary lubricating ability of full-length recombinant human PRG4 (rhPRG4), and (2) cartilage boundary lubricating ability of purified rhPRG4, both alone and in combination with hyaluronan (HA). rhPRG4 adsorption onto articular cartilage explants was assessed by immunohistochemistry and dot blot. An in vitro cartilage-cartilage friction test was used to assess rhPRG4's cartilage boundary lubricating ability compared to bovine PRG4, and that of purified rhPRG4 both alone and in combination with HA. rhPRG4 was able to adsorb to the articular surface, as well as the cut surface, of cartilage explants. The kinetic coefficient of friction of rhPRG4 was similar to that of PRG4 (p = 0.16) and lower than phosphate-buffered saline (p < 0.05), while that of purified rhPRG4 + HA was significantly lower than rhPRG4 alone (p < 0.05). This study demonstrates that rhPRG4 can adsorb to an intact articular cartilage surface and functions as an effective boundary lubricant, both alone and with HA, and provides the foundation for in vivo evaluation of this clinically relevant full-length rhPRG4 for treatment of osteoarthritis.

  2. Investigation of polarization-sensitive optical coherence tomography towards the study of microstructure of articular cartilage

    Science.gov (United States)

    Kasaragod, Deepa; Lu, Zenghai; Le Maitre, Christine; Wilkinson, J. Mark; Matcher, Stephen

    2013-03-01

    This paper highlights the extended Jones matrix calculus based multi-angle study carried out to understand the depth dependent structural orientation of the collagen fibers in articular cartilage using polarization-sensitive optical coherence tomography (PS-OCT). A 3D lamellar model for the collagen fiber orientation, with a quadratic profile for the arching of the collagen fibers in transitional zone which points towards an ordered arrangement of fibers in that zone is the basis of the organization architecture of collagen fibers in articular cartilage. Experimental data for both ex-vivo bovine fetlock and human patellar cartilage samples are compared with theoretical predictions, with a good quantitative agreement for bovine and a reasonable qualitative agreement for human articular cartilage samples being obtained

  3. Chondrocyte-seeded type I/III collagen membrane for autologous chondrocyte transplantation: prospective 2-year results in patients with cartilage defects of the knee joint.

    Science.gov (United States)

    Niemeyer, Philipp; Lenz, Philipp; Kreuz, Peter C; Salzmann, Gian M; Südkamp, Norbert P; Schmal, Hagen; Steinwachs, Matthias

    2010-08-01

    We report the 2-year clinical results and identify prognostic factors in patients treated with autologous chondrocyte transplantation by use of a collagen membrane to seed the chondrocytes (ACT-CS). This is a prospective study of 59 patients who were treated with ACT-CS and followed up for 24 months. Clinical function was assessed by International Knee Documentation Committee (IKDC-2000), objective International Cartilage Repair Society, and Lysholm scores before surgery and at 6, 12, and 24 months after surgery. On the basis of objective International Cartilage Repair Society (ICRS) rating, the percentage of patients rated A (normal) and B (nearly normal) increased from 33.9% preoperatively to 92.5% at 24 months after ACT-CS. IKDC and Lysholm scores increased from 50.1 points (SD, 13.4) and 60.5 points (SD, 9.4), respectively, to 76.1 points (SD, 15.2) (P < .001) and 82.5 points (SD, 13.7) (P < .001), respectively, at 24 months. The failure rate was highest, at 26.7% at 2 years' follow-up, in the subgroup of patients who underwent ACT-CS as a salvage procedure. The rate of failures in patients with isolated cartilage defects was 5.9%. ACT-CS represents a technical modification of membrane-associated autologous chondrocyte transplantation that combines easy handling and attractive application properties with reliable clinical results 24 months after surgery, especially in patients with isolated cartilage defects. Even though the failure rate was higher in patients with kissing lesions or mild osteoarthritis, ACT-CS also seems to improve function in a large proportion of such patients. Level IV, prospective case series. 2010 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

  4. Supplementation of calves with stabilized orthosilicic acid. Effect on the Si, Ca, Mg, and P concentrations in serum and the collagen concentration in skin and cartilage.

    Science.gov (United States)

    Calomme, M R; Vanden Berghe, D A

    1997-02-01

    The bioavailability of silicon in stabilized orthosilicic acid was investigated in a double blind, placebo controlled supplementation study of calves maintained on a normal diet. The total dietary Si intake was increased by 4.9% in the form of stabilized orthosilicic acid. After 23 wk of Si supplementation, the serum Si concentration increased (p = 0.0001, n = 29) by 70% compared to control animals in spite of the low Si dose administered and the Si adequate diet. The individually administered Si dose was significantly associated with the serum Si concentration (r = 0.44, p = 0.016, n = 29). The collagen concentration in dermis was significantly higher (p = 0.019, n = 4) in the Si group and a positive correlation (r = 0.72, p = 0.018, n = 9) was found between the Si concentration in serum and the collagen concentration in cartilage. The calcium (Ca) and phosphorus (P) concentrations in serum were marginally higher for animals supplemented with Si compared to control animals. In serum, a significant linear relationship was found between the Si and the Ca concentration (r = 0.31, p = 0.019, n = 59), whereas the magnesium concentration correlated marginally with the Si concentration (r = 0.25, p = 0.068, n = 59). In summary, increasing the total dietary Si intake by 4.9% in the form of stabilized orthosilicic acid resulted in a 70% higher Si concentration in serum indicating a high bioavailability of Si in this supplement. The positive correlation between the serum Si concentration and the collagen concentration in cartilage and the serum Ca concentration, respectively, suggest the involvement of Si both in the formation of extracellular matrix components and in Ca metabolism.

  5. Viscoelastic behavior of discrete human collagen fibrils

    DEFF Research Database (Denmark)

    2010-01-01

    Whole tendon and fibril bundles display viscoelastic behavior, but to the best of our knowledge this property has not been directly measured in single human tendon fibrils. In the present work an atomic force microscopy (AFM) approach was used for tensile testing of two human patellar tendon...... on the strain. The slope of the viscous response showed a strain rate dependence corresponding to a power function of powers 0.242 and 0.168 for the two patellar tendon fibrils, respectively. In conclusion, the present work provides direct evidence of viscoelastic behavior at the single fibril level, which has...

  6. Upregulation of Bone Morphogenetic Protein-2 Synthesis and Consequent Collagen II Expression in Leptin-stimulated Human Chondrocytes.

    Science.gov (United States)

    Chang, Shun-Fu; Hsieh, Rong-Ze; Huang, Kuo-Chin; Chang, Cheng Allen; Chiu, Fang-Yao; Kuo, Hsing-Chun; Chen, Cheng-Nan; Su, Yu-Ping

    2015-01-01

    Bone morphogenetic proteins (BMPs) play positive roles in cartilage development, but they can barely be detected in healthy articular cartilage. However, recent evidence has indicated that BMPs could be detected in osteoarthritic and damaged cartilage and their precise roles have not been well defined. Extremely high amounts of leptin have been reported in obese individuals, which can be associated with osteoarthritis (OA) development. The aim of this study was to investigate whether BMPs could be induced in human primary chondrocytes during leptin-stimulated OA development and the underlying mechanism. We found that expression of BMP-2 mRNA, but not BMP-4, BMP-6, or BMP-7 mRNA, could be increased in human primary chondrocytes under leptin stimulation. Moreover, this BMP-2 induction was mediated through transcription factor-signal transducer and activator of transcription (STAT) 3 activation via JAK2-ERK1/2-induced Ser727-phosphorylation. Of note, histone deacetylases (HDACs) 3 and 4 were both involved in modulating leptin-induced BMP-2 mRNA expression through different pathways: HDAC3, but not HDAC4, associated with STAT3 to form a complex. Our results further demonstrated that the role of BMP-2 induction under leptin stimulation is to increase collagen II expression. The findings in this study provide new insights into the regulatory mechanism of BMP-2 induction in leptin-stimulated chondrocytes and suggest that BMP-2 may play a reparative role in regulating leptin-induced OA development.

  7. Upregulation of Bone Morphogenetic Protein-2 Synthesis and Consequent Collagen II Expression in Leptin-stimulated Human Chondrocytes.

    Directory of Open Access Journals (Sweden)

    Shun-Fu Chang

    Full Text Available Bone morphogenetic proteins (BMPs play positive roles in cartilage development, but they can barely be detected in healthy articular cartilage. However, recent evidence has indicated that BMPs could be detected in osteoarthritic and damaged cartilage and their precise roles have not been well defined. Extremely high amounts of leptin have been reported in obese individuals, which can be associated with osteoarthritis (OA development. The aim of this study was to investigate whether BMPs could be induced in human primary chondrocytes during leptin-stimulated OA development and the underlying mechanism. We found that expression of BMP-2 mRNA, but not BMP-4, BMP-6, or BMP-7 mRNA, could be increased in human primary chondrocytes under leptin stimulation. Moreover, this BMP-2 induction was mediated through transcription factor-signal transducer and activator of transcription (STAT 3 activation via JAK2-ERK1/2-induced Ser727-phosphorylation. Of note, histone deacetylases (HDACs 3 and 4 were both involved in modulating leptin-induced BMP-2 mRNA expression through different pathways: HDAC3, but not HDAC4, associated with STAT3 to form a complex. Our results further demonstrated that the role of BMP-2 induction under leptin stimulation is to increase collagen II expression. The findings in this study provide new insights into the regulatory mechanism of BMP-2 induction in leptin-stimulated chondrocytes and suggest that BMP-2 may play a reparative role in regulating leptin-induced OA development.

  8. Bioreactor based engineering of large-scale human cartilage grafts for joint resurfacing.

    Science.gov (United States)

    Santoro, Rosaria; Olivares, Andy L; Brans, Gerben; Wirz, Dieter; Longinotti, Cristina; Lacroix, Damien; Martin, Ivan; Wendt, David

    2010-12-01

    Apart from partial or total joint replacement, no surgical procedure is currently available to treat large and deep cartilage defects associated with advanced diseases such as osteoarthritis. In this work, we developed a perfusion bioreactor system to engineer human cartilage grafts in a size with clinical relevance for unicompartmental resurfacing of human knee joints (50 mm diameter × 3 mm thick). Computational fluid dynamics models were developed to optimize the flow profile when designing the perfusion chamber. Using the developed system, human chondrocytes could be seeded throughout large 50 mm diameter scaffolds with a uniform distribution. Following two weeks culture, tissues grown in the bioreactor were viable and homogeneously cartilaginous, with biomechanical properties approaching those of native cartilage. In contrast, tissues generated by conventional manual production procedures were highly inhomogeneous and contained large necrotic regions. The unprecedented engineering of human cartilage tissues in this large-scale opens the practical perspective of grafting functional biological substitutes for the clinical treatment for extensive cartilage defects, possibly in combination with surgical or pharmacological therapies to support durability of the implant. Ongoing efforts are aimed at integrating the up-scaled bioreactor based processes within a fully automated and closed manufacturing system for safe, standardized, and GMP compliant production of large-scale cartilage grafts. Copyright © 2010 Elsevier Ltd. All rights reserved.

  9. Cartilage oligomeric matrix protein deficiency promotes early onset and the chronic development of collagen-induced arthritis

    DEFF Research Database (Denmark)

    Geng, Hui; Carlsen, Stefan; Nandakumar, Kutty

    2008-01-01

    -collagen II and anti-COMP antibodies as well as serum COMP levels in arthritic and wild-type mice were measured by enzyme-linked immunosorbent assay. RESULTS: COMP-deficient mice showed a significant early onset and increase in the severity of CIA in the chronic phase, whereas collagen II-antibody titers were...

  10. Serum- and growth-factor-free three-dimensional culture system supports cartilage tissue formation by promoting collagen synthesis via Sox9-Col2a1 interaction.

    Science.gov (United States)

    Ahmed, Nazish; Iu, Jonathan; Brown, Chelsea E; Taylor, Drew Wesley; Kandel, Rita A

    2014-08-01

    One of the factors preventing clinical application of regenerative medicine to degenerative cartilage diseases is a suitable source of cells. Chondrocytes, the only cell type of cartilage, grown in vitro under culture conditions to expand cell numbers lose their phenotype along with the ability to generate hyaline cartilaginous tissue. In this study we determine that a serum- and growth-factor-free three-dimensional (3D) culture system restores the ability of the passaged chondrocytes to form cartilage tissue in vitro, a process that involves sox9. Bovine articular chondrocytes were passaged twice to allow for cell number expansion (P2) and cultured at high density on 3D collagen-type-II-coated membranes in high glucose content media supplemented with insulin and dexamethasone (SF3D). The cells were characterized after monolayer expansion and following 3D culture by flow cytometry, gene expression, and histology. The early changes in signaling transduction pathways during redifferentiation were characterized. The P2 cells showed a progenitor-like antigen profile of 99% CD44(+) and 40% CD105(+) and a gene expression profile suggestive of interzone cells. P2 in SF3D expressed chondrogenic genes and accumulated extracellular matrix. Downregulating insulin receptor (IR) with HNMPA-(AM3) or the PI-3/AKT kinase pathway (activated by insulin treatment) with Wortmannin inhibited collagen synthesis. HNMPA-(AM3) reduced expression of Col2, Col11, and IR genes as well as Sox6 and -9. Co-immunoprecipitation and chromatin immunoprecipitation analyses of HNMPA-(AM3)-treated cells showed binding of the coactivators Sox6 and Med12 with Sox9 but reduced Sox9-Col2a1 binding. We describe a novel culture method that allows for increase in the number of chondrocytes and promotes hyaline-like cartilage tissue formation in part by insulin-mediated Sox9-Col2a1 binding. The suitability of the tissue generated via this approach for use in joint repair needs to be examined in vivo.

  11. Human Adipose-Derived Mesenchymal Progenitor Cells Engraft into Rabbit Articular Cartilage

    Directory of Open Access Journals (Sweden)

    Wen Wang

    2015-05-01

    Full Text Available Mesenchymal stem cells (MSCs are known to have the potential for articular cartilage regeneration, and are suggested for the treatment of osteoarthritis (OA. Here, we investigated whether intra-articular injection of xenogeneic human adipose-derived mesenchymal progenitor cells (haMPCs promoted articular cartilage repair in rabbit OA model and engrafted into rabbit articular cartilage. The haMPCs were cultured in vitro, and phenotypes and differentiation characteristics of cells were evaluated. OA was induced surgically by anterior cruciate ligament transection (ACLT and medical meniscectomy of knee joints. At six weeks following surgery, hyaluronic acid (HA or haMPCs was injected into the knee joints, the contralateral knee served as normal control. All animals were sacrificed at the 16th week post-surgery. Assessments were carried out by macroscopic examination, hematoxylin/eosin (HE and Safranin-O/Fast green stainings and immunohistochemistry. The data showed that haMPC treatment promoted cartilage repair. Signals of human mitochondrial can be directly detected in haMPC treated cartilage. The haMPCs expressed human leukocyte antigen I (HLA-I but not HLA-II-DR in vivo. These results suggest that intra-articular injection of haMPCs promotes regeneration of articular cartilage in rabbit OA model, and support the notion that MPCs are transplantable between HLA-incompatible individuals.

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

    Directory of Open Access Journals (Sweden)

    Jeremy S Bredfeldt

    2014-01-01

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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    The growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis stimulates collagen synthesis in tendon and skeletal muscle, but no studies have investigated the effect of reducing IGF-I on collagen synthesis in healthy humans.......The growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis stimulates collagen synthesis in tendon and skeletal muscle, but no studies have investigated the effect of reducing IGF-I on collagen synthesis in healthy humans....

  14. Effects of ionizing radiation and preservation on biomechanical properties of human costal cartilage.

    Science.gov (United States)

    Martinho, A C; Rosifini Alves-Claro, A P; Pino, E S; Machado, L D B; Herson, M R; Santin, S P; Mathor, M B

    2013-03-01

    Tissue banks around the world store human cartilage obtained from cadaveric donors for use in diverse reconstructive surgical procedures. To ensure this tissue is sterile at the time of distribution, tissues may be sterilized by ionizing radiation. In this work, we evaluate the physical changes in deep frozen costal cartilage (-70 °C) or costal cartilage preserved in high concentrations of glycerol (>98 %) followed by a terminal sterilization process using ionizing radiation, at 3 different doses (15, 25 and 50 kGy). Tension and compression tests were carried out to determine the mechanical changes related both to the different preservation methods and irradiation doses. For both methods of preservation, tension strength was increased by about 24 %, when cartilage tissue was irradiated with 15 kGy. Deep frozen samples, when irradiated with 25 or 50 kGy, had a decrease in their mechanical performance, albeit to a lesser extent than when tissues were preserved in high concentration of glycerol and equally irradiated. In conclusion, processing in high concentration of glycerol did not increase tissue protection against radiation damage; while cartilage preserved in high concentrations of glycerol withstands radiation up to 25 kGy, deep frozen human costal cartilage may be sterilized with a doses up to 50 kGy without significant mechanical impact.

  15. Effect of Age-Related Cartilage Turnover on Serum C-Telopeptide of Collagen Type II and Osteocalcin Levels in Growing Rabbits with and without Surgically Induced Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Chung-Cheng Huang

    2014-01-01

    Full Text Available This study aims to determine the effect of age-related cartilage turnover on the serum C-telopeptide of type II collagen (CTX-II and osteocalcin (OC levels in growing rabbits with and without surgically induced osteoarthritis. Twenty-four New Zealand male 3-month-old rabbits were randomized into three operated groups (n = 6 per group, with surgically induced osteroarthritis in the right knee; after blood sampling, the knees were harvested following euthanization at 2, 3, and 6 months after surgery and a control group (n = 6, blood samples were obtained monthly between 3 and 15 months. Histomorphologically, the medial femoral condyles, particularly the central parts, harbored the most severe osteoarthritic changes among the operated rabbits. The serum levels of CTX-II and OC decreased in the controls from 3 to 11 months and then remained stable. No significant differences in the serum CTX-II and OC levels between the osteoarthritic rabbits and controls were observed. The osteoarthritic-to-normal ratios (ONRs, the ratios of serum CTX-II or OC levels in osteoarthritic rabbits to those of the controls at same ages enabled an overall assessment of osteoarthritis and age-related cartilage turnover. Elevated CTX-II ONRs were observed in rabbits with mild to advanced osteoarthritis. However, the OC ONRs were unhelpful in assessing osteoarthritic growing rabbits.

  16. Time-of-flight secondary ion mass spectrometry-based molecular distribution distinguishing healthy and osteoarthritic human cartilage

    CERN Document Server

    Cillero-Pastor, Berta; Kiss, Andras; Blanco, Francisco J; Heeren, Ron M A

    2013-01-01

    Osteoarthritis (OA) is a pathology that ultimately causes joint destruction. The cartilage is one of the principal affected tissues. Alterations in the lipid mediators and an imbalance in the metabolism of cells that form the cartilage (chondrocytes) have been described as contributors to the OA development. In this study, we have studied the distribution of lipids and chemical elements in healthy and OA human cartilage. Time of flight-secondary ion mass spectrometry (TOF-SIMS) allows us to study the spatial distribution of molecules at a high resolution on a tissue section. TOF-SIMS revealed a specific peak profile that distinguishes healthy from OA cartilages. The spatial distribution of cholesterol-related peaks exhibited a remarkable difference between healthy and OA cartilages. A distinctive colocalization of cholesterol and other lipids in the superficial area of the cartilage was found. A higher intensity of oleic acid and other fatty acids in the OA cartilages exhibited a similar localization. On the ...

  17. New approaches and recent results concerning human-tissue collagen synthesis.

    Science.gov (United States)

    Smith, Ken; Rennie, Michael J

    2007-09-01

    Knowledge of the physiological regulation of human-tissue collagen metabolism in vivo is poor, due to the lack of appropriately robust methods. Recent application of stable isotope tracer techniques to measure human collagen synthesis has provided some insights into the role of nutrition and exercise on collagen turnover in the extracellular matrix of the musculoskeletal system. Collagen turnover in the musculoskeletal system is faster than previously thought. Bone collagen synthesis is increased by feeding, whereas both muscle collagen and tendon are unresponsive. Exercise stimulates collagen synthesis in both muscle and tendon in an apparently coordinated manner. There are also sex differences and normal aging is associated with increased muscle collagen synthesis and reductions in bone collagen synthesis, particularly in mature bone collagen. Collagen turnover appears to be faster than previously thought and is regulated by feeding and exercise, in a tissue-specific manner. Further application of these approaches, coupled with measures of gene and protein expression, to measure the acute regulation of collagen, will lead to a better understanding of the physiology and pathophysiology of human collagen turnover. This is particularly important for developing new therapies to improve bone health and minimize tissue fibrosis.

  18. Enhanced cartilage formation via three-dimensional cell engineering of human adipose-derived stem cells.

    Science.gov (United States)

    Yoon, Hee Hun; Bhang, Suk Ho; Shin, Jung-Youn; Shin, Jaehoon; Kim, Byung-Soo

    2012-10-01

    Autologous chondrocyte implantation is an effective treatment for damaged articular cartilage. However, this method involves surgical procedures that may cause further cartilage degeneration, and in vitro expansion of chondrocytes can result in dedifferentiation. Adipose-derived stem cells (ADSCs) may be an alternative autologous cell source for cartilage regeneration. In this study, we developed an effective method for large-scale in vitro chondrogenic differentiation, which is the procedure that would be required for clinical applications, and the subsequent in vivo cartilage formation of human ADSCs (hADSCs). The spheroid formation and chondrogenic differentiation of hADSCs were induced on a large scale by culturing hADSCs in three-dimensional suspension bioreactors (spinner flasks). In vitro chondrogenic differentiation of hADSCs was enhanced by a spheroid culture compared with a monolayer culture. The enhanced chondrogenesis was probably attributable to hypoxia-related cascades and enhanced cell-cell interactions in hADSC spheroids. On hADSCs loading in fibrin gel and transplantation into subcutaneous space of athymic mice for 4 weeks, the in vivo cartilage formation was enhanced by the transplantation of spheroid-cultured hADSCs compared with that of monolayer-cultured hADSCs. This study shows that the spheroid culture may be an effective method for large-scale in vitro chondrogenic differentiation of hADSCs and subsequent in vivo cartilage formation.

  19. Limited Immunogenicity of Human Induced Pluripotent Stem Cell-Derived Cartilages

    Science.gov (United States)

    Kimura, Takeshi; Yamashita, Akihiro; Ozono, Keiichi

    2016-01-01

    Articular cartilage damage does not spontaneously heal and could ultimately result in a loss of joint function. Damaged cartilage can be repaired with cell/tissue sources that are transplanted, however, autologous chondrocytes are limited in number as a cell source. Induced pluripotent stem cells (iPSCs) are a relatively new and abundant cell source and can be made from the patient, but at a considerable cost. Because cartilage is immunoprivileged tissue, allogeneic cartilages have been transplanted effectively without matching for human leukocyte antigen (HLA), but are difficult to acquire due to scarcity of donors. In this study, we examined the immunogenicity of human iPSC-derived cartilages (hiPS-Carts) in vitro to evaluate whether allogeneic hiPS-Carts can be a new cell/tissue source. The cells in hiPS-Carts expressed limited amounts of major histocompatibility complex (MHC) class I (HLA-ABC) and MHC class II (HLA-DRDQDP). Treatment with interferon γ (IFNγ) induced the expression of MHC class I, but not MHC class II in hiPS-Carts. A mixed lymphocyte reaction assay showed that hiPS-Carts stimulated the proliferation of neither T cells nor the activation of NK cells. Furthermore, hiPS-Carts suppressed the proliferation of T cells stimulated with interleukin 2 and phytohemagglutinin (PHA). Together with previously reported findings, these results suggest that hiPS-Carts are no more antigenic than human cartilage. Additionally, in combination with the fact that iPSCs are unlimitedly expandable and thus can supply unlimited amounts of iPS-Carts from even one iPSC line, they suggest that allogeneic hiPS-Carts are a candidate source for transplantation to treat articular cartilage damage. PMID:27762664

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

    DEFF Research Database (Denmark)

    Langberg, Henning; Rosendal, L; Kjaer, M

    2001-01-01

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

  1. Development of an Electromechanical Grade to Assess Human Knee Articular Cartilage Quality.

    Science.gov (United States)

    Sim, Sotcheadt; Hadjab, Insaf; Garon, Martin; Quenneville, Eric; Lavigne, Patrick; Buschmann, Michael D

    2017-10-01

    Quantitative assessments of articular cartilage function are needed to aid clinical decision making. Our objectives were to develop a new electromechanical grade to assess quantitatively cartilage quality and test its reliability. Electromechanical properties were measured using a hand-held electromechanical probe on 200 human articular surfaces from cadaveric donors and osteoarthritic patients. These data were used to create a reference electromechanical property database and to compare with visual arthroscopic International Cartilage Repair Society (ICRS) grading of cartilage degradation. The effect of patient-specific and location-specific characteristics on electromechanical properties was investigated to construct a continuous and quantitative electromechanical grade analogous to ICRS grade. The reliability of this novel grade was assessed by comparing it with ICRS grades on 37 human articular surfaces. Electromechanical properties were not affected by patient-specific characteristics for each ICRS grade, but were significantly different across the articular surface. Electromechanical properties varied linearly with ICRS grade, leading to a simple linear transformation from one scale to the other. The electromechanical grade correlated strongly with ICRS grade (r = 0.92, p < 0.0001). Additionally, the electromechanical grade detected lesions that were not found visually. This novel grade can assist the surgeon in assessing human knee cartilage by providing a quantitative and reliable grading system.

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

    Science.gov (United States)

    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.

  3. Automated bioreactor system for cartilage tissue engineering of human primary nasal septal chondrocytes.

    Science.gov (United States)

    Princz, Sascha; Wenzel, Ulla; Tritschler, Hanna; Schwarz, Silke; Dettmann, Christian; Rotter, Nicole; Hessling, Martin

    2017-10-26

    An automated bioreactor system for three-dimensional (3D) cultivation of facial cartilage replacement matrices (e.g. whole human auricles) with automatised medium exchange, gas flow and temperature control was developed. The measurement of O2 saturation and pH value in the medium was performed with a non-invasive optical method. The whole system can be observed via remote monitoring worldwide. First results demonstrated that the complete system remained sterile throughout a period of 42 days. Human chondrocytes migrated into the employed cartilage replacement matrix consisting of decellularised porcine nasoseptal cartilage (pNSC). Furthermore, an improved migration and new synthesis of aggrecan was detected. A first evaluation of the system was conducted by comparison of the results from laboratory analysis with computational fluid dynamics (CFD).

  4. Intra-articular Recombinant Human Proteoglycan 4 Mitigates Cartilage Damage After Destabilization of the Medial Meniscus in the Yucatan Minipig.

    Science.gov (United States)

    Waller, Kimberly A; Chin, Kaitlyn E; Jay, Gregory D; Zhang, Ling X; Teeple, Erin; McAllister, Scott; Badger, Gary J; Schmidt, Tannin A; Fleming, Braden C

    2017-06-01

    Lubricin, or proteoglycan 4 (PRG4), is a glycoprotein responsible for joint boundary lubrication. PRG4 has been shown previously to be down-regulated after traumatic joint injury such as a meniscal tear. Preliminary evidence suggests that intra-articular injection of PRG4 after injury will reduce cartilage damage in rat models of surgically induced posttraumatic osteoarthritis. To determine the efficacy of intra-articular injection of full-length recombinant human lubricin (rhPRG4) for reducing cartilage damage after medial meniscal destabilization (DMM) in a preclinical large animal model. Controlled laboratory study. Unilateral DMM was performed in 29 Yucatan minipigs. One week after DMM, animals received 3 weekly intra-articular injections (3 mL per injection): (1) rhPRG4 (1.3 mg/mL; n = 10); (2) rhPRG4+hyaluronan (1.3 mg/mL rhPRG4 and 3 mg/mL hyaluronan [~950 kDA]; n = 10); and (3) phosphate-buffered saline (PBS; n = 9). Hindlimbs were harvested 26 weeks after surgery. Cartilage integrity was evaluated by use of macroscopic (India ink) and microscopic (safranin O-fast green and hematoxylin and eosin) scoring systems. Secondary outcomes evaluated via enzyme-linked immunosorbent assay (ELISA) included PRG4 levels in synovial fluid, carboxy-terminal telepeptide of type II collagen (CTX-II) concentrations in urine and serum, and interleukin 1β (IL-1β) levels in synovial fluid and serum. The rhPRG4 group had significantly less macroscopic cartilage damage in the medial tibial plateau compared with the PBS group ( P = .002). No difference was found between the rhPRG4+hyaluronan and PBS groups ( P = .23). However, no differences in microscopic damage scores were observed between the 3 groups ( P = .70). PRG4 production was elevated in the rhPRG4 group synovial fluid compared with the PBS group ( P = .033). The rhPRG4 group presented significantly lower urinary CTX-II levels, but not serum levels, when compared with the PBS ( P = .013) and rhPRG4+hyaluronan ( P

  5. SMAD3 Is Upregulated in Human Osteoarthritic Cartilage Independent of the Promoter DNA Methylation.

    Science.gov (United States)

    Aref-Eshghi, Erfan; Liu, Ming; Razavi-Lopez, Seyd Babak; Hirasawa, Kensuke; Harper, Patricia E; Martin, Glynn; Furey, Andrew; Green, Roger; Sun, Guang; Rahman, Proton; Zhai, Guangju

    2016-02-01

    To compare SMAD3 gene expression between human osteoarthritic and healthy cartilage and to examine whether expression is regulated by the promoter DNA methylation of the gene. Human cartilage samples were collected from patients undergoing total hip/knee joint replacement surgery due to primary osteoarthritis (OA), and from patients with hip fractures as controls. DNA/RNA was extracted from the cartilage tissues. Real-time quantitative PCR was performed to measure gene expression, and Sequenom EpiTyper was used to assay DNA methylation. Mann-Whitney test was used to compare the methylation and expression levels between OA cases and controls. Spearman rank correlation coefficient was calculated to examine the association between the methylation and gene expression. A total of 58 patients with OA (36 women, 22 men; mean age 64 ± 9 yrs) and 55 controls (43 women, 12 men; mean age 79 ± 10 yrs) were studied. SMAD3 expression was on average 83% higher in OA cartilage than in controls (p = 0.0005). No difference was observed for DNA methylation levels in the SMAD3 promoter region between OA cases and controls. No correlation was found between SMAD3 expression and promoter DNA methylation. Our study demonstrates that SMAD3 is significantly overexpressed in OA. This overexpression cannot be explained by DNA methylation in the promoter region. The results suggest that the transforming growth factor-β/SMAD3 pathway may be overactivated in OA cartilage and has potential in developing targeted therapies for OA.

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

    NARCIS (Netherlands)

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

    1995-01-01

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

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

    NARCIS (Netherlands)

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

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

  8. Hypoxia Is a Critical Parameter for Chondrogenic Differentiation of Human Umbilical Cord Blood Mesenchymal Stem Cells in Type I/III Collagen Sponges.

    Science.gov (United States)

    Gómez-Leduc, Tangni; Desancé, Mélanie; Hervieu, Magalie; Legendre, Florence; Ollitrault, David; de Vienne, Claire; Herlicoviez, Michel; Galéra, Philippe; Demoor, Magali

    2017-09-08

    Umbilical cord blood (UCB) is an attractive alternative to bone marrow for isolation of mesenchymal stem cells (MSCs) to treat articular cartilage defects. Here, we set out to determine the growth factors (bone morphogenetic protein 2 (BMP-2) and transforming growth factor-β (TGF-β1)) and oxygen tension effects during chondrogenesis of human UCB-MSCs for cartilage engineering. Chondrogenic differentiation was induced using 3D cultures in type I/III collagen sponges with chondrogenic factors in normoxia (21% O₂) or hypoxia (<5% O₂) for 7, 14 and 21 days. Our results show that UCB-MSCs can be committed to chondrogenesis in the presence of BMP-2+TGF-β1. Normoxia induced the highest levels of chondrocyte-specific markers. However, hypoxia exerted more benefit by decreasing collagen X and matrix metalloproteinase-13 (MMP13) expression, two chondrocyte hypertrophy markers. However, a better chondrogenesis was obtained by switching oxygen conditions, with seven days in normoxia followed by 14 days in hypoxia, since these conditions avoid hypertrophy of hUCB-MSC-derived chondrocytes while maintaining the expression of chondrocyte-specific markers observed in normoxia. Our study demonstrates that oxygen tension is a key factor for chondrogenesis and suggests that UBC-MSCs 3D-culture should begin in normoxia to obtain a more efficient chondrocyte differentiation before placing them in hypoxia for chondrocyte phenotype stabilization. UCB-MSCs are therefore a reliable source for cartilage engineering.

  9. Biochemical and metabolic abnormalities in normal and osteoarthritic human articular cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, J.; Treadwell, B.V.; Mankin, H.J.

    1984-01-01

    Incorporation of radioactive precursors into macromolecules was studied with human normal and osteoarthritic articular cartilage organ culture. Analysis of the salt extracted matrix components separated by cesium chloride buoyant density gradient centrifugation showed an increase in the specific activities of all gradient fractions prepared from the osteoarthritic cartilage. Further analysis of these fractions showed the osteoarthritic cartilage contained 5 times as much sulfate incorporated into proteoglycans, and an even greater amount of 3H-glucosamine incorporated into material sedimenting to the middle of the gradient. Greater than half of this radioactive middle fraction appears to be hyaluronate, as judged by the position it elutes from a DEAE column and its susceptibility to hyaluronidase digestion. This study supports earlier findings showing increased rates of macromolecular synthesis in osteoarthritis, and in addition, an even greater synthetic rate for hyaluronic acid is demonstrated.

  10. Indian Hedgehog in Synovial Fluid Is a Novel Marker for Early Cartilage Lesions in Human Knee Joint

    Directory of Open Access Journals (Sweden)

    Congming Zhang

    2014-04-01

    Full Text Available To determine whether there is a correlation between the concentration of Indian hedgehog (Ihh in synovial fluid (SF and the severity of cartilage damage in the human knee joints, the knee cartilages from patients were classified using the Outer-bridge scoring system and graded using the Modified Mankin score. Expression of Ihh in cartilage and SF samples were analyzed with immunohistochemistry (IHC, western blot, and enzyme-linked immunosorbent assay (ELISA. Furthermore, we detected and compared Ihh protein levels in rat and mice cartilages between normal control and surgery-induced osteoarthritis (OA group by IHC and fluorescence molecular tomography in vivo respectively. Ihh expression was increased 5.2-fold in OA cartilage, 3.1-fold in relative normal OA cartilage, and 1.71-fold in OA SF compared to normal control samples. The concentrations of Ihh in cartilage and SF samples was significantly increased in early-stage OA samples when compared to normal samples (r = 0.556; p < 0.001; however, there were no significant differences between normal samples and late-stage OA samples. Up-regulation of Ihh protein was also an early event in the surgery-induced OA models. Increased Ihh is associated with the severity of OA cartilage damage. Elevated Ihh content in human knee joint synovial fluid correlates with early cartilage lesions.

  11. In vivo effect of opticin deficiency in cartilage in a surgically induced mouse model of osteoarthritis.

    Science.gov (United States)

    Farrán, Aina; Valverde-Franco, Gladys; Tío, Laura; Lussier, Bertrand; Fahmi, Hassan; Pelletier, Jean-Pierre; Bishop, Paul N; Monfort, Jordi; Martel-Pelletier, Johanne

    2018-01-11

    The SLRP opticin (OPTC) has been demonstrated to be produced and degraded in osteoarthritic (OA) human cartilage. Here, we investigated the in vivo effect of OPTC deficiency in OA cartilage. OA was induced in 10-week-old Optc -/- and Optc +/+ mice. Ten weeks post-surgery, cartilage was processed for histology and immunohistochemistry. SLRP expression was determined in non-operated mouse cartilage. OA Optc -/- demonstrated significant protection against cartilage degradation. Data revealed that in non-operated Optc -/- cartilage, expression of SLRPs lumican and epiphycan was up-regulated at day 3 and in 10-week-olds (p ≤ 0.039), and fibromodulin down-regulated in 10-week-olds (p = 0.001). Immunohistochemistry of OA mice showed a similar pattern. In OA Optc -/- cartilage, markers of degradation and complement factors were all down-regulated (p ≤ 0.038). In OA Optc -/- cartilage, collagen fibers were thinner and better organized (p = 0.038) than in OA Optc +/+ cartilage. The protective effect of OPTC deficiency during OA results from an overexpression of lumican and epiphycan, known to bind and protect collagen fibers, and a decrease in fibromodulin, contributing to a reduction in the complement activation/inflammatory process. This work suggests that the evaluation of the composition of the different SLRPs in OA cartilage could be applied as a new tool for OA prognosis classification.

  12. Immunohistochemistry Evaluation of TGF-β1, SOX-9, Type II Collagen and Aggrecan in Cartilage Lesions Treated with Conditioned Medium of Umbilical Cord Mesencyhmal Stem Cells in Wistar Mice (Rattus novergicus

    Directory of Open Access Journals (Sweden)

    Bintang Soetjahjo

    2018-01-01

    Full Text Available Currently, umbilical cord mesenchymal stem cells have the potential to be used as treatment options for any cartilage lesion. This research aimed to evaluate the effects of conditioned medium from umbilical cord mesenchymal stem cells (UC-MSC on damaged cartilage through the expression of proteins TGF-β1, SOX-9, type II collagen and aggrecan, which are known to be related to chondrogenesis. UC-MSC were isolated from 19-days-pregnant Wistar mice and were cultured using the standard procedure to obtain 80% confluence. Subsequently, the culture was confirmed through a microscopic examination that was driven to be an embryoid body to obtain a pre-condition medium. This research utilized 3-month-old male Wistar mice and was categorized into 6 groups (3 control and 3 treatment groups. Each animal had surgery performed to create a femur condyle cartilage defect. The treatment groups were administered a dose of stem cells at 1 mL/kg. Next, immunohistochemical (IHC staining was performed to examine the expression of TGF-β1, SOX-9, type II collagen and aggrecan in the 2nd, 3rd, and 4th month of evaluation. The results were analyzed statistically using ANOVA test. For each of the treatment groups, there was increased expression (p < 0.05 in all proteins TGF-β1, SOX-9, type II collagen and aggrecan when compared with control groups at the 2nd, 3rd, and 4th month of evaluation. Pre-conditioned medium from UC-MSC potentially increases the expression of TGF-β1, SOX-9, type II collagen and aggrecan in the damaged cartilage of Wistar mice.

  13. Human corneal fibrillogenesis. Collagen V structural analysis and fibrillar assembly by stromal fibroblasts in culture.

    Science.gov (United States)

    Ruggiero, F; Burillon, C; Garrone, R

    1996-08-01

    The stroma of the developing cornea is a highly organized extracellular matrix formed essentially by uniform, small-diameter collagen fibrils with constant interfibrillar spacing. Unlike the fibrillogenesis of chicken cornea, the assembly and maturation of human corneal fibrils have been poorly investigated. In the current study, the authors aimed to ascertain the heterotypic organization (collagens I and V) of the human corneal fibrils at the supramolecular level. To gain more insight into the molecular structure of collagen V, its cellular source, and its role in fibrillogenesis, the authors used cultured human corneal fibroblasts. The structure of human corneal stroma after brief homogenization of the tissue was analyzed by immunogold labeling using specific polyclonal antibodies and rotary shadowing. Biochemical, electron microscopic, and immunolabeling approaches were used to investigate the collagen fibril formation and the extracellular matrix synthesis using human corneal fibroblasts grown in culture as a model system. The authors showed that in human corneal stroma, collagen I is distributed uniformly along the striated fibrils, in contrast to collagen V, which could be identified only at sites at which the fibrils partially were disrupted. Rotary shadowing observations of the homogenate revealed that collagen VI, a major component of the human cornea, was associated closely with the collagen fibril surface. Corneal fibroblasts synthesize and deposit a collagenous matrix with fibrils resembling those of the human cornea in appearance and collagen composition. Biochemical data indicate that a high concentration (20% to 30%) of collagen V is synthesized by stromal fibroblasts and that collagen V molecules are processed similarly to matrix forms in which the extension peptides are retained on the molecules. The heterotypic nature (collagens I and V) of human corneal fibrils was determined. Results indicate that human corneal fibroblasts synthesize the major

  14. Layer-by-layer assembly of type I collagen and chondroitin sulfate on aminolyzed PU for potential cartilage tissue engineering application

    Energy Technology Data Exchange (ETDEWEB)

    He Xianyun [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China); Wang Yingjun, E-mail: imwangyj@163.com [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China) and National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China) and Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China); Wu Gang, E-mail: imwugang@scut.edu.cn [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006 (China)

    2012-10-01

    Highlights: Black-Right-Pointing-Pointer A novel biodegradable polyurethane (PU) was successfully synthesized. Black-Right-Pointing-Pointer Surface aminolyzing of the PU was performed by reacting it with 1,3-propanediamine. Black-Right-Pointing-Pointer Collagen and chondroitin sulfate were deposited alternately on the PU surface. - Abstract: In this paper, a two-step method was used to synthesize a biodegradable polyurethane (PU) composed of L-lysine ethyl ester diisocyanate (LDI), poly({epsilon}-caprolactone) diols (PCL-diol) and 1,4:3,6-dianhydro-D-sorbitol (isosorbide). Amino groups were introduced onto the surface of the PU membrane by an amination reacting with 1,3-propanediamine to produce polycationic substratum. And then, type I collagen (Col) and chondroitin sulfate (CS) were deposited alternately on the polycationic substratum through layer-by-layer (LBL) assembly technology. The FTIR and {sup 1}H NMR results showed that the polyurethane was successfully synthesized. Rhodamine B isothiocyanate (RBITC) fluorescence spectrum indicated that amino groups were successfully introduced onto the PU surface. The results of quartz-crystal microbalance (QCM) and RBITC-Col fluorescence spectroscopy monitoring the LBL assemble process presented that the Col/CS deposited alternately on the PU surface. X-ray photoelectron spectroscopy (XPS) results displayed that the CS deposited on the PU surface as well. The surface of the assembled PU became even smoother observed from the surface morphology by atomic force microscopy (AFM) imaging. The hydrophilicity of the PU membrane was greatly enhanced though the modification of LBL assembly. The PU modified with the adsorption of Col/CS may be a potential application for cartilage tissue engineering due to its created mimicking chondrogenic environment.

  15. Interstitial collagen is increased in the non-infarcted human myocardium after myocardial infarction

    NARCIS (Netherlands)

    Volders, P. G.; Willems, I. E.; Cleutjens, J. P.; Arends, J. W.; Havenith, M. G.; Daemen, M. J.

    1993-01-01

    In this study we report the changes of interstitial collagen in the human non-infarcted interventricular septum after a myocardial infarction as well as in hypertrophic human hearts with or without hypertension. The collagen amount was determined with the Sirius Red morphometry technique, which

  16. Recognizing different tissues in human fetal femur cartilage by label-free Raman microspectroscopy

    Science.gov (United States)

    Kunstar, Aliz; Leijten, Jeroen; van Leuveren, Stefan; Hilderink, Janneke; Otto, Cees; van Blitterswijk, Clemens A.; Karperien, Marcel; van Apeldoorn, Aart A.

    2012-11-01

    Traditionally, the composition of bone and cartilage is determined by standard histological methods. We used Raman microscopy, which provides a molecular "fingerprint" of the investigated sample, to detect differences between the zones in human fetal femur cartilage without the need for additional staining or labeling. Raman area scans were made from the (pre)articular cartilage, resting, proliferative, and hypertrophic zones of growth plate and endochondral bone within human fetal femora. Multivariate data analysis was performed on Raman spectral datasets to construct cluster images with corresponding cluster averages. Cluster analysis resulted in detection of individual chondrocyte spectra that could be separated from cartilage extracellular matrix (ECM) spectra and was verified by comparing cluster images with intensity-based Raman images for the deoxyribonucleic acid/ribonucleic acid (DNA/RNA) band. Specific dendrograms were created using Ward's clustering method, and principal component analysis (PCA) was performed with the separated and averaged Raman spectra of cells and ECM of all measured zones. Overall (dis)similarities between measured zones were effectively visualized on the dendrograms and main spectral differences were revealed by PCA allowing for label-free detection of individual cartilaginous zones and for label-free evaluation of proper cartilaginous matrix formation for future tissue engineering and clinical purposes.

  17. Local tissue properties of human osteoarthritic cartilage correlate with magnetic resonance T(1) rho relaxation times.

    Science.gov (United States)

    Tang, Simon Y; Souza, Richard B; Ries, Michael; Hansma, Paul K; Alliston, Tamara; Li, Xiaojuan

    2011-09-01

    The objective of this study is to examine the local relationship between T(1ρ) relaxation times and the mechanical behavior of human osteoarthritic articular cartilage using high-resolution magnetic resonance imaging (MRI) and local in situ microindentation. Seven human tibial plateaus were obtained from patients who underwent total knee arthroplasty due to severe osteoarthritis (OA). Three to six sites were selected from each sample for visual classification using the ICRS Outerbridge scale (a total of 36 sites). Samples were imaged by MR, and the local distribution of T(1ρ) relaxation times were obtained at these selected sites. The elastic and viscoelastic characteristics of the tissue were quantified nondestructively using dynamic microindentation to measure peak dynamic modulus, energy dissipation, and phase angle. Measured Outerbridge scores, MR T(1ρ) relaxation times, and mechanical properties were highly heterogeneous across each cartilage surface. Site-specific measures of T(1ρ) relaxation times correlated significantly with the phase angle (p properties in highly heterogeneous OA cartilage. These findings suggest that MRI T(1ρ) can provide a functional assessment of articular cartilage. Copyright © 2011 Orthopaedic Research Society.

  18. Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2.

    Directory of Open Access Journals (Sweden)

    Alexander J Neumann

    Full Text Available Articular cartilage progenitor cells (ACPCs represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs. This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2. hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1 concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase. To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs

  19. Immunohistochemical analysis of collagen expression in human corpora lutea during the menstrual cycle and early pregnancy.

    Science.gov (United States)

    Iwahashi, Masaaki; Muragaki, Yasuteru; Ooshima, Akira; Umesaki, Naohiko

    2006-04-01

    To investigate the characteristic structure and function of human corpora lutea (CL), various types of collagen expression were determined in the CL tissues during the menstrual cycle and early pregnancy. In vitro experiment. Department of obstetrics and gynecology at a medical university. Regulatory cycling women and pregnant women with ovarian tumor and ectopic pregnancy who underwent adnexectomy. Immunohistochemistry for human type I, III, and IV collagen with specific monoclonal antibodies was used for analysis. Expression of type I, III, and IV collagen. Immunohistochemical staining for type I and III collagen revealed intense staining of the CL stroma during early pregnancy, as compared with those in the menstrual cycle. Moreover, pericellular intense immunostaining for type IV collagen was observed around the luteal cells, especially luteal granulosa cells, of early pregnancy. These results suggest that alterations in distribution of collagen might play an important role in determining the physiology and structure of the CL during the menstrual cycle and early pregnancy.

  20. Tendon collagen synthesis declines with immobilization in elderly humans

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  1. [Distribution of collagen types III and IV in human placental villi].

    Science.gov (United States)

    Nanaev, A K; Rukosuev, V S; Milovanov, A P; Fokin, E I; Shirinskiĭ, V P

    1989-02-01

    Immunofluorescent examination showed more significant accumulation of interstitial collagen type III in the stroma of mature placenta compared with immature one. Localization of membrane collagen type IV was found neither in basal membranes of epithelium and villous vessels of mature term placenta, nor in their stroma. The described patterns of distribution of collagen types III and IV in human placenta villi were proved by immunoelectronmicroscopic method.

  2. Computational biodynamics of human knee joint in gait: from muscle forces to cartilage stresses.

    Science.gov (United States)

    Adouni, M; Shirazi-Adl, A; Shirazi, R

    2012-08-09

    Using a validated finite element model of the intact knee joint we aim to compute muscle forces and joint response in the stance phase of gait. The model is driven by reported in vivo kinematics-kinetics data and ground reaction forces in asymptomatic subjects. Cartilage layers and menisci are simulated as depth-dependent tissues with collagen fibril networks. A simplified model with less refined mesh and isotropic depth-independent cartilage is also considered to investigate the effect of model accuracy on results. Muscle forces and joint detailed response are computed following an iterative procedure yielding results that satisfy kinematics/kinetics constraints while accounting at deformed configurations for muscle forces and passive properties. Predictions confirm that muscle forces and joint response alter substantially during the stance phase and that a simplified joint model may accurately be used to estimate muscle forces but not necessarily contact forces/areas, tissue stresses/strains, and ligament forces. Predictions are in general agreement with results of earlier studies. Performing the analyses at 6 periods from beginning to the end (0%, 5%, 25%, 50%, 75% and 100%), hamstrings forces peaked at 5%, quadriceps forces at 25% whereas gastrocnemius forces at 75%. ACL Force reached its maximum of 343 N at 25% and decreased thereafter. Contact forces reached maximum at 5%, 25% and 75% periods with the medial compartment carrying a major portion of load and experiencing larger relative movements and cartilage strains. Much smaller contact stresses were computed at the patellofemoral joint. This novel iterative kinematics-driven model is promising for the joint analysis in altered conditions. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Characterization of the collagen component of cartilage repair tissue of the talus with quantitative MRI: comparison of T2 relaxation time measurements with a diffusion-weighted double-echo steady-state sequence (dwDESS)

    Energy Technology Data Exchange (ETDEWEB)

    Kretzschmar, M.; Hainc, N.; Studler, U. [University Hospital Basel, Department of Radiology, Basel (Switzerland); Bieri, O. [University Hospital Basel, Division of Radiological Physics, Basel (Switzerland); Miska, M. [University Hospital, Department of Orthopedics, Heidelberg (Germany); Wiewiorski, M.; Valderrabano, V. [University Hospital Basel, Department of Orthopedic Surgery, Basel (Switzerland)

    2015-04-01

    The purpose of this study was to characterize the collagen component of repair tissue (RT) of the talus after autologous matrix-induced chondrogenesis (AMIC) using quantitative T2 and diffusion-weighted imaging. Mean T2 values and diffusion coefficients of AMIC-RT and normal cartilage of the talus of 25 patients with posttraumatic osteochondral lesions and AMIC repair were compared in a cross-sectional design using partially spoiled steady-state free precession (pSSFP) for T2 quantification, and diffusion-weighted double-echo steady-state (dwDESS) for diffusion measurement. RT and cartilage were graded with modified Noyes and MOCART scores on morphological sequences. An association between follow-up interval and quantitative MRI measures was assessed using multivariate regression, after stratifying the cohort according to time interval between surgery and MRI. Mean T2 of the AMIC-RT and cartilage were 43.1 ms and 39.1 ms, respectively (p = 0.26). Mean diffusivity of the RT (1.76 μm{sup 2}/ms) was significantly higher compared to normal cartilage (1.46 μm{sup 2}/ms) (p = 0.0092). No correlation was found between morphological and quantitative parameters. RT diffusivity was lowest in the subgroup with follow-up >28 months (p = 0.027). Compared to T2-mapping, dwDESS demonstrated greater sensitivity in detecting differences in the collagen matrix between AMIC-RT and cartilage. Decreased diffusivity in patients with longer follow-up times may indicate an increased matrix organization of RT. (orig.)

  4. Hydrocortisone regulates types I and III collagen gene expression and collagen synthesis in human marrow stromal cells.

    Science.gov (United States)

    Fernández, M; Minguell, J J

    1997-01-01

    Hematopoiesis is the resultant of the orderly molecular and cellular interactions between progenitor cells and stroma. In vitro studies (Dexter-type cultures) have shown that initiation of hematopoiesis only occurs after establishment of a hydrocortisone-dependent layer of stromal cells. Although the molecular basis for the requirement of hydrocortisone are not well understood, data have shown that synthesis/assembly of extracellular matrix molecules (proteoglycans and fibronectin) is regulated by hydrocortisone. Since interstitial collagens are abundantly expressed in the marrow stroma, we investigated whether hydrocortisone may also modulate the expression of collagen types I and III. For these studies, human bone marrow fibroblast cultures were grown in standard culture medium either in the absence or presence of 10(-7) M hydrocortisone. Under both conditions, bone marrow fibroblasts synthesized collagen types I and III, and expressed the respective genes. However, hydrocortisone produced a decrease in the synthesis of interstitial collagens and also in the relative abundance of pro-alpha 1(I) and pro-alpha 1(III) mRNAs. The results of this study are consistent with the assumption that glucocorticoids regulate the expression of several extracellular matrix molecules in the marrow stroma and thus permit in vitro hematopoiesis to occur.

  5. Insulin-like growth factor I enhances collagen synthesis in engineered human tendon tissue

    DEFF Research Database (Denmark)

    Herchenhan, Andreas; Bayer, Monika L.; Eliasson, Pernilla

    2015-01-01

    investigated the influence of insulin-like growth factor I (IGF-I) on tendon construct formation in 3D cell culture. DESIGN: Tendon constructs were grown in 0.5 or 10% FBS with or without IGF-I (250 mg/ml) supplementation. Collagen content (fluorometric), mRNA levels (PCR) and fibril diameter (transmission...... of fibril diameter (day 14), collagen content (at days 21 and 28) and mRNA expression for collagen, tenomodulin and scleraxis. CONCLUSION: IGF-I supplementation promotes early onset of tensile load induced collagen formation and tendon structural arrangement, whereas the FBS concentration routinely used......OBJECTIVE: Isolated human tendon cells form 3D tendon constructs that demonstrate collagen fibrillogenesis and feature structural similarities to tendon when cultured under tensile load. The exact role of circulating growth factors for collagen formation in tendon is sparsely examined. We...

  6. Differential expression of type XIV collagen/undulin by human mammary gland intralobular and interlobular fibroblasts.

    Science.gov (United States)

    Atherton, A J; Warburton, M J; O'Hare, M J; Monaghan, P; Schuppan, D; Gusterson, B A

    1998-03-01

    Immunolocalisation of type XIV collagen/undulin in the human mammary gland revealed greater deposition in the interlobular stroma than in the intralobular stroma. The interlobular stroma is located between the breast lobules and their associated intralobular stroma. Fibroblasts isolated from the interlobular stroma synthesised 3- to 5-fold more type XIV collagen/undulin than intralobular fibroblasts, but synthesised type I and type IV collagens in similar amounts. The differential expression of type XIV collagen/undulin was maintained with passage in culture. The results suggest a role for type XIV collagen/undulin in stabilising dense collagen fibrils. The maintenance of two types of structurally distinct stromas may be important during developmental processes in the mammary gland.

  7. Second Harmonic Generation Imaging Analysis of Collagen Arrangement in Human Cornea.

    Science.gov (United States)

    Park, Choul Yong; Lee, Jimmy K; Chuck, Roy S

    2015-08-01

    To describe the horizontal arrangement of human corneal collagen bundles by using second harmonic generation (SHG) imaging. Human corneas were imaged with an inverted two photon excitation fluorescence microscope. The excitation laser (Ti:Sapphire) was tuned to 850 nm. Backscatter signals of SHG were collected through a 425/30-nm bandpass emission filter. Multiple, consecutive, and overlapping image stacks (z-stacks) were acquired to generate three dimensional data sets. ImageJ software was used to analyze the arrangement pattern (irregularity) of collagen bundles at each image plane. Collagen bundles in the corneal lamellae demonstrated a complex layout merging and splitting within a single lamellar plane. The patterns were significantly different in the superficial and limbal cornea when compared with deep and central regions. Collagen bundles were smaller in the superficial layer and larger in deep lamellae. By using SHG imaging, the horizontal arrangement of corneal collagen bundles was elucidated at different depths and focal regions of the human cornea.

  8. Determination of markers for collagen type I turnover in peritendinous human tissue by microdialysis

    DEFF Research Database (Denmark)

    Olesen, J L; Langberg, Henning; Heinemeier, K M

    2006-01-01

    Previous results from our group have shown that loading of human tendon elevates tendinous type I collagen production measured by microdialysis. However, exclusion of the observed elevation as a response to trauma from inserting the microdialysis catheters or a possible influence from the collagen...

  9. Label-free characterization of articular cartilage in osteoarthritis model mice by Raman spectroscopy

    Science.gov (United States)

    Oshima, Yusuke; Akehi, Mayu; Kiyomatsu, Hiroshi; Miura, Hiromasa

    2017-02-01

    Osteoarthritis (OA) is very common joint disease in the aging population. Main symptom of OA is accompanied by degenerative changes of articular cartilage. Cartilage contains mostly type II collagen and proteoglycans, so it is difficult to access the quality and morphology of cartilage tissue in situ by conventional diagnostic tools (X-ray, MRI and echography) directly or indirectly. Raman spectroscopy is a label-free technique which enables to analyze molecular composition in degenerative cartilage. In this study, we generated an animal OA model surgically induced by knee joint instability, and the femurs were harvested at two weeks after the surgery. We performed Raman spectroscopic analysis for the articular cartilage of distal femurs in OA side and unaffected side in each mouse. In the result, there is no gross findings in the surface of the articular cartilage in OA. On the other hand, Raman spectral data of the articular cartilage showed drastic changes in comparison between OA and control side. The major finding of this study is that the relative intensity of phosphate band (960 cm-1) increases in the degenerative cartilage. This may be the result of exposure of subchondral bone due to thinning of the cartilage layer. In conclusion, Raman spectroscopic technique is sufficient to characterize articular cartilage in OA as a pilot study for Raman application in cartilage degeneration and regeneration using animal models and human subjects.

  10. Tissue-engineered recombinant human collagen-based corneal substitutes for implantation: performance of type I versus type III collagen.

    Science.gov (United States)

    Merrett, Kimberley; Fagerholm, Per; McLaughlin, Christopher R; Dravida, Subhadra; Lagali, Neil; Shinozaki, Naoshi; Watsky, Mitchell A; Munger, Rejean; Kato, Yasuhiro; Li, Fengfu; Marmo, Christopher J; Griffith, May

    2008-09-01

    To compare the efficacies of recombinant human collagens types I and III as corneal substitutes for implantation. Recombinant human collagen (13.7%) type I or III was thoroughly mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. The final homogenous solution was either molded into sheets for in vitro studies or into implants with the appropriate corneal dimensions for transplantation into minipigs. Animals with implants were observed for up to 12 months after surgery. Clinical examinations of the cornea included detailed slit lamp biomicroscopy, in vivo confocal microscopy, and fundus examination. Histopathologic examinations were also performed on corneas harvested after 12 months. Both cross-linked recombinant collagens had refractive indices of 1.35, with optical clarity similar to that in human corneas. Their chemical and mechanical properties were similar, although RHC-III implants showed superior optical clarity. Implants into pig corneas over 12 months show comparably stable integration, with regeneration of corneal cells, tear film, and nerves. Optical clarity was also maintained in both implants, as evidenced by fundus examination. Both RHC-I and -III implants can be safely and stably integrated into host corneas. The simple cross-linking methodology and recombinant source of materials makes them potentially safe and effective future corneal matrix substitutes.

  11. Fetal mesenchymal stromal cells differentiating towards chondrocytes acquire a gene expression profile resembling human growth plate cartilage.

    Directory of Open Access Journals (Sweden)

    Sandy A van Gool

    Full Text Available We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs differentiating towards chondrocytes as an alternative model for the human growth plate (GP. Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.

  12. Fetal mesenchymal stromal cells differentiating towards chondrocytes acquire a gene expression profile resembling human growth plate cartilage.

    Science.gov (United States)

    van Gool, Sandy A; Emons, Joyce A M; Leijten, Jeroen C H; Decker, Eva; Sticht, Carsten; van Houwelingen, Johannes C; Goeman, Jelle J; Kleijburg, Carin; Scherjon, Sicco A; Gretz, Norbert; Wit, Jan Maarten; Rappold, Gudrun; Post, Janine N; Karperien, Marcel

    2012-01-01

    We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.

  13. Compositional and structural studies of the bone-cartilage interface using PIXE and SAXS techniques

    Energy Technology Data Exchange (ETDEWEB)

    Kaabar, W., E-mail: W.kaabar@surrey.ac.u [Department of Physics, University of Surrey, Guildford, GU2 7XH (United Kingdom); Laklouk, A. [Al-Fateh University, Tripoli-Libya (Libyan Arab Jamahiriya); Bunk, O. [Swiss Light Source, Paul Scherrer Institute, 5232 Villigen (Switzerland); Baily, M. [Department of Medical Physics and Applied Radiation Sciences, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada, L8S 4K1 (Canada); Farquharson, M.J. [Surrey Ion Beam Centre, University of Surrey, Guildford, GU2 7XH (United Kingdom); Bradley, David [Department of Physics, University of Surrey, Guildford, GU2 7XH (United Kingdom)

    2010-07-21

    Micro-proton-induced X-ray emission ({mu}-PIXE) analysis has been employed in investigating the presence of number of essential anions and cations in thin sections of diseased human articular cartilage affected by osteoarthritis (OA). Distribution maps for Ca, P, K and S in diseased sections show marked alterations in the concentrations of these at the bone-cartilage interface compared to normal tissue. For a decalcified section of human articular cartilage, organisational changes of the collagen network were investigated by small-angle X-ray scattering (SAXS). The established gradual reorientation of collagen fibres from vertical to the surface of the joint to normal to the bone-cartilage interface is observed to be heavily disrupted in OA.

  14. Cartilage Regeneration in Human with Adipose Tissue-Derived Stem Cells: Current Status in Clinical Implications

    Directory of Open Access Journals (Sweden)

    Jaewoo Pak

    2016-01-01

    Full Text Available Osteoarthritis (OA is one of the most common debilitating disorders among the elderly population. At present, there is no definite cure for the underlying causes of OA. However, adipose tissue-derived stem cells (ADSCs in the form of stromal vascular fraction (SVF may offer an alternative at this time. ADSCs are one type of mesenchymal stem cells that have been utilized and have demonstrated an ability to regenerate cartilage. ADSCs have been shown to regenerate cartilage in a variety of animal models also. Non-culture-expanded ADSCs, in the form of SVF along with platelet rich plasma (PRP, have recently been used in humans to treat OA and other cartilage abnormalities. These ADSCs have demonstrated effectiveness without any serious side effects. However, due to regulatory issues, only ADSCs in the form of SVF are currently allowed for clinical uses in humans. Culture-expanded ADSCs, although more convenient, require clinical trials for a regulatory approval prior to uses in clinical settings. Here we present a systematic review of currently available clinical studies involving ADSCs in the form of SVF and in the culture-expanded form, with or without PRP, highlighting the clinical effectiveness and safety in treating OA.

  15. Cartilage constructs from human cord blood stem cells seeded in structurally-graded polycaprolactone scaffolds

    DEFF Research Database (Denmark)

    Munir, Samir; Koch, Thomas Gadegaard; Foldager, Casper Bindzus

    stimulation. This study demonstrated the chondrogenic potential of human cord blood-derived Multi-Lineage Progenitor Cells (MLPCs) under normoxic and hypoxic culture conditions. Second, MLPCs were seeded in a novel, structurally graded polycaprolactone (SGS-PCL) scaffold and chondrogenesis was evaluated......, safranin O), glycosaminoglycan (GAG) protein secretion, and gene expression of cartilage markers. Based on this data, MLPCs were seeded in SGS-PCL scaffolds and cultured under optimal oxygen tension for 21 days followed by chondrogenic evaluation as above. Porous SGS-PCL scaffolds were provided by i...... tensions. Histological sections revealed a cartilaginous struc¬ture as recognized by chondrocyte-like cells embedded in lacunae. Histological sections of control pellets did not stain for GAG nor show a cartilage-like morphology. Gene expression analyses (qRT-PCR), GAG protein secretion, and histology...

  16. Age variations in the properties of human tibial trabecular bone and cartilage

    DEFF Research Database (Denmark)

    Ding, Ming

    2000-01-01

    such as osteoarthrosis and osteoporosis, and for the design, fixation and durability of total joint prosthesis. The specific aims of the present studies were: 1) to investigate normal age-related variations in the mechanical, physical/compositional, and structural properties of human tibial trabecular bone; and 2...... in the properties of trabecular bone and the cartilage-bone complex, and osteoarthrotic specimens were used for the investigation of changes in the mechanical properties of the cartilage-bone complex induced by this disease process. The mechanical properties and physical/compositional properties of trabecular bone...... in the microstructural properties had the same trends for both medial and lateral condyles of the tibia. The observed increase of anisotropy may be interpreted as the consequence of structural adaptation secondary to age-induced bone loss. The aging trabeculae align more strongly to the primary direction, which...

  17. Image registration improves human knee cartilage T1 mapping with delayed gadolinium-enhanced MRI of cartilage (dGEMRIC)

    NARCIS (Netherlands)

    E.E. Bron (Esther); J. van Tiel (Jasper); H.A. Smit (Henk); D.H.J. Poot; W.J. Niessen (Wiro); G.P. Krestin (Gabriel); H.H. Weinans (Harrie); E.H.G. Oei (Edwin); G. Kotek (Gyula); S. Klein (Stefan)

    2013-01-01

    textabstractObjectives: To evaluate the effect of automated registration in delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) of the knee on the occurrence of movement artefacts on the T1 map and the reproducibility of region-of-interest (ROI)-based measurements. Methods: Eleven patients with

  18. Viability of human chondrocytes in an ex vivo model in relation to temperature and cartilage depth.

    Science.gov (United States)

    Drobnic, M; Mars, T; Alibegović, A; Bole, V; Balazic, J; Grubic, Z; Brecelj, J

    2005-01-01

    Chondrocytes in human articular cartilage remain viable post-mortem. It has however not been established yet how the storage temperature affects their survival, which is essential information when post-mortem cartilage is used for toxicologic studies. Our aim was to construct a simple model of explanted knee cartilage and to test the influences of time and temperature on the viability of chondrocytes in the ex vivo conditions. Osteochondral cylinders were procured from the cadaveric femoral condyles. The cylinders were embedded in water-tight rubber tubes, which formed separate chondral and osteal compartments. Tubes were filled with normal saline, without additives, to keep chondrocytes under close-to-normal conditions. The samples were divided into two groups stored at 4 degrees C and 35 degrees C, respectively. Three samples of each of these two groups were analysed at the time of removal, and then three and nine days later. Images of Live-Dead staining were scanned by a confocal laser microscope. Count of viable chondrocytes in four regions, from surface to bone, was obtained using image analysis software. The regression model revealed that the number of viable chondrocytes decreased every day by 19% and that an increase in temperature by 1 degree C decreased their viability by 5.8%. The temperature effect fell by 0.2 percentage points for every 100 microm from the surface to the bone. Herein we demonstrate that chondrocytes remain viable in the ex vivo model of human knee cartilage long enough to be able to serve as a model for toxicologic studies. Their viability is, however, significantly influenced by time and temperature.

  19. Electromechanical Assessment of Human Knee Articular Cartilage with Compression-Induced Streaming Potentials.

    Science.gov (United States)

    Becher, Christoph; Ricklefs, Marcel; Willbold, Elmar; Hurschler, Christof; Abedian, Reza

    2016-01-01

    To assess the electromechanical properties of human knee articular cartilage with compression-induced streaming potentials for reliability among users and correlation with macroscopic and histological evaluation tools and sulfated glycosaminoglycan (sGAG) content. Streaming potentials are induced in cartilage in response to loading when mobile positive ions in the interstitial fluid temporarily move away from negatively charged proteoglycans. Streaming potential integrals (SPIs) were measured with an indentation probe on femoral condyles of 10 human knee specimens according to a standardized location scheme. Interobserver reliability was measured using an interclass correlation coefficient (ICC). The learning curves of 3 observers were evaluated by regression analysis. At each SPI measurement location the degradation level of the tissue was determined by means of the International Cartilage Repair Society (ICRS) score, Mankin score, and sGAG content. The computed ICC was 0.77 (0.70-0.83) indicating good to excellent linear agreement of SPI values among the 3 users. A significant positive linear correlation of the learning index values was observed for 2 of the 3 users. Statistically significant negative correlations between SPI and both ICRS and Mankin scores were observed (r = 0.502, P < 0.001, and r = 0.255, P = 0.02, respectively). No correlation was observed between SPI and sGAG content (r = 0.004, P = 0.973). SPI values may be used as a quantitative means of cartilage evaluation with sufficient reliability among users. Due to the significant learning curve, adequate training should be absolved before routine use of the technique.

  20. Influence of caffeine and hyaluronic acid on collagen biosynthesis in human skin fibroblasts.

    Science.gov (United States)

    Donejko, Magdalena; Przylipiak, Andrzej; Rysiak, Edyta; Głuszuk, Katarzyna; Surażyński, Arkadiusz

    2014-01-01

    The aim of this study was to evaluate the effect of caffeine on collagen biosynthesis in human skin fibroblasts and the influence of hyaluronic acid (HA) on this process. Collagen, [(3)H]-thymidine incorporation, and prolidase activity were measured in confluent human skin fibroblast cultures that had been treated with 1, 2, and 5 mM caffeine and with caffeine and 500 μg/mL HA. Western immunoblot analysis was performed to evaluate expression of β1-integrin receptor, insulin-like growth factor receptor phospho-Akt protein and mitogen-activated protein kinase (phospho-extracellular signal-regulated kinase). Caffeine inhibited collagen biosynthesis in a dose-dependent manner. The mechanism of this process was found at the level of prolidase activity. Caffeine significantly inhibited the enzyme activity. The addition of HA had no effect on collagen biosynthesis or prolidase activity in fibroblasts incubated with caffeine. Caffeine also had an inhibitory effect on DNA biosynthesis. HA, however, did not have any significant effect on this process. The inhibition of the expression of β1-integrin and insulin-like growth factor receptor in fibroblasts incubated with the caffeine indicates a possible mechanism of inhibition of collagen biosynthesis. Caffeine reduces collagen synthesis in human cultured skin fibroblasts. HA did not have any significant protective effect on this process. This is the first study to our knowledge that reports caffeine-induced inhibition of collagen synthesis in human skin fibroblasts.

  1. Second Harmonic Generation Imaging Analysis of Collagen Arrangement in Human Cornea

    OpenAIRE

    Park, Choul Yong; Lee, Jimmy K.; Chuck, Roy S.

    2015-01-01

    In this paper, we imaged human cornea using a second harmonic generation imaging technique. The horizontal collagen bundle arrangement of corneal stroma as a function of depth and location was analyzed.

  2. The normal human chondro-osseous junctional region: evidence for contact of uncalcified cartilage with subchondral bone and marrow spaces

    Directory of Open Access Journals (Sweden)

    Stoddart Robert W

    2006-06-01

    Full Text Available Abstract Background The chondro-osseous junctional region of diarthrodial joints is peculiarly complex and may be considered to consist of the deepest layer of non-calcified cartilage, the tidemark, the layer of calcified cartilage, a thin cement line (between the calcified cartilage and the subchondral bone and the subchondral bone. A detailed knowledge of the structure, function and pathophysiology of the normal chondro-osseous junction is essential for an understanding of the pathogenesis of osteoarthrosis. Methods Full thickness samples from human knee joints were processed and embedded in paraffin wax. One hundred serial sections (10 μm thick were taken from the chondro-osseous junctional region of a block from the medial tibial plateau of a normal joint. They were stained with haematoxylin and eosin and photographed. For a simple physical reconstruction images of each 10th sequential tissue section were printed and the areas of the photomicrographs containing the chondro-osseous junctional region were cut out and then overlaid so as to create a three-dimensional (3D model of this region. A 3D reconstruction was also made using computer modelling. Results Histochemical staining revealed some instances where prolongations of uncalcified cartilage, delineated by the tidemark, dipped into the calcified cartilage and, in places, abutted onto subchondral bone and marrow spaces. Small areas of uncalcified cartilage containing chondrocytes (virtual islands were seen, in two-dimensional (2D sections, to be apparently entombed in calcified matrix. The simple physical 3D reconstruction confirmed that these prolongations of uncalcified cartilage were continuous with the cartilage of zone IV and demonstrated that the virtual islands of uncalcified cartilage were cross-sections of these prolongations. The computer-generated 3D reconstructions clearly demonstrated that the uncalcified prolongations ran through the calcified cartilage to touch bone and

  3. In vivo articular cartilage deformation: noninvasive quantification of intratissue strain during joint contact in the human knee

    Science.gov (United States)

    Chan, Deva D.; Cai, Luyao; Butz, Kent D.; Trippel, Stephen B.; Nauman, Eric A.; Neu, Corey P.

    2016-01-01

    The in vivo measurement of articular cartilage deformation is essential to understand how mechanical forces distribute throughout the healthy tissue and change over time in the pathologic joint. Displacements or strain may serve as a functional imaging biomarker for healthy, diseased, and repaired tissues, but unfortunately intratissue cartilage deformation in vivo is largely unknown. Here, we directly quantified for the first time deformation patterns through the thickness of tibiofemoral articular cartilage in healthy human volunteers. Magnetic resonance imaging acquisitions were synchronized with physiologically relevant compressive loading and used to visualize and measure regional displacement and strain of tibiofemoral articular cartilage in a sagittal plane. We found that compression (of 1/2 body weight) applied at the foot produced a sliding, rigid-body displacement at the tibiofemoral cartilage interface, that loading generated subject- and gender-specific and regionally complex patterns of intratissue strains, and that dominant cartilage strains (approaching 12%) were in shear. Maximum principle and shear strain measures in the tibia were correlated with body mass index. Our MRI-based approach may accelerate the development of regenerative therapies for diseased or damaged cartilage, which is currently limited by the lack of reliable in vivo methods for noninvasive assessment of functional changes following treatment.

  4. Three step derivation of cartilage like tissue from human embryonic stem cells by 2D-3D sequential culture in vitro and further implantation in vivo on alginate/PLGA scaffolds.

    Science.gov (United States)

    Bai, Hai Yan; Chen, Gui An; Mao, Gen Hong; Song, Tian Ran; Wang, Yan Xia

    2010-08-01

    In this study a three step culture system, 2D-3D sequential culture in vitro and further implantation in vivo was developed to induce human embryonic stem cells (hESCs) into cartilage like tissues. Five-day-old embryoid bodies were plated for chondrogenic induction for 27 days (step1), then the cells were suspended in alginate and seeded onto polylactic-co-glycolic acid (PLGA) scaffolds for 3D cultivation for 7 days (step 2) and the cells/alginate/PLGA complexes were further transplanted into nude mice for 8 weeks (step 3). At same time, some of complexes were cultured in vitro up to 8 weeks. At the end of step 1, cells exhibited fibroblast-like morphology and expressed chondrocyte-specific markers, Sox 9 and collagen II. During the following 8 weeks of 3D cultivation in vitro, cells displayed spherical morphology, decreased immunoreactivity to Sox-9 and increased one to collagen II, demonstrated further differentiation to mature chondrocyte. In implanted grafts, not only cells appeared typical chondrocytes shape and markers but also cartilage like tissues were formed. These results indicate that 2D-3D sequential culture in vitro is an efficient protocol to induce hESCs differentiates into chondrocytes, while the three step culture system may be an appropriate procedure to derive cartilage like tissues from hESCs. (c) 2010 Wiley Periodicals, Inc.

  5. Study of ionizing radiation effects in human costal cartilage by thermogravimetry and optical coherence tomography; Estudo dos efeitos da radiacao ionizante em cartilagem costal humana por meio de termogravimetria e tomografia por coerencia optica

    Energy Technology Data Exchange (ETDEWEB)

    Martinho Junior, Antonio Carlos

    2012-07-01

    Tissue Banks around the world have stored human cartilages obtained from post mortem donors for use in several kinds of reconstructive surgeries. To ensure that such tissues are not contaminated, they have been sterilized with ionizing radiation. However, high doses of gamma radiation may cause undesirable changes in the tissues. In this work, we evaluated the possibility of use Optical Coherence Tomography (OCT) and Thermogravimetric Analysis (TGA) to identify possible structural modifications caused by both preservation methods of cartilage and gamma irradiation doses. Cartilages were obtained from cadaveric donors and were frozen at -70 deg C or preserved in glycerol. Irradiation was performed by {sup 60}Co source with doses of 15, 25 and 50 kGy. Our TGA results showed that glycerolized cartilages irradiated with different doses of radiation does not presented statistical differences when compared to the control group for the dehydration rate. However, the same was not observed for deep-frozen cartilages irradiated with 15 kGy. The results of OCT associated to total optical attenuation coefficient showed that doses of 15 kGy promote cross-link between collagen fibrils, corroborating the results obtained from TGA. Moreover, total optical attenuation coefficient values are proportional to stress at break of cartilages, what will be very useful in a near future to predict the quality of the allografts, without unnecessary loss of biological tissue, once OCT is a nondestructive technique. By PS-OCT images, we found that high doses of ionizing radiation does not promote sufficient impairments to promote complete loss of tissue birefringence. Thus, TGA and OCT are techniques that can be used for tissue banks to verify tissue quality before its transplant. (author)

  6. Quantification of collagen organization in the peripheral human cornea at micron-scale resolution.

    Science.gov (United States)

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

    2011-07-06

    The collagen microstructure of the peripheral cornea is important in stabilizing corneal curvature and refractive status. However, the manner in which the predominantly orthogonal collagen fibrils of the central cornea integrate with the circumferential limbal collagen is unknown. We used microfocus wide-angle x-ray scattering to quantify the relative proportion and orientation of collagen fibrils over the human corneolimbal interface at intervals of 50 μm. Orthogonal fibrils changed direction 1-1.5 mm before the limbus to integrate with the circumferential limbal fibrils. Outside the central 6 mm, additional preferentially aligned collagen was found to reinforce the cornea and limbus. The manner of integration and degree of reinforcement varied significantly depending on the direction along which the limbus was approached. We also employed small-angle x-ray scattering to measure the average collagen fibril diameter from central cornea to limbus at 0.5 mm intervals. Fibril diameter was constant across the central 6 mm. More peripherally, fibril diameter increased, indicative of a merging of corneal and scleral collagen. The point of increase varied with direction, consistent with a scheme in which the oblique corneal periphery is reinforced by chords of scleral collagen. The results have implications for the cornea's biomechanical response to ocular surgeries involving peripheral incision. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2011-01-01

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

  8. Image registration improves human knee cartilage T1 mapping with delayed gadolinium-enhanced MRI of cartilage (dGEMRIC).

    Science.gov (United States)

    Bron, Esther E; van Tiel, Jasper; Smit, Henk; Poot, Dirk H J; Niessen, Wiro J; Krestin, Gabriel P; Weinans, Harrie; Oei, Edwin H G; Kotek, Gyula; Klein, Stefan

    2013-01-01

    To evaluate the effect of automated registration in delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) of the knee on the occurrence of movement artefacts on the T1 map and the reproducibility of region-of-interest (ROI)-based measurements. Eleven patients with early-stage knee osteoarthritis and ten healthy controls underwent dGEMRIC twice at 3 T. Controls underwent unenhanced imaging. ROIs were manually drawn on the femoral and tibial cartilage. T1 calculation was performed with and without registration of the T1-weighted images. Automated three-dimensional rigid registration was performed on the femur and tibia cartilage separately. Registration quality was evaluated using the square root Cramér-Rao lower bound (CRLB(σ)). Additionally, the reproducibility of dGEMRIC was assessed by comparing automated registration with manual slice-matching. Automated registration of the T1-weighted images improved the T1 maps as the 90% percentile of the CRLB(σ) was significantly (P registration of the re-imaged T1 map gave comparable intraclass correlation coefficients of respectively 0.89/0.90 (patients) and 0.85/0.85 (controls). Registration in dGEMRIC reduces movement artefacts on T1 maps and provides a good alternative to manual slice-matching in longitudinal studies.

  9. Enzyme replacement in a human model of mucopolysaccharidosis IVA in vitro and its biodistribution in the cartilage of wild type mice.

    Directory of Open Access Journals (Sweden)

    Melita Dvorak-Ewell

    Full Text Available Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS, an enzyme that degrades keratan sulfate (KS. Currently no therapy for MPS IVA is available. We produced recombinant human (rhGALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active ( approximately 2 U/mg and pure (>or=97% rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (K(uptake = 2.5 nM, thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for

  10. Immunocytochemical localisation of collagens (I-V) in the human iris.

    Science.gov (United States)

    Konstas, A G; Marshall, G E; Lee, W R

    1990-01-01

    In this study we investigated the distribution of collagen types I-V in the human iris at the fine-structural level using cryoultramicrotomy and London Resin White plastic embedding. Collagen type I was shown to be present in the basement membrane of iris vessels, in contrast to type III, which was absent; both types I and III were present in the iris stroma. Collagen type IV was a major component of basement membranes of vascular cells, myoepithelial cells, fibroblasts and epithelial cells. Types II and V were absent. Both cryo and plastic embedding techniques produced closely comparable results.

  11. Repair of Avascular Meniscus Tears with Electrospun Collagen Scaffolds Seeded with Human Cells

    Science.gov (United States)

    Baek, Jihye; Sovani, Sujata; Glembotski, Nicholas E.; Du, Jiang; Jin, Sungho; Grogan, Shawn P.

    2016-01-01

    The self-healing capacity of an injured meniscus is limited to the vascularized regions and is especially challenging in the inner avascular regions. As such, we investigated the use of human meniscus cell-seeded electrospun (ES) collagen type I scaffolds to produce meniscal tissue and explored whether these cell-seeded scaffolds can be implanted to repair defects created in meniscal avascular tissue explants. Human meniscal cells (derived from vascular and avascular meniscal tissue) were seeded on ES scaffolds and cultured. Constructs were evaluated for cell viability, gene expression, and mechanical properties. To determine potential for repair of meniscal defects, human meniscus avascular cells were seeded and cultured on aligned ES collagen scaffolds for 4 weeks before implantation. Surgical defects resembling “longitudinal tears” were created in the avascular zone of bovine meniscus and implanted with cell-seeded collagen scaffolds and cultured for 3 weeks. Tissue regeneration and integration were evaluated by histology, immunohistochemistry, mechanical testing, and magentic resonance imaging. Ex vivo implantation with cell-seeded collagen scaffolds resulted in neotissue that was significantly better integrated with the native tissue than acellular collagen scaffolds or untreated defects. Human meniscal cell-seeded ES collagen scaffolds may therefore be useful in facilitating meniscal repair of avascular meniscus tears. PMID:26842062

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    Mechanical loading of tissue is known to influence local collagen synthesis, and microdialysis studies indicate that mechanical loading of human tendon during exercise elevates tendinous type I collagen production. Transforming growth factor-beta1 (TGF-beta1), a potent stimulator of type I collagen...

  13. Fibrillar collagen inhibits cholesterol biosynthesis in human aortic smooth muscle cells.

    Science.gov (United States)

    Ferri, Nicola; Roncalli, Elisa; Arnaboldi, Lorenzo; Fenu, Simone; Andrukhova, Olena; Aharinejad, Seyedhossein; Camera, Marina; Tremoli, Elena; Corsini, Alberto

    2009-10-01

    Integrin-mediated cell adhesion to type I fibrillar collagen regulates gene and protein expression, whereas little is known of its effect on lipid metabolism. In the present study, we examined the effect of type I fibrillar collagen on cholesterol biosynthesis in human aortic smooth muscle cells (SMCs). SMCs were cultured on either fibrillar or monomer collagen for 48 hours and [(14)C]-acetate incorporation into cholesterol was evaluated. Fibrillar collagen reduced by 72.9+/-2.6% cholesterol biosynthesis without affecting cellular cholesterol levels. Fibrillar collagen also reduced 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) promoter activity (-72.6+/-7.3%), mRNA (-58.7+/-6.4%), protein levels (-35.5+/-8.5%), and enzyme activity (-37.7+/-2.2%). Intracellular levels of the active form of sterol regulatory element binding proteins (SREBP) 1a was decreased by 60.7+/-21.7% in SMCs cultured on fibrillar collagen, whereas SREBP2 was not significantly affected (+12.1+/-7.1%). The overexpression of the active form of SREBP1a rescued the downregulation of fibrillar collagen on HMG-CoA reductase levels. Blocking antibody to alpha2 integrin partially reversed the downregulation of HMG-CoA reductase mRNA expression. Finally, fibrillar collagen led to an intracellular accumulation of unprenylated Ras. Our study demonstrated that alpha2 beta 1 integrin interaction with fibrillar collagen affected the expression of HMG-CoA reductase, which led to the inhibition of cholesterol biosynthesis in human SMCs.

  14. Influence of caffeine and hyaluronic acid on collagen biosynthesis in human skin fibroblasts

    Directory of Open Access Journals (Sweden)

    Donejko M

    2014-10-01

    Full Text Available Magdalena Donejko,1 Andrzej Przylipiak,1 Edyta Rysiak,2 Katarzyna Głuszuk,2 Arkadiusz Surażyński2 1Department of Esthetic Medicine, 2Department of Medicinal Chemistry, Faculty of Pharmacy, Medical University of Białystok, Białystok, Poland Aim: The aim of this study was to evaluate the effect of caffeine on collagen biosynthesis in human skin fibroblasts and the influence of hyaluronic acid (HA on this process. Materials and methods: Collagen, [3H]-thymidine incorporation, and prolidase activity were measured in confluent human skin fibroblast cultures that had been treated with 1, 2, and 5 mM caffeine and with caffeine and 500 µg/mL HA. Western immunoblot analysis was performed to evaluate expression of ß1-integrin receptor, insulin-like growth factor receptor phospho-Akt protein and mitogen-activated protein kinase (phospho-extracellular signal-regulated kinase. Results: Caffeine inhibited collagen biosynthesis in a dose-dependent manner. The mechanism of this process was found at the level of prolidase activity. Caffeine significantly inhibited the enzyme activity. The addition of HA had no effect on collagen biosynthesis or prolidase activity in fibroblasts incubated with caffeine. Caffeine also had an inhibitory effect on DNA biosynthesis. HA, however, did not have any significant effect on this process. The inhibition of the expression of ß1-integrin and insulin-like growth factor receptor in fibroblasts incubated with the caffeine indicates a possible mechanism of inhibition of collagen biosynthesis. Conclusion: Caffeine reduces collagen synthesis in human cultured skin fibroblasts. HA did not have any significant protective effect on this process. This is the first study to our knowledge that reports caffeine-induced inhibition of collagen synthesis in human skin fibroblasts. Keywords: collagen, caffeine, hyaluronic acid, fibroblast

  15. New insight into the shortening of the collagen fibril D-period in human cornea.

    Science.gov (United States)

    Jastrzebska, Maria; Tarnawska, Dorota; Wrzalik, Roman; Chrobak, Artur; Grelowski, Michal; Wylegala, Edward; Zygadlo, Dorota; Ratuszna, Alicja

    2017-02-01

    Collagen fibrils type I display a typical banding pattern, so-called D-periodicity, of about 67 nm, when visualized by atomic force or electron microscopy imaging. Herein we report on a significant shortening of the D-period for human corneal collagen fibrils type I (21 ± 4 nm) upon air-drying, whereas no changes in the D-period were observed for human scleral collagen fibrils type I (64 ± 4 nm) measured under the same experimental conditions as the cornea. It was also found that for the corneal stroma fixed with glutaraldehyde and air-dried, the collagen fibrils show the commonly accepted D-period of 61 ± 8 nm. We used the atomic force microscopy method to image collagen fibrils type I present in the middle layers of human cornea and sclera. The water content in the cornea and sclera samples was varying in the range of .066-.085. Calculations of the D-period using the theoretical model of the fibril and the FFT approach allowed to reveal the possible molecular mechanism of the D-period shortening in the corneal collagen fibrils upon drying. It was found that both the decrease in the shift and the simultaneous reduction in the distance between tropocollagen molecules can be responsible for the experimentally observed effect. We also hypothesize that collagen type V, which co-assembles with collagen type I into heterotypic fibrils in cornea, could be involved in the observed shortening of the corneal D-period.

  16. The relationship between ultra-short telomeres, aging of articular cartilage and the development of human hip osteoarthritis

    DEFF Research Database (Denmark)

    Harbo, M; Delaisse, J M; Kjaersgaard-Andersen, P

    2013-01-01

    Ultra-short telomeres caused by stress-induced telomere shortening are suggested to induce chondrocyte senescence in human osteoarthritic knees. Here we have further investigated the role of ultra-short telomeres in the development of osteoarthritis (OA) and in aging of articular cartilage in human...

  17. Determination of Collagen Fiber Orientation in Human Tissue by Use of Polarization Measurement of Molecular Second-Harmonic-Generation Light

    Science.gov (United States)

    Yasui, Takeshi; Tohno, Yoshiyuki; Araki, Tsutomu

    2004-05-01

    Based on the reflection-type polarization measurement of second-harmonic-generation (SHG) light induced by collagen molecules, we are able to determine the collagen fiber orientation in human tissues taken from a cadaver. The resulting SHG radar graph shows the direction of the absolute orientation and the degree of organization of collagen fibers. To evaluate the probing sensitivity to the collagen orientation, we compared the proposed method with other polarimetric methods. Use of the proposed method revealed characteristic orientation differences among collagen fibers and demonstrated significant inhomogeneity with respect to the distribution of collagen orientation in human dentin. The proposed method provides a powerful research and diagnostic tool for examining the collagen orientation in human tissues.

  18. Comparable Senescence Induction in Three-dimensional Human Cartilage Model by Exposure to Therapeutic Doses of X-rays or C-ions

    Energy Technology Data Exchange (ETDEWEB)

    Hamdi, Dounia Houria; Chevalier, François [Laboratoire d' Accueil et de Recherche avec les Ions Accélérés (LARIA), Institut de Radiobiologie Cellulaire et Moléculaire (IRCM), Direction de la Recherche Fondamentale - DRF, Commissariat à l' Energie Atomique et aux Energies Alternatives, Caen (France); Groetz, Jean-Emmanuel [UMR6249, Université de Franche-Comté, Besançon (France); Durantel, Florent [UMR6252, Centre de Recherche sur les Ions, les Matériaux et la Photonique (CIMAP), Direction de la Recherche Fondamentale (DRF), Commissariat à l' Energie Atomique et aux Energies Alternatives, Caen (France); Thuret, Jean-Yves; Mann, Carl [FRE3377, Service de Biologie Intégrative et Génétique Moléculaire SBIGeM, Institut de Biologie et de Technologies de Saclay (iBiTec-S), Direction de la Recherche Fondamentale (DRF), Commissariat à l' Energie Atomique et aux Energies Alternatives, Gif-sur-Yvette (France); Institut de Biologie Intégrative de la Cellule I2BC / Université Paris Saclay, Gif-sur-Yvette (France); and others

    2016-05-01

    Purpose: Particle therapy using carbon ions (C-ions) has been successfully used in the treatment of tumors resistant to conventional radiation therapy. However, the potential side effects to healthy cartilage exposed to lower linear energy transfer (LET) ions in the beam track before the tumor have not been evaluated. The aim of the present study was to assess the extent of damage after C-ion irradiation in a 3-dimensional (3D) cartilage model close to human homeostasis. Methods and Materials: Primary human articular chondrocytes from a healthy donor were cultured in a collagen scaffold to construct a physioxic 3D cartilage model. A 2-dimensional (2D) culture was used as a reference. The cells were irradiated with a single dose of a monoenergetic C-ion beam with a LET of approximatively 30 keV/μm. This LET corresponds to the entrance channel of C-ions in the shallow healthy tissues before the spread-out Bragg peak (∼100 keV/μm) during hadron therapy protocols. The same dose of X-rays was used as a reference. Survival, cell death, and senescence assays were performed. Results: As expected, in the 2D culture, C-ions were more efficient than X-rays in reducing cell survival with a relative biological effectiveness of 2.6. This correlated with stronger radiation-induced senescence (two-fold) but not with higher cell death induction. This differential effect was not reflected in the 3D culture. Both ionizing radiation types induced a comparable rate of senescence induction in the 3D model. Conclusions: The greater biological effectiveness of C-ions compared with low LET radiation when evaluated in treatment planning systems might be misevaluated using 2D culture experiments. Radiation-induced senescence is an important factor of potential cartilage attrition. The present data should encourage the scientific community to use relevant models and beams to improve the use of charged particles with better safety for patients.

  19. Quantitative analysis of collagen and collagen subtypes I, III, and V in human pancreatic cancer, tumor-associated chronic pancreatitis, and alcoholic chronic pancreatitis.

    Science.gov (United States)

    Imamura, T; Iguchi, H; Manabe, T; Ohshio, G; Yoshimura, T; Wang, Z H; Suwa, H; Ishigami, S; Imamura, M

    1995-11-01

    The collagen content in human pancreatic cancer tissue, tissue of tumor-associated chronic pancreatitis (TACP), and normal pancreatic tissue was determined in 14 patients with pancreatic cancer by measuring the amount of 4-hydroxyproline. Four patients with alcoholic chronic pancreatitis (AlCP) were also analyzed. The mean collagen content in both pancreatic cancer tissue and TACP tissue was approximately threefold higher than in normal pancreatic tissue. Cyanogen bromide peptides of type I, III, and V collagens from invasive ductal carcinomatous tissue of the pancreas and from TACP tissue of eight patients were analyzed sequentially using high-performance liquid chromatography with ion-exchange and gel-permeation columns. No difference in the proportion of type I, III, and V collagens was detected between pancreatic cancer tissue and TACP tissue. The mean collagen content in AlCP tissue was significantly lower than that in TACP tissue, but no difference in the proportion of type I, III, and V collagens was detected between these two tissues. These results indicate a similar quantity and distribution pattern of fibrillar collagen in human pancreatic cancer and TACP.

  20. Type I collagen synthesis and degradation in peritendinous tissue after exercise determined by microdialysis in humans

    DEFF Research Database (Denmark)

    Langberg, Henning; Skovgaard, D; Petersen, L J

    1999-01-01

    1. Physical activity is known to increase type I collagen synthesis measured as the concentration of biomarkers in plasma. By the use of microdialysis catheters with a very high molecular mass cut-off value (3000 kDa) we aimed to determine local type I collagen synthesis and degradation...... catheters were placed in the peritendinous space ventral to the Achilles' tendon under ultrasound guidance and perfused with a Ringer-acetate solution containing 3H-labelled human type IV collagen and [15-3H(N)]PGE2 for in vivo recovery determination. Relative recovery was 37-59 % (range of the s...... increased in blood during running, and returned to baseline in the recovery period, whereas interstitial PGE2 concentration was elevated in the early recovery phase. 4. The findings of the present study indicate that acute exercise induces increased formation of type I collagen in peritendinous tissue...

  1. Tensile properties of human collagen fibrils and fascicles are insensitive to environmental salts

    DEFF Research Database (Denmark)

    Svensson, René B; Hassenkam, Tue; Grant, Colin A

    2010-01-01

    loading direction of tendon is along its longitudinal axis. Thus, in this study, we focus on the tensile mechanical properties of two hierarchical levels from human patellar tendon, namely: individual collagen fibrils and fascicles. Investigations on collagen fibrils and fascicles were made at pH 7.......4 in solutions of phosphate-buffered saline at three different concentrations as well as two HEPES buffered solutions containing NaCl or NaCl + CaCl2. An atomic force microscope technique was used for tensile testing of individual collagen fibrils. Only a slight increase in relative energy dissipation...... was observed at the highest phosphate-buffered saline concentration for both the fibrils and fascicles, indicating a stabilizing effect of ionic screening, but changes were much less than reported for radial compression. Due to the small magnitude of the effects, the tensile mechanical properties of collagen...

  2. Trypsin-mediated enzymatic degradation of type II collagen in the human vitreous

    Science.gov (United States)

    van Deemter, Mariëlle; Kuijer, Roel; Harm Pas, Hendri; Jacoba van der Worp, Roelofje; Hooymans, Johanna Martina Maria

    2013-01-01

    Purpose Aging of the vitreous body can result in sight-threatening pathology. One aspect of vitreous aging is liquefaction, which results from the vanishing of collagen fibrils. We investigated the possibility that trypsins are involved in vitreous type II collagen degradation. Methods Immunohistochemistry and western blotting were used for detecting and locating trypsin isoforms in the vitreous and retina of human donor eyes. The capability of the retina to produce these trypsins was analyzed with polymerase chain reaction. Whether the different trypsins degraded type II collagen was tested in vitro. The sizes of the in vitro induced type II collagen degradation products were compared to those present in the vitreous of human eyes of different ages. Results Trypsin-1 and trypsin-2 were detected in the vitreous. In the retina, messenger ribonucleic acid (mRNA) coding for trypsin-2, -3, and -4 was present. Using immunohistochemistry, trypsin-2 was detected in microglial cells located in the vitreous and the retina. All trypsin isoforms degraded type II collagen and produced degradation products of similar sizes as those present in the vitreous. Conclusions Trypsin-1 and trypsin-2 appear to have a function in the degradation of vitreous type II collagen. They could therefore have a role in the development of vitreous liquefaction. PMID:23882137

  3. The collagenic structure of human digital skin seen by scanning electron microscopy after Ohtani maceration technique.

    Science.gov (United States)

    Sangiorgi, Simone; Manelli, Alessandro; Protasoni, Marina; Ronga, Mario; Raspanti, Mario

    2005-03-01

    We performed a morphological scanning electron microscope (SEM) study to describe the fine structure and disposition of collagenous tissue in the human toe. After therapeutic amputation of a human right leg, we applied the Othani maceration technique to the skin of three toes surgically explanted from the foot. We distinguished eight cutaneous regions and focused on some specialized collagenous structures differing in the thickness of the skin. The eight areas investigated were: the dorsal skin, the eponychium, the perionychium, the hyponychium, the region under the visible nail, the nail root, the plantar skin and finally the toe tip. Each of these areas is characterized by a distinctive collagenous surface disposition, with some peculiar features mostly related to dermal papillae. At high magnification, we observed the spatial arrangement of the collagen fibers constituting the top of the dermal papillae that represents the attachment site of the proliferative basal layer of the epidermis. We also noted an impressive density of collagen fibers throughout the thickness of the dermal layer, organized in specialized structures and constituting the skeleton of dermal thermoreceptorial corpuscles or sweat glands. A combination of SEM and Ohtani technique disclosed the three-dimensional architecture of the collagenous matrix of tarsal skin under physiologic conditions, giving a detailed description of the most reactive tissue during pathologic processes.

  4. Mechanical Stimulation Protocols of Human Derived Cells in Articular Cartilage Tissue Engineering - A Systematic Review.

    Science.gov (United States)

    Khozoee, Baktash; Mafi, Pouya; Mafi, Reza; Khan, Wasim S

    2017-01-01

    Mechanical stimulation is a key factor in articular cartilage generation and maintenance. Bioreactor systems have been designed and built in order to deliver specific types of mechanical stimulation. The focus has been twofold, applying a type of preconditioning in order to stimulate cell differentiation, and to simulate in vivo conditions in order to gain further insight into how cells respond to different stimulatory patterns. Due to the complex forces at work within joints, it is difficult to simulate mechanical conditions using a bioreactor. The aim of this review is to gain a deeper understanding of the complexities of mechanical stimulation protocols by comparing those employed in bioreactors in the context of tissue engineering for articular cartilage, and to consider their effects on cultured cells. Allied and Complementary Medicine 1985 to 2016, Ovid MEDLINE[R] 1946 to 2016, and Embase 1974 to 2016 were searched using key terms. Results were subject to inclusion and exclusion criteria, key findings summarised into a table and subsequently discussed. Based on this review it is overwhelmingly clear that mechanical stimulation leads to increased chondrogenic properties in the context of bioreactor articular cartilage tissue engineering using human cells. However, given the variability and lack of controlled factors between research articles, results are difficult to compare, and a standardised method of evaluating stimulation protocols proved challenging. With improved standardisation in mechanical stimulation protocol reporting, bioreactor design and building processes, along with a better understanding of joint behaviours, we hope to perform a meta-analysis on stimulation protocols and methods. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Effect of indomethacin and lactoferrin on human tenocyte proliferation and collagen formation in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yaonan [Centre for Nanohealth, College of Medicine, Swansea University, Singleton Park, Swansea, UK SA2 8PP (United Kingdom); Department of Orthopaedic, Beijing Hospital of Ministry of Public Health, Beijing, China 100730 (China); Wang, Xiao; Qiu, Yiwei [Centre for Nanohealth, College of Medicine, Swansea University, Singleton Park, Swansea, UK SA2 8PP (United Kingdom); Cornish, Jillian [Department of Medicine, University of Auckland, Private Bag 92019, Auckland (New Zealand); Carr, Andrew J. [Centre for Nanohealth, College of Medicine, Swansea University, Singleton Park, Swansea, UK SA2 8PP (United Kingdom); Xia, Zhidao, E-mail: z.xia@swansea.ac.uk [Centre for Nanohealth, College of Medicine, Swansea University, Singleton Park, Swansea, UK SA2 8PP (United Kingdom)

    2014-11-14

    Highlights: • Indomethacin, a classic NSAID, inhibited human tenocyte proliferation at high concentration (100 µM). • Lactoferrin at 50-100 µg/ml promoted human tenocyte survival, proliferation and collagen synthesis. • Lactoferrin is anabolic to human tenocytes in vitro and reverses potential inhibitory effects of NSAIDs on human tenocytes. - Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in patients with injuries and inflammation of tendon and ligament, and as post-surgical analgesics. The aim of this study is to investigate the effect of indomethacin, a classic NSAID and its combinational effect with an anabolic agent of skeletal tissue, lactoferrin, on the proliferation and collagen formation of human tenocytes in vitro. A factorial experimental design was employed to study the dose-dependent effect of the combination of indomethacin and lactoferrin. The results showed that indomethacin at high concentration (100 μM) inhibited human tenocyte proliferation in culture medium with 1–10% fetal bovine serum (FBS) in vitro. Also, high dose of indomethacin inhibited the collagen formation of human tenocytes in 1% FBS culture medium. Lactoferrin at 50–100 μg/ml promoted human tenocyte survival in serum-free culture medium and enhanced proliferation and collagen synthesis of human tenocytes in 1% FBS culture medium. When 50–100 μg/ml lactoferrin was used in combination with 100–200 μM indomethacin, it partially rescued the inhibitory effects of indomethacin on human tenocyte proliferation, viability and collagen formation. To our knowledge, it is the first evidence that lactoferrin is anabolic to human tenocytes in vitro and reverses potential inhibitory effects of NSAIDs on human tenocytes.

  6. Asiaticoside induces cell proliferation and collagen synthesis in human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Linda Yulianti

    2015-08-01

    Full Text Available Asiatiocoside, a saponin component isolated from Centella asiatica can improve wound healing by promoting the proliferation of human dermal fibroblasts (HDF and synthesis of collagen. The skin-renewing cells and type I and III collagen synthesis decrease with aging, resulting in the reduction of skin elasticity and delayed wound healing. Usage of natural active compounds from plants in wound healing should be evaluated and compared to retinoic acid as an active agent that regulates wound healing. The aim of this study was to compare and evaluate the effect of asiaticoside and retinoic acid to induce greater cell proliferation and type I and III collagen synthesis in human dermal fibroblast. Methods Laboratory experiments were conducted using human dermal fibroblasts (HDF isolated from human foreskin explants. Seven passages of HDF were treated with asiaticoside and retinoic acid at several doses and incubated for 24 and 48 hours. Cell viability in all groups was tested with the MTT assay to assess HDF proliferation. Type I and III collagen synthesis was examined using the respective ELISA kits. Analysis of variance was performed to compare the treatment groups. Results Asiaticoside had significantly stronger effects on HDF proliferation than retinoic acid (p<0.05. The type III collagen production was significantly greater induction with asiaticoside compared to retinoic acid (p<0.05. Conclusion Asiaticoside induces HDF proliferation and type I and III collagen synthesis in a time- and dose-dependent pattern. Asiaticoside has a similar effect as retinoic acid on type I and type III collagen synthesis.

  7. A novel bioreactor system for biaxial mechanical loading enhances the properties of tissue-engineered human cartilage.

    Science.gov (United States)

    Meinert, Christoph; Schrobback, Karsten; Hutmacher, Dietmar W; Klein, Travis J

    2017-12-05

    The ex vivo engineering of autologous cartilage tissues has the potential to revolutionize the clinical management of joint disorders. Yet, high manufacturing costs and variable outcomes associated with tissue-engineered implants are still limiting their application. To improve clinical outcomes and facilitate a wider use of engineered tissues, automated bioreactor systems capable of enhancing and monitoring neotissues are required. Here, we developed an innovative system capable of applying precise uni- or biaxial mechanical stimulation to developing cartilage neotissues in a tightly controlled and automated fashion. The bioreactor allows for simple control over the loading parameters with a user-friendly graphical interface and is equipped with a load cell for monitoring tissue maturation. Applying our bioreactor, we demonstrate that human articular chondrocytes encapsulated in hydrogels composed of gelatin methacryloyl (GelMA) and hyaluronic acid methacrylate (HAMA) respond to uni- and biaxial mechanical stimulation by upregulation of hyaline cartilage-specific marker genes. We further demonstrate that intermittent biaxial mechanostimulation enhances accumulation of hyaline cartilage-specific extracellular matrix. Our study underlines the stimulatory effects of mechanical loading on the biosynthetic activity of human chondrocytes in engineered constructs and the need for easy-to-use, automated bioreactor systems in cartilage tissue engineering.

  8. In vitro phagocytosis of collagens by immortalised human retinal Muller cells

    NARCIS (Netherlands)

    Ponsioen, Theodorus Leonardus; van Luyn, Marja Johanna Adriana; van der Worp, Roelofje Jacoba; Nolte, Ilja Maria; Hooymans, Johanna Martina Maria; Los, Leonoor Inge

    Purpose: This study is a first step to investigate phagocytosis of collagens by human retinal Muller cells, since Muller cells could be involved in remodelling of the vitreous and vitreoretinal interface in the human eye. Methods: Muller cells in culture were exposed to 2.0 mu m fluorescent latex

  9. Effect of indomethacin and lactoferrin on human tenocyte proliferation and collagen formation in vitro.

    Science.gov (United States)

    Zhang, Yaonan; Wang, Xiao; Qiu, Yiwei; Cornish, Jillian; Carr, Andrew J; Xia, Zhidao

    2014-11-14

    Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in patients with injuries and inflammation of tendon and ligament, and as post-surgical analgesics. The aim of this study is to investigate the effect of indomethacin, a classic NSAID and its combinational effect with an anabolic agent of skeletal tissue, lactoferrin, on the proliferation and collagen formation of human tenocytes in vitro. A factorial experimental design was employed to study the dose-dependent effect of the combination of indomethacin and lactoferrin. The results showed that indomethacin at high concentration (100 μM) inhibited human tenocyte proliferation in culture medium with 1-10% fetal bovine serum (FBS) in vitro. Also, high dose of indomethacin inhibited the collagen formation of human tenocytes in 1% FBS culture medium. Lactoferrin at 50-100 μg/ml promoted human tenocyte survival in serum-free culture medium and enhanced proliferation and collagen synthesis of human tenocytes in 1% FBS culture medium. When 50-100 μg/ml lactoferrin was used in combination with 100-200 μM indomethacin, it partially rescued the inhibitory effects of indomethacin on human tenocyte proliferation, viability and collagen formation. To our knowledge, it is the first evidence that lactoferrin is anabolic to human tenocytes in vitro and reverses potential inhibitory effects of NSAIDs on human tenocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Chondrogenic differentiation of mesenchymal stem cells in a leakproof collagen sponge

    Energy Technology Data Exchange (ETDEWEB)

    Chen Guoping [Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan)], E-mail: Guoping.CHEN@nims.go.jp; Akahane, Daisuke [Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan); Division of Bioengineering and Bioinformatics, Graduate School of Information Science and Technology, Hokkaido University (Japan); Kawazoe, Naoki [Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan); Yamamoto, Katsuyuki [Division of Bioengineering and Bioinformatics, Graduate School of Information Science and Technology, Hokkaido University (Japan); Tateishi, Tetsuya [Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan); Division of Bioengineering and Bioinformatics, Graduate School of Information Science and Technology, Hokkaido University (Japan)

    2008-01-10

    A three-dimensional culture of mesenchymal stem cells (MSCs) in a porous scaffold has been developed as a promising strategy for cartilage tissue engineering. The chondrogenic differentiation of MSCs derived from human bone marrow was studied by culturing the cells in a novel scaffold constructed of leakproof collagen sponge. All the surfaces of the collagen sponge except the top were wrapped with a membrane that has pores smaller than the cells to protect against cell leakage during cell seeding. The cells adhered to the collagen, distributed evenly, and proliferated to fill the spaces in the sponge. Cell seeding efficiency was greater than 95%. The MSCs cultured in the collagen sponge in the presence of TGF-{beta}3 and BMP6 expressed a high level of genes encoding type II and type X collagen, sox9, and aggrecan. Histological examination by HE staining indicated that the differentiated cells showed a round morphology. The extracellular matrices were positively stained by safranin O and toluidine blue. Immunostaining with anti-type II collagen and anti-cartilage proteoglycan showed that type II collagen and cartilage proteoglycan were detected around the cells. These results suggest the chondrogenic differentiation of MSCs when cultured in the collagen sponge in the presence of TGF-{beta}3 and BMP6.

  11. Platelet rich plasma (PRP) induces chondroprotection via increasing autophagy, anti-inflammatory markers, and decreasing apoptosis in human osteoarthritic cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Moussa, Mayssam, E-mail: Moussa-mayssam@hotmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Lajeunesse, Daniel, E-mail: daniel.lajeunesse@umontreal.ca [Research Centre in Osteoarthritis, Research Centre in Monteral University (Canada); Hilal, George, E-mail: George2266@gmail.com [Cancer and metabolism Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); El Atat, Oula, E-mail: oulaatat@hotmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Haykal, Gaby, E-mail: Gaby.haykal@hdf.usj.edu.lb [Hotel Dieu de France, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Serhal, Rim, E-mail: rim.basbous@gmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Chalhoub, Antonio, E-mail: Mava.o@hotmail.com [Carantina Hospital, Beirut (Lebanon); Khalil, Charbel, E-mail: charbelk3@hotmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon); Alaaeddine, Nada, E-mail: Nada.aladdin@gmail.com [Regenerative medicine and inflammation Laboratory, Faculty of Medicine, Saint-Joseph University, Beirut (Lebanon)

    2017-03-01

    Objectives: Autophagy constitutes a defense mechanism to overcome aging and apoptosis in osteoarthritic cartilage. Several cytokines and transcription factors are linked to autophagy and play an important role in the degradative cascade in osteoarthritis (OA). Cell therapy such as platelet rich plasma (PRP) has recently emerged as a promising therapeutic tool for many diseases including OA. However, its mechanism of action on improving cartilage repair remains to be determined. The purpose of this study is to investigate the effect of PRP on osteoarthritic chondrocytes and to elucidate the mechanism by which PRP contributes to cartilage regeneration. Methods: Osteoarthritic chondrocytes were co-cultured with an increasing concentration of PRP obtained from healthy donors. The effect of PRP on the proliferation of chondrocytes was performed using cell counting and WST8 proliferation assays. Autophagy, apoptosis and intracellular level of IL-4, IL-10, and IL-13 were determined using flow cytometry analyses. Autophagy markers BECLIN and LC3II were also determined using quantitative polymerase chain reaction (qPCR). qPCR and ELISA were used to measure the expression of ADAMDTS-5, MMP3, MMP13, TIMP-1–2–3, aggregan, Collagen type 2, TGF-β, Cox-2, Il-6, FOXO1, FOXO3, and HIF-1 in tissues and co-cultured media. Results: PRP increased significantly the proliferation of chondrocytes, decreased apoptosis and increased autophagy and its markers along with its regulators FOXO1, FOXO3 and HIF-1 in osteoarthritic chondrocytes. Furthermore, PRP caused a dose-dependent significant decrease in MMP3, MMP13, and ADAMTS-5, IL-6 and COX-2 while increasing TGF-β, aggregan, and collagen type 2, TIMPs and intracellular IL-4, IL-10, IL-13. Conclusion: These results suggest that PRP could be a potential therapeutic tool for the treatment of OA. - Highlights: • Platelet Rich Plasma is suggested as a new treatment for osteoarthritis. • The proposed therapeutic effect is

  12. Reference genes for normalization of gene expression studies in human osteoarthritic articular cartilage

    Directory of Open Access Journals (Sweden)

    Gomez-Reino Juan J

    2008-01-01

    Full Text Available Abstract Background Assessment of gene expression is an important component of osteoarthritis (OA research, greatly improved by the development of quantitative real-time PCR (qPCR. This technique requires normalization for precise results, yet no suitable reference genes have been identified in human articular cartilage. We have examined ten well-known reference genes to determine the most adequate for this application. Results Analyses of expression stability in cartilage from 10 patients with hip OA, 8 patients with knee OA and 10 controls without OA were done with classical statistical tests and the software programs geNorm and NormFinder. Results from the three methods of analysis were broadly concordant. Some of the commonly used reference genes, GAPDH, ACTB and 18S RNA, performed poorly in our analysis. In contrast, the rarely used TBP, RPL13A and B2M genes were the best. It was necessary to use together several of these three genes to obtain the best results. The specific combination depended, to some extent, on the type of samples being compared. Conclusion Our results provide a satisfactory set of previously unused reference genes for qPCR in hip and knee OA This confirms the need to evaluate the suitability of reference genes in every tissue and experimental situation before starting the quantitative assessment of gene expression by qPCR.

  13. Nanoscale helium ion microscopic analysis of collagen fibrillar changes following femtosecond laser dissection of human cornea.

    Science.gov (United States)

    Riau, Andri K; Poh, Rebekah; Pickard, Daniel S; Park, Chris H J; Chaurasia, Shyam S; Mehta, Jodhbir S

    2014-08-01

    Over the last decade, femtosecond lasers have emerged as an important tool to perform accurate and fine dissections with minimal collateral damage in biological tissue. The most common surgical procedure in medicine utilizing femtosecond laser is LASIK. During the femtosecond laser dissection process, the corneal collagen fibers inevitably undergo biomechanical and thermal changes on a sub-micro- or even a nanoscale level, which can potentially lead to post-surgical complications. In this study, we utilized helium ion microscopy, complemented with transmission electron microscopy to examine the femtosecond laser-induced collagen fibrillar damage in ex vivo human corneas. We found that the biomechanical damage induced by laser etching, generation of tissue bridges, and expansion of cavitation bubble and its subsequent collapse, created distortion to the surrounding collagen lamellae. Femtosecond laser-induced thermal damage was characterized by collapsed collagen lamellae, loss of collagen banding, collagen coiling, and presence of spherical debris. Our findings have shown the ability of helium ion microscopy to provide high resolution images with unprecedented detail of nanoscale fibrillar morphological changes in order to assess a tissue damage, which could not be resolved by conventional scanning electron microscopy previously. This imaging technology has also given us a better understanding of the tissue-laser interactions in a nano-structural manner and their possible effects on post-operative wound recovery.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2015-10-01

    The structural and optical properties of clinically biocompatible, cell-free hydrogels comprised of synthetically cross-linked and moulded recombinant human collagen type III (RHCIII) with and without the incorporation of 2-methacryloyloxyethyl phosphorylcholine (MPC) were assessed using transmission electron microscopy (TEM), X-ray scattering, spectroscopy and refractometry. These findings were examined alongside similarly obtained data from 21 human donor corneas. TEM demonstrated the presence of loosely bundled aggregates of fine collagen filaments within both RHCIII and RHCIII-MPC implants, which X-ray scattering showed to lack D-banding and be preferentially aligned in a uniaxial orientation throughout. This arrangement differs from the predominantly biaxial alignment of collagen fibrils that exists in the human cornea. By virtue of their high water content (90%), very fine collagen filaments (2-9 nm) and lack of cells, the collagen hydrogels were found to transmit almost all incident light in the visible spectrum. They also transmitted a large proportion of UV light compared to the cornea which acts as an effective UV filter. Patients implanted with these hydrogels should be cautious about UV exposure prior to regrowth of the epithelium and in-growth of corneal cells into the implants. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  17. Collagen cross-linking in sun-exposed and unexposed sites of aged human skin

    Science.gov (United States)

    Yamauchi, M.; Prisayanh, P.; Haque, Z.; Woodley, D. T.

    1991-01-01

    A recently described nonreducible, acid-heat stable compound, histidinohydroxylysinonorleucine (HHL), is a collagen cross-link isolated from mature skin tissue. Its abundance is related to chronologic aging of skin. The present communication describes the quantity of HHL from aged human skin of the same individuals in sun-exposed (wrist) and unexposed (buttock) sites. Punch biopsies were obtained from these sites from nine people of age 60 or older. HHL contents (moles/mole of collagen) at these sites were for wrist 0.13 +/- 0.07 and for buttock 0.69 +/- 0.17 (mean +/- SD, p less than 0.001). In addition, it was found that acute irradiation of the cross-linked peptides with UVA (up to 250 J/cm2) and UVB (up to 1 J/cm2) had no effect on HHL structure. The same treatment significantly degraded another nonreducible, stable collagen cross-link, pyridinoline. The results suggest that chronic sunlight exposure may be associated with an impediment to normal maturation of human dermal collagen resulting in tenuous amount of HHL. Thus, the process of photoaging in dermal collagen is different from that of chronologic aging in human skin.

  18. Innervation of tissue-engineered recombinant human collagen-based corneal substitutes : A comparative in vivo confocal microscopy study

    OpenAIRE

    Lagali, Neil; Griffith, May; Fagerholm, Per; Merrett, Kimberley; Huynh, Melissa; Munger, Rejean

    2008-01-01

    PURPOSE. To compare reinnervation in recombinant human collagen-based corneal substitutes with allografts during a 1-year postimplantation follow-up period in pigs. A retrospective comparison to innervation in porcine collagen-based biosynthetic grafts was also performed. METHODS. Pigs received a corneal allograft or a substitute made of either recombinant human type-I or -III collagen. In vivo confocal microscopic examination of the central cornea of surgical and untouched control eyes befor...

  19. The effect of protease inhibitors on the induction of osteoarthritis-related biomarkers in bovine full-depth cartilage explants

    DEFF Research Database (Denmark)

    He, Yi; Zheng, Qinlong; Jiang, Mengmeng

    2015-01-01

    Objective The specific degradation of type II collagen and aggrecan by matrix metalloproteinase (MMP)-9, -13 and ADAMTS-4 and -5 (aggrecanase-1 and -2) in the cartilage matrix is a critical step in pathology of osteoarthritis (OA). The aims of this study were: i) To investigate the relative...... contribution of ADAMTS-4 and ADAMTS-5 to cartilage degradation upon catabolic stimulation; ii) To investigate the effect of regulating the activities of key enzymes by mean of broad-spectrum inhibitors. Methods Bovine full-depth cartilage explants stimulated with tumor necrosis factor alpha (TNF...... protease for the generation of 374ARGS aggrecan fragment in the TNF-α/OSM stimulated bovine cartilage explants. This study addresses the need to determine the roles of ADAMTS-4 and ADAMTS-5 in human articular degradation in OA and hence identify the attractive target for slowing down human cartilage...

  20. SOX trio decrease in the articular cartilage with the advancement of osteoarthritis.

    Science.gov (United States)

    Lee, Jai-Sun; Im, Gun-Il

    2011-01-01

    SOX trio (SOX-5, SOX-6, and SOX-9) maintain the chondrocytic phenotypes and are vital for chondrogenesis in embryonic development. The purpose of this study is to investigate the change in the expression of SOX trio with the advancement of osteoarthritis (OA) in human articular cartilage (AC). Human OA samples from eight patients were obtained from the distal femoral condyles during total knee arthroplasty. Minimally OA cartilage taken from areas with no obvious surface defects on lateral condyles was compared with advanced OA cartilage obtained from areas within 1 cm of overt lesion located on medial condyle surface. SOX-5, SOX-6, and SOX-9 gene expressions significantly decreased by 41% (p = 0.047), 46% (p = 0.047), and 56% (p = 0.029) in advanced OA area compared with the minimally OA area. There was a significant decrease in aggrecan and type II collagen (COL2A1) gene expressions by 73% (p = 0.029) and 65% (p = 0.029), respectively, in advanced OA area compared with the minimally OA area. From Western blotting and immunohistochemistry, SOX-5, SOX-6, SOX-9, type II collagen, and aggrecan protein expressions also significantly decreased in advanced OA cartilage compared with minimally OA cartilage. DNA methylation study of SOX-9 promoter regions revealed no difference in the epigenetic status between the two areas. It is concluded that SOX trio gene and protein decreased with advancement of OA in human articular cartilage.

  1. The effect of acute exercise on collagen turnover in human tendons

    DEFF Research Database (Denmark)

    Mørch, Lina Steinrud; Pingel, Jessica; Boesen, Mikael

    2013-01-01

    Mechanical loading of human tendon stimulates collagen synthesis, but the relationship between acute loading responses and training status of the tendon is not clear. We tested the effect of prolonged load deprivation on the acute loading-induced collagen turnover in human tendons, by applying...... the same absolute load to a relative untrained Achilles tendon (2-week immobilization period prior to acute loading) and a habitually loaded contra-lateral Achilles tendon, respectively, within the same individuals. Eight untrained, healthy males had one lower limb totally immobilized for 2 weeks, whereas...... the contra-lateral leg was used habitually. Following the procedure both Achilles tendons and calf muscles were loaded with the same absolute load during a 1-h treadmill run. Tissue collagen turnover was measured by microdialysis performed post-immobilization but pre-exercise around both Achilles tendons...

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

    DEFF Research Database (Denmark)

    Nielsen, Rie H; Doessing, Simon; Goto, K.

    2011-01-01

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

  3. Suppression of human cartilage proteoglycan synthesis by rheumatoid synovial fluid mononuclear cells activated with mycobacterial 60-kd heat-shock protein

    NARCIS (Netherlands)

    Wilbrink, B.; Holewijn, M.; Bijlsma, J. W.; van Roy, J. L.; den Otter, W.; van Eden, W.

    1993-01-01

    To examine whether T cell reactivity toward heat-shock proteins (HSP) contributes to cartilage destruction in rheumatoid arthritis (RA). An in vitro system was used, in which human cartilage explants were cocultured with hsp60-activated synovial fluid mononuclear cells (SFMC) from patients with RA,

  4. Collagen crosslinks in chondromalacia of the patella.

    Science.gov (United States)

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

    1998-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Miyatake, Kazumasa [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); Tsuji, Kunikazu, E-mail: ktsuji.gcoe@tmd.ac.jp [International Research Center for Molecular Science in Tooth and Bone Diseases (Global Center of Excellence Program), Tokyo Medical and Dental University, Tokyo (Japan); Yamaga, Mika; Yamada, Jun; Matsukura, Yu; Abula, Kahaer [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); Sekiya, Ichiro [Section of Cartilage Regeneration, Tokyo Medical and Dental University, Tokyo (Japan); Muneta, Takeshi [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); International Research Center for Molecular Science in Tooth and Bone Diseases (Global Center of Excellence Program), Tokyo Medical and Dental University, Tokyo (Japan)

    2013-02-01

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

  6. Metabolic activity and collagen turnover in human tendon in response to physical activity

    DEFF Research Database (Denmark)

    Kjaer, M; Langberg, H; Miller, B F

    2005-01-01

    Connective tissue of the human tendon plays an important role in force transmission. The extracellular matrix turnover of tendon is influenced by physical activity. Blood flow, oxygen demand, and the level of collagen synthesis and matrix metalloproteinases increase with mechanical loading. Gene ...

  7. Osteogenic differentiation of human periosteal-derived cells in a three-dimensional collagen scaffold.

    Science.gov (United States)

    Ryu, Young-Mo; Hah, Young-Sool; Park, Bong-Wook; Kim, Deok Ryong; Roh, Gu Seob; Kim, Jong-Ryoul; Kim, Uk-Kyu; Rho, Gyu-Jin; Maeng, Geun-Ho; Byun, June-Ho

    2011-06-01

    This study examined the osteogenic differentiation of cultured human periosteal-derived cells grown in a three dimensional collagen-based scaffold. Periosteal explants with the appropriate dimensions were harvested from the mandible during surgical extraction of lower impacted third molar. Periosteal-derived cells were introduced into cell culture. After passage 3, the cells were divided into two groups and cultured for 28 days. In one group, the cells were cultured in two-dimensional culture dishes with osteogenic inductive medium containing dexamethasone, ascorbic acid, and β-glycerophosphate. In the other group, the cells were seeded onto a three-dimensional collagen scaffold and cultured under the same conditions. We examined the bioactivity of alkaline phosphatase (ALP), the RT-PCR analysis for ALP and osteocalcin, and measurements of the calcium content in the periosteal-derived cells of two groups. Periosteal-derived cells were successfully differentiated into osteoblasts in the collagen-based scaffold. The ALP activity in the periosteal-derived cells was appreciably higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The levels of ALP and osteocalcin mRNA in the periosteal-derived cells was also higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The calcium level in the periosteal-derived cells seeded onto three-dimensional collagen scaffolds showed a 5.92-fold increase on day 7, 3.28-fold increase on day 14, 4.15-fold increase on day 21, and 2.91-fold increase on day 28, respectively, compared with that observed in two-dimensional culture dishes. These results suggest that periosteal-derived cells have good osteogenic capacity in a three-dimensional collagen scaffold, which provides a suitable environment for the osteoblastic differentiation of these cells.

  8. Influence of caffeine and hyaluronic acid on collagen biosynthesis in human skin fibroblasts

    OpenAIRE

    Donejko M; Przylipiak A; Rysiak E; Głuszuk K; Surażyński A

    2014-01-01

    Magdalena Donejko,1 Andrzej Przylipiak,1 Edyta Rysiak,2 Katarzyna Głuszuk,2 Arkadiusz Surażyński2 1Department of Esthetic Medicine, 2Department of Medicinal Chemistry, Faculty of Pharmacy, Medical University of Białystok, Białystok, Poland Aim: The aim of this study was to evaluate the effect of caffeine on collagen biosynthesis in human skin fibroblasts and the influence of hyaluronic acid (HA) on this process. Materials and methods: Collagen, [3H]-thymidine incorporation, an...

  9. Engineered collagen hydrogels for the sustained release of biomolecules and imaging agents: promoting the growth of human gingival cells.

    Science.gov (United States)

    Choi, Jonghoon; Park, Hoyoung; Kim, Taeho; Jeong, Yoon; Oh, Myoung Hwan; Hyeon, Taeghwan; Gilad, Assaf A; Lee, Kwan Hyi

    2014-01-01

    We present here the in vitro release profiles of either fluorescently labeled biomolecules or computed tomography contrast nanoagents from engineered collagen hydrogels under physiological conditions. The collagen constructs were designed as potential biocompatible inserts into wounded human gingiva. The collagen hydrogels were fabricated under a variety of conditions in order to optimize the release profile of biomolecules and nanoparticles for the desired duration and amount. The collagen constructs containing biomolecules/nanoconstructs were incubated under physiological conditions (ie, 37°C and 5% CO2) for 24 hours, and the release profile was tuned from 20% to 70% of initially loaded materials by varying the gelation conditions of the collagen constructs. The amounts of released biomolecules and nanoparticles were quantified respectively by measuring the intensity of fluorescence and X-ray scattering. The collagen hydrogel we fabricated may serve as an efficient platform for the controlled release of biomolecules and imaging agents in human gingiva to facilitate the regeneration of oral tissues.

  10. Mechanical properties of cross-linked collagen meshes after human adipose derived stromal cells seeding.

    Science.gov (United States)

    Ochoa, Ignacio; Peña, Estefania; Andreu, Enrique J; Pérez-Ilzarbe, Maitane; Robles, Jose E; Alcaine, Clara; López, Tania; Prósper, Felipe; Doblaré, Manuel

    2011-02-01

    The main goal of this study was to evaluate the potential of collagen meshes derived from porcine dermis as scaffolds for repairing pelvic organ prolapses. Mechanical properties of collagen meshes with different cross-linking percentages before and after Adipose Derived Stromal Cells (ADSC) seeding were studied as well as the cell-scaffold interaction. Uniaxial tensile tests of the collagen meshes with three different cross-linking percentages (full-, partial-, and noncross-linked) were carried out along orthogonal directions. Their mechanical properties were studied with the same tests before and after seeding with human derived adipose stem cells (ADSC) after 1 and 7 days. Histological analyses were performed to determine adhesion and proliferation of ADSC. Significant differences in mechanical properties of the unseeded meshes were observed between each orthogonal direction independently of the cross-linking percentage. A better cell adhesion rate was observed in the cross-linked meshes. An increase in the mechanical properties after cell seeding was observed with a direct relation with the degree of cross-linking. All meshes analyzed showed a marked anisotropy that should be taken into account during the surgical procedure. The cross-linking treatment increased cell adhesion and the mechanical properties of the collagen meshes after seeding. These results suggest that the mechanical properties of this type of collagen mesh could be useful as scaffolds for repair of pelvic organ prolapse. 2010 Wiley Periodicals, Inc.

  11. Chondrogenic Differentiation of Defined Equine Mesenchymal Stem Cells Derived from Umbilical Cord Blood for Use in Cartilage Repair Therapy

    Directory of Open Access Journals (Sweden)

    Mélanie Desancé

    2018-02-01

    Full Text Available Cartilage engineering is a new strategy for the treatment of cartilage damage due to osteoarthritis or trauma in humans. Racehorses are exposed to the same type of cartilage damage and the anatomical, cellular, and biochemical properties of their cartilage are comparable to those of human cartilage, making the horse an excellent model for the development of cartilage engineering. Human mesenchymal stem cells (MSCs differentiated into chondrocytes with chondrogenic factors in a biomaterial appears to be a promising therapeutic approach for direct implantation and cartilage repair. Here, we characterized equine umbilical cord blood-derived MSCs (eUCB-MSCs and evaluated their potential for chondrocyte differentiation for use in cartilage repair therapy. Our results show that isolated eUCB-MSCs had high proliferative capacity and differentiated easily into osteoblasts and chondrocytes, but not into adipocytes. A three-dimensional (3D culture approach with the chondrogenic factors BMP-2 and TGF-β1 potentiated chondrogenic differentiation with a significant increase in cartilage-specific markers at the mRNA level (Col2a1, Acan, Snorc and the protein level (type II and IIB collagen without an increase in hypertrophic chondrocyte markers (Col10a1 and Mmp13 in normoxia and in hypoxia. However, these chondrogenic factors caused an increase in type I collagen, which can be reduced using small interfering RNA targeting Col1a2. This study provides robust data on MSCs characterization and demonstrates that eUCB-MSCs have a great potential for cartilage tissue engineering.

  12. Rapid multicomponent T2 analysis of the articular cartilage of the human knee joint at 3.0T.

    Science.gov (United States)

    Liu, Fang; Chaudhary, Rajeev; Hurley, Samuel A; Munoz Del Rio, Alejandro; Alexander, Andrew L; Samsonov, Alexey; Block, Walter F; Kijowski, Richard

    2014-05-01

    To determine the feasibility of using multicomponent-driven equilibrium single-shot observation of T1 and T2 (mcDESPOT) for evaluating the human knee joint at 3.0T and to investigate depth-dependent and regional-dependent variations in multicomponent T2 parameters within articular cartilage. mcDESPOT was performed on the knee joint of 10 asymptomatic volunteers at 3.0T. Single-component T2 relaxation time (T2single ), multicomponent T2 relaxation time for water tightly bound to proteoglycan (T2PG ) and bulk water loosely bound to the macromolecular matrix (T2BW ), and fraction of water tightly bound to proteoglycan (FPG ) were measured in eight cartilage subsections and within the superficial and deep layers of patellar cartilage. Statistical analysis was used to investigate depth-dependent and regional-dependent variations in parameters. There was lower (P = 0.001) T2single and T2PG and higher (P Multicomponent T2 parameters of the articular cartilage of the human knee joint can be measured at 3.0T using mcDESPOT and show depth-dependent and regional-dependent variations. Copyright © 2013 Wiley Periodicals, Inc.

  13. Se-methylselenocysteine alters collagen gene and protein expression in human prostate cells.

    Science.gov (United States)

    Hurst, Rachel; Elliott, Ruan M; Goldson, Andrew J; Fairweather-Tait, Susan J

    2008-09-28

    The anti-cancer activity of selenium is dose-dependent and species-specific but the mechanism is unclear. Se-methylselenocysteine (MSC), found in selenium-enriched alliums, is one of the most potent forms. We exposed two human prostate cell lines (LNCaP clone FGC and PNT1A) to nutritionally relevant doses of MSC and selenite, ranging from deficient to the equivalent of selenium supplementation in humans. The cells were adapted for one month to attain steady-state selenium status. Two microarray platforms, an in-house printed microarray (14,000 genes) and the Affymetrix U133A array (22,000 genes) were used to probe the molecular effects of selenium dose and form and several selenium-responsive genes were identified, many of which have been ascribed to cancer cell growth and progression. In response to MSC supplementation, the expression of 23 genes changed significantly, including several collagen genes. Quantitative RT-PCR assays were designed and optimized for four of the collagen genes to validate array data. Significant decreases in expression of collagen type I alpha 1 (COL1A1), COL1A2 and COL7A1 genes were observed in cells adapted to MSC supplementation compared to the control and selenite exposed cells. There were significant increases in genes encoding other types of collagen, including significant increases in COL6A1 and COL4A5 in response to MSC dose. Functional changes in collagen type I protein expression in response to MSC were confirmed by ELISA. This study reveals for the first time that MSC can alter the expression of several types of collagen and thus potentially modulate the extracellular matrix and stroma, which may at least partially explain the anti-cancer activity of MSC.

  14. Quantitative Mapping of Collagen Fiber Orientation in Non-glaucoma and Glaucoma Posterior Human Sclerae

    OpenAIRE

    Pijanka, Jacek K.; Coudrillier, B.; Ziegler, K; Sorensen, T.; Meek, Keith Michael Andrew; Nguyen, T. D.; Quigley, H A; Boote, Craig

    2012-01-01

    Purpose. The posterior sclera has a major biomechanical influence on the optic nerve head, and may therefore be important in glaucoma. Scleral material properties are influenced significantly by collagen fiber architecture. Here we quantitatively map fiber orientation in non-glaucoma and glaucoma posterior human sclerae.\\ud Methods. Wide-angle x-ray scattering quantified fiber orientation at 0.5-mm intervals across seven non-glaucoma post-mortem human sclerae, and five sclerae with glaucoma h...

  15. Micromechanical properties and collagen composition of ruptured human achilles tendon

    DEFF Research Database (Denmark)

    Hansen, Philip; Kovanen, Vuokko; Hölmich, Per

    2013-01-01

    The Achilles tendon is one of the strongest tendons in the human body, and yet it frequently ruptures, which is a substantial clinical problem. However, the cause of ruptures remains elusive.......The Achilles tendon is one of the strongest tendons in the human body, and yet it frequently ruptures, which is a substantial clinical problem. However, the cause of ruptures remains elusive....

  16. Participation of collagen types I, III, IV, V, and fibronectin in the formation of villi fibrosis in human term placenta.

    Science.gov (United States)

    Rukosuev, V S; Nanaev, A K; Milovanov, A P

    1990-01-01

    The indirect immunofluorescence method was used to study the human term placenta in pathological pregnancy for the distribution of collagen types I, III, IV, V, and fibronectin in fibrosis stromatis villi. All collagen types and fibronectin were shown to participate in fibrosis villorum formation. Fibronectin was also detected in the fibrinoid that surrounded villi at stroma. The presence of free cytotrophoblast cells in the fibrinoid was accompanied by a noticeable increase in fibronectin fluorescence. A significant amount of collagen types IV and V and a less amount of collagen types I and III were identified.

  17. Confocal and conventional immunofluorescent and immunogold electron microscopic localization of collagen types III and IV in human placenta.

    Science.gov (United States)

    Nanaev, A K; Rukosuev, V S; Shirinsky, V P; Milovanov, A P; Domogatsky, S P; Duance, V C; Bradbury, F M; Yarrow, P; Gardiner, L; d'Lacey, C

    1991-01-01

    Confocal and conventional indirect immunofluorescence and immunogold electron microscopic methods were applied to examine the distribution of extracellular matrix constituents (collagens types III and IV) in the villi of immature and term human placentae. The immunofluorescence study revealed that collagen type III is more distinct in the villous stroma of term placenta as compared with that of the first trimester. Collagen type IV was detected mainly in endothelial and epithelial basement membranes and interestingly also to a certain extent in the stroma. Results obtained using immunoelectron microscopy support the proposal that collagen types III and IV are characteristic of stromal and basement membranes, respectively. Stromal collagen type IV is apparently localized in association with the interstitial types of collagen (I and III), in the villous stroma of term placenta.

  18. Effects of HEMA on type I collagen protein in human gingival fibroblasts.

    Science.gov (United States)

    Falconi, M; Teti, G; Zago, M; Pelotti, S; Breschi, L; Mazzotti, G

    2007-09-01

    The cytotoxicity of dental composites has been attributed to the release of residual monomers from polymerized adhesive systems due to degradation processes or the incomplete polymerization of materials. 2-Hydroxyethyl methacrylate (HEMA) is one of the major components released from dental adhesives. Cytotoxic effects due to high concentrations of HEMA have already been investigated, but the influence of minor toxic concentrations on specific proteins such as type I collagen has not been studied in depth. The objective of this project was to study the effect of minor toxic concentrations of HEMA on human gingival fibroblasts (HGFs), investigating modification in cell morphology, cell viability, and the influence on type I collagen protein. Primary lines of human gingival fibroblasts were exposed to 3 mmol/L HEMA for different periods of time (24 h, 72 h, 96 h). The cell vitality was determined by MTT assay, and high-resolution scanning electron microscopy analysis was performed to evaluate differences in cell morphology before and after treatment. The presence and localization of type I collagen was determined by immunofluorescence in HGFs treated with HEMA for the same period of time. The vitality of the cells decreased after 72 h of exposure. The HGFs grown in monolayer and observed by field emission in-lens scanning electron microscopy demonstrated a preserved surface morphology after 24 h of treatment, while they showed an altered morphology after 96 h of treatment. Immunofluorescence demonstrated a reduction of type I collagen due to HEMA exposure after 96 h. From these results, we conclude that low concentrations of HEMA can significantly alter the morphology of human gingival fibroblasts and interfere with the presence of type I collagen protein.

  19. Quantitative analysis of immunogold labellings of collagen types I, III, IV and VI in healthy and pathological human corneas.

    Science.gov (United States)

    Delaigue, O; Arbeille, B; Rossazza, C; Lemesle, M; Roingeard, P

    1995-06-01

    We studied the distribution of collagen types I, III, IV and VI in one healthy human cornea and in seven pathological human corneas, in which the disorders were three cases of pseudophakic bullous keratopathy (two severe, one moderate) and one case each of stage IV keratoconus, chronic ulcer, vascularized cornea and disciform keratitis. Transmission electron microscopy examinations were performed on post-embedding immunogold-labelled sections. The staining was evaluated by gold particle count in the different tissues. The presence or absence of a given antigen was determined by statistical analysis, using a d-value test. Our results on healthy corneal tissues corroborate the data available from previous studies, except for collagen type VI, which we found to be absent in Bowman's layer. In pathological corneas with a collagenous layer posterior to Descemet's membrane, collagen types I, III and especially IV were detected in this collagenous layer. Collagen types I, III and VI were detected in the anterior healed stroma of other pathological corneas, except for the keratoconus cornea, in which intense collagen III staining was observed. The presence of collagen types I and III in the posterior collagenous layer of our pseudophakic bullous keratopathy corneas suggests that this layer corresponds to scar tissue secreted by stimulated endothelial cells.

  20. The Role of Collagen Crosslinking in Differentiation of Human Mesenchymal Stem Cells and MC3T3-E1 Cells

    NARCIS (Netherlands)

    Fernandes, Hugo; Dechering, Koen; Van Someren, Eugene; Steeghs, Ilse; Apotheker, Marion; Leusink, Anouk; Bank, Ruud; Janeczek, Karolina; Van Blitterswijk, Clemens; de Boer, Jan

    2009-01-01

    Collagen is the main protein component of the extracellular matrix of bone, and it has structural and instructive properties. Collagen undergoes many post-translational modifications, including extensive crosslinking. Although defective crosslinking has been implicated in human syndromes (e.g.,

  1. The role of collagen crosslinking in differentiation of human mesenchymal stem cells and MC3T3-E1 cells

    NARCIS (Netherlands)

    Fernandes, H.A.M.; Dechering, Koen; Someren, Eugene; Steeghs, Ilse; Apotheker, Marion; Mentink-Leusink, Anouk; Bank, Ruud; Portalska, K.K.; van Blitterswijk, Clemens; de Boer, Jan

    2009-01-01

    Collagen is the main protein component of the extracellular matrix of bone, and it has structural and instructive properties. Collagen undergoes many post-translational modifications, including extensive crosslinking. Although defective crosslinking has been implicated in human syndromes (e.g.,

  2. Innervation of tissue-engineered recombinant human collagen-based corneal substitutes: a comparative in vivo confocal microscopy study.

    Science.gov (United States)

    Lagali, Neil; Griffith, May; Fagerholm, Per; Merrett, Kimberley; Huynh, Melissa; Munger, Rejean

    2008-09-01

    To compare reinnervation in recombinant human collagen-based corneal substitutes with allografts during a 1-year postimplantation follow-up period in pigs. A retrospective comparison to innervation in porcine collagen-based biosynthetic grafts was also performed. Pigs received a corneal allograft or a substitute made of either recombinant human type-I or -III collagen. In vivo confocal microscopic examination of the central cornea of surgical and untouched control eyes before surgery and at 2, 6, and 12 months after surgery was performed to quantify the number, density, and diameter of nerves at various corneal depths. By 12 months after surgery, the number and density of regenerated nerves in the anterior and deep anterior corneal stroma recovered to preoperative and control levels in both types of substitute grafts and in the allografts. In the subepithelial and subbasal regions, however, significantly fewer nerves were detected relative to those in control subjects at 12 months, regardless of graft type (P collagen-based biosynthetic grafts. An absence of thick stromal nerve trunks (diameter, >10 mum) in all grafts, irrespective of material type, indicated that nerve regeneration in grafts was accompanied by persistent morphologic changes. Nerve regeneration in recombinant human collagen-based biosynthetic corneal grafts proceeded similarly to that in allograft tissue, demonstrating the suitability of recombinant human collagen constructs as nerve-friendly corneal substitutes. Furthermore, only minor differences were noted between type-I and -III collagen grafts, indicating an insensitivity of nerve regeneration to initial collagen type.

  3. 3D Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications.

    Science.gov (United States)

    Markstedt, Kajsa; Mantas, Athanasios; Tournier, Ivan; Martínez Ávila, Héctor; Hägg, Daniel; Gatenholm, Paul

    2015-05-11

    The introduction of 3D bioprinting is expected to revolutionize the field of tissue engineering and regenerative medicine. The 3D bioprinter is able to dispense materials while moving in X, Y, and Z directions, which enables the engineering of complex structures from the bottom up. In this study, a bioink that combines the outstanding shear thinning properties of nanofibrillated cellulose (NFC) with the fast cross-linking ability of alginate was formulated for the 3D bioprinting of living soft tissue with cells. Printability was evaluated with concern to printer parameters and shape fidelity. The shear thinning behavior of the tested bioinks enabled printing of both 2D gridlike structures as well as 3D constructs. Furthermore, anatomically shaped cartilage structures, such as a human ear and sheep meniscus, were 3D printed using MRI and CT images as blueprints. Human chondrocytes bioprinted in the noncytotoxic, nanocellulose-based bioink exhibited a cell viability of 73% and 86% after 1 and 7 days of 3D culture, respectively. On the basis of these results, we can conclude that the nanocellulose-based bioink is a suitable hydrogel for 3D bioprinting with living cells. This study demonstrates the potential use of nanocellulose for 3D bioprinting of living tissues and organs.

  4. Nanomechanical assessment of human and murine collagen fibrils via atomic force microscopy cantilever-based nanoindentation.

    Science.gov (United States)

    Andriotis, Orestis G; Manuyakorn, Wiparat; Zekonyte, Jurgita; Katsamenis, Orestis L; Fabri, Sebastien; Howarth, Peter H; Davies, Donna E; Thurner, Philipp J

    2014-11-01

    The nanomechanical assessment of collagen fibrils via atomic force microscopy (AFM) is of increasing interest within the biomedical research community. In contrast to conventional nanoindentation there exists no common standard for conducting experiments and analysis of data. Currently used analysis approaches vary between studies and validation of quantitative results is usually not performed, which makes comparison of data from different studies difficult. Also there are no recommendations with regards to the maximum indentation depth that should not be exceeded to avoid substrate effects. Here we present a methodology and analysis approach for AFM cantilever-based nanoindentation experiments that allows efficient use of captured data and relying on a reference sample for determination of tip shape. Further we show experimental evidence that maximum indentation depth on collagen fibrils should be lower than 10-15% of the height of the fibril to avoid substrate effects and we show comparisons between our and other approaches used in previous works. While our analysis approach yields similar values for indentation modulus compared to the Oliver-Pharr method we found that Hertzian analysis yielded significantly lower values. Applying our approach we successfully and efficiently indented collagen fibrils from human bronchi, which were about 30 nm in size, considerably smaller compared to collagen fibrils obtained from murine tail-tendon. In addition, derived mechanical parameters of collagen fibrils are in agreement with data previously published. To establish a quantitative validation we compared indentation results from conventional and AFM cantilever-based nanoindentation on polymeric samples with known mechanical properties. Importantly we can show that our approach yields similar results when compared to conventional nanoindentation on polymer samples. Introducing an approach that is reliable, efficient and taking into account the AFM tip shape, we anticipate

  5. Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells

    NARCIS (Netherlands)

    Fernandes, H.; Mentink, A.; Bank, R.; Stoop, R.; Blitterswijk, C. van; Boer, J. de

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  6. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, H.A.M.; Mentink-Leusink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  7. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, Hugo; Mentink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  8. Treatment with recombinant interferon-beta reduces inflammation and slows cartilage destruction in the collagen-induced arthritis model of rheumatoid arthritis

    NARCIS (Netherlands)

    van Holten, Judith; Reedquist, Kris; Sattonet-Roche, Pascale; Smeets, Tom J. M.; Plater-Zyberk, Christine; Vervoordeldonk, Margriet J.; Tak, Paul P.

    2004-01-01

    We investigated the therapeutic potential and mechanism of action of IFN-beta protein for the treatment of rheumatoid arthritis (RA). Collagen-induced arthritis was induced in DBA/1 mice. At the first clinical sign of disease, mice were given daily injections of recombinant mouse IFN-beta or saline

  9. Expression of BMP-receptor type 1A correlates with progress of osteoarthritis in human knee joints with focal cartilage lesions.

    Science.gov (United States)

    Schmal, Hagen; Pilz, Ingo H; Mehlhorn, Alexander T; Dovi-Akue, David; Kirchhoff, Christina; Südkamp, Norbert P; Gerlach, Ulrike; Niemeyer, Philipp

    2012-08-01

    Bone morphogenetic protein-2 (BMP-2) and its receptor type 1A (BMPR-1A) play significant roles in cartilage metabolism. The aim of this study was to evaluate a possible correlation between intra-articular expression of these proteins and the degree of osteoarthritis (OA) in human knees. Biopsies of synovia and debrided cartilage were taken in 15 patients undergoing autologous chondrocyte implantation. Expression of BMP-2 and BMPR-1A was evaluated semi-quantitatively by immunohistologic staining. These data were complemented by grading of cartilage lesions according to International Cartilage Repair Society (ICRS), defect size, duration of complaints, knee osteoarthritis scoring system (KOSS) and Henderson and Kellgren-Lawrence scores. General histologic stainings were used to determine Mankin, Pritzker and Krenn scores. The expression of BMPR-1A but not of BMP-2 was significantly higher in cartilage biopsies taken in type 3 lesions with intact subchondral layer compared with type 4 defects (P < 0.05). In cartilage areas of bordering sectors, the intensity of immunohistologic staining of BMPR-1A was statistically significantly higher in mature cartilage compared with repair zones (P < 0.05). Expression of BMP-2 and its receptor 1A correlated in the cartilage biopsies (P < 0.02) but not in the synovia. The degree of OA was scored in all biopsies according to Mankin and Pritzker, and these scores correlated statistically significantly with BMPR-1A expression in the synovia (P < 0.05). In patients with an osteochondritis dissecans, the degree of OA was higher compared with other causes of chondromalacia, as evaluated by defect size, ICRS score, duration of complaints, Pritzker score and expression of BMPR-1A in cartilage (P < 0.05). These data support the role of BMPR-1A as an indicator of OA progression in human knees with circumscribed cartilage lesions.

  10. MCF-7 human mammary adenocarcinoma cells exhibit augmented responses to human insulin on a collagen IV surface

    DEFF Research Database (Denmark)

    Listov-Saabye, Nicolai; Jensen, Marianne Blirup; Kiehr, Benedicte

    2009-01-01

    was significantly more mitogenic than native insulin, validating the ability of the assay to identify hypermitogenic human insulin analogs. With MCF-7 cells on a collagen IV surface, the ranking of mitogens was maintained, but fold mitogenic responses and dynamic range and steepness of dose-response curves were...... increased. Also, PI3K pathway activation by insulin was enhanced on a collagen IV surface. This study provided the first determination and ranking of the mitogenic potencies of standard reference compounds in an optimized MCF-7 assay. The optimized MCF-7 assay described here is of relevance for in vitro...... toxicological testing and carcinogenicity safety assessment of new insulin compounds....

  11. Dependence of optical attenuation coefficient and mechanical tension of irradiated human cartilage measured by optical coherence tomography.

    Science.gov (United States)

    Martinho Junior, A C; Freitas, A Z; Raele, M P; Santin, S P; Soares, F A N; Herson, M R; Mathor, M B

    2015-03-01

    As banked human tissues are not widely available, the development of new non-destructive and contactless techniques to evaluate the quality of allografts before distribution for transplantation is very important. Also, tissues will be processed accordingly to standard procedures and to minimize disease transmission most tissue banks will include a decontamination or sterilization step such as ionizing radiation. In this work, we present a new method to evaluate the internal structure of frozen or glycerol-processed human cartilages, submitted to various dosis of irradiation, using the total optical attenuation coefficient retrieved from optical coherence tomography (OCT) images. Our results show a close relationship between tensile properties and the total optical attenuation coefficient of cartilages. Therefore, OCT associated with the total optical attenuation coefficient open a new window to evaluate quantitatively biological changes in processed tissues.

  12. Mechanical properties and structure-function relationships of human chondrocyte-seeded cartilage constructs after in vitro culture.

    Science.gov (United States)

    Middendorf, Jill M; Griffin, Darvin J; Shortkroff, Sonya; Dugopolski, Caroline; Kennedy, Stephen; Siemiatkoski, Joseph; Cohen, Itai; Bonassar, Lawrence J

    2017-10-01

    Autologous Chondrocyte Implantation (ACI) is a widely recognized method for the repair of focal cartilage defects. Despite the accepted use, problems with this technique still exist, including graft hypertrophy, damage to surrounding tissue by sutures, uneven cell distribution, and delamination. Modified ACI techniques overcome these challenges by seeding autologous chondrocytes onto a 3D scaffold and securing the graft into the defect. Many studies on these tissue engineered grafts have identified the compressive properties, but few have examined frictional and shear properties as suggested by FDA guidance. This study is the first to perform three mechanical tests (compressive, frictional, and shear) on human tissue engineered cartilage. The objective was to understand the complex mechanical behavior, function, and changes that occur with time in these constructs grown in vitro using compression, friction, and shear tests. Safranin-O histology and a DMMB assay both revealed increased sulfated glycosaminoglycan (sGAG) content in the scaffolds with increased maturity. Similarly, immunohistochemistry revealed increased lubricin localization on the construct surface. Confined compression and friction tests both revealed improved properties with increased construct maturity. Compressive properties correlated with the sGAG content, while improved friction coefficients were attributed to increased lubricin localization on the construct surfaces. In contrast, shear properties did not improve with increased culture time. This study suggests the various mechanical and biological properties of tissue engineered cartilage improve at different rates, indicating thorough mechanical evaluation of tissue engineered cartilage is critical to understanding the performance of repaired cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2298-2306, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  13. Human type VII collagen: cDNA cloning and chromosomal mapping of the gene

    Energy Technology Data Exchange (ETDEWEB)

    Parente, M.G.; Chung, L.C.; Ryynaenen, J.; Monli Chu; Uitto, J. (Thomas Jefferson Univ., Philadelphia, PA (United States)); Woodley, D.T.; Wynn, K.C.; Bauer, E.A. (Stanford Univ., CA (United States)); Mattei, M.G. (Institute National de la Sante et de la Recherche Medicale, Marseille (France))

    1991-08-15

    A human keratinocyte cDNA expression library in bacteriophage {lambda}gt11 was screened with the purified IgG fraction of serum from a patient with epidermolysis bullosa acquisita, which had a high titer of anti-type VII collagen antibodies. Screening of {approx}3 {times} 10{sup 5} plaques identified 8 positive clones, the largest one (K-131) being {approx}1.9 kilobases in size. Dideoxynucleotide sequencing of K-131 indicated that it consisted of 1875 base pairs and contained an open reading frame coding for a putative N-terminal noncollagenous domain of 439 amino acids and a collagenous domain was characterized by repeating Gly-Xaa-Yaa sequences that were interrupted in several positions by insertions or deletions of 1-3 amino acids. The deduced amino acid sequence also revealed a peptide segment that had a high degree of identity with a published type VII collagen protein sequence. The results mapped the COL7A1 to the locus 3p21. The cDNA clones characterized in this study will be valuable for understanding the protein structure and gene expression of type VII collagen present in anchoring fibrils and its aberrations in the dystrophic forms of heritable epidermolysis bullosa.

  14. Photobiomodulation on the proliferation and collagen synthesis of normal human skin fibroblast cells

    Science.gov (United States)

    Cheng, Lei; Liu, Timon Cheng-Yi; Chi, Jin-Quan; Li, Yan; Jin, Hua

    2006-01-01

    Background and Objective: Cultured normal human skin fibroblast cells (HSFs) were once used to study the mechanism of the effects of low intensity He-Ne laser irradiation (LHNL) on wound healing. The proliferation and collagen synthesis of HFSs were modulated by LHNL in different papers, respectively, and both of them are studied in this paper. Study Design/Materials and Methods: The dosage was studied for the same radiation time 300s. The proliferation and collagen synthesis were measured by 3-[4,5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay and the spectrophotometric method for the determination of hydroxyproline, respectively. Results: The dose zones were called dose 1, dose 2 and dose 3 from low dose on so that HSF proliferation was inhibited in dose 1 (16, 24 mJ/cm2), and promoted in dose 2 (298, 503, 597mJ/cm2), and the collagen synthesis was inhibited in dose 2 (401, 526 mJ/cm2), and promoted in dose 3 (714, 926, 1539, 1727mJ/cm2), which supports our biological model of photobiomodulation. It was found there is the linear relationship of the effect with dose with dose in each dose zone. Conclusions: The photobiomodulation on the proliferation and collagen synthesis of HSFs might be linearly dose-dependent in limited dosage with radiation time kept constant, which provides a foundation to discuss photobiomodulation on wound healing.

  15. Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

    DEFF Research Database (Denmark)

    Rasmussen, Camilla Holzmann; Petersen, Dorthe Roenn; Møller, Jonas Bech

    2015-01-01

    Human embryonic stem cells have the ability to generate all cell types in the body and can potentially provide an unlimited source of cells for cell replacement therapy to treat degenerative diseases such as diabetes. Current differentiation protocols of human embryonic stem cells towards insulin...... embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed...... and consistent differentiation of stem cells to definitive endoderm. The results shed light on the importance of extracellular matrix proteins for differentiation and also points to a cost effective and easy method to improve differentiation....

  16. Immunogold fine structural localization of extracellular matrix components in aged human cornea. II. Collagen types V and VI.

    Science.gov (United States)

    Marshall, G E; Konstas, A G; Lee, W R

    1991-01-01

    Using immunogold immunocytochemical techniques we studied the distribution of collagen types V and VI in corneal tissue from seven enucleated human eyes (age range, 63-78 years). Results obtained by cryoultramicrotomy were marginally more intense than those obtained using London Resin white (LR white) embedding. Type V collagen was present in the striated collagen fibrils in Bowman's layer, in the stroma and in a thin, non-banded anterior zone of Descemet's membrane. Our results suggest that types I, III and V collagen co-distribute in striated collagen fibrils. By contrast, type VI collagen was located in fine filaments in the interfibrillar matrix of the stroma, in Bowman's layer and in the anchoring plaques of the sub-epithelial basement-membrane complex. This implies an importance in epithelial adhesion which was previously unsuspected. Keratocyte bodies were electron-dense, amorphous extracellular deposits of matrix-like material, and these were labelled with types III, V and VI collagen antibodies. Long-spacing collagen was observed in the corneal stroma, and this deposit did not contain any of the collagen types studied.

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

    Science.gov (United States)

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

    2015-08-01

    We studied the effect of fibril formation of fish scale collagen on the osteoblastic differentiation of human mesenchymal stem cells (hMSCs). We found that hMSCs adhered easily to tilapia scale collagen, which remarkably accelerated the early stage of osteoblastic differentiation in hMSCs during in vitro cell culture. Osteoblastic markers such as ALP activity, osteopontin, and bone morphogenetic protein 2 were markedly upregulated when the hMSCs were cultured on a tilapia collagen surface, especially in the early osteoblastic differentiation stage. We hypothesized that this phenomenon occurs due to specific fibril formation of tilapia collagen. Thus, we examined the time course of collagen fibril formation using high-speed atomic force microscopy. Moreover, to elucidate the effect of the orientation of fibril formation on the differentiation of hMSCs, we measured ALP activity of hMSCs cultured on two types of tilapia scale collagen membranes with different degrees of fibril formation. The ALP activity in hMSCs cultured on a fibrous collagen membrane was significantly higher than on a non-fibrous collagen membrane even before adding osteoblastic differentiation medium. These results showed that the degree of the fibril formation of tilapia collagen was essential for the osteoblastic differentiation of hMSCs. © 2014 Wiley Periodicals, Inc.

  18. Adherens junction proteins are expressed in collagen corneal equivalents produced in vitro with human cells

    OpenAIRE

    Giasson, Claude J.; Deschambeault, Alexandre; Carrier, Patrick; Germain, Lucie

    2014-01-01

    Purpose To test whether adherens junction proteins are present in the epithelium and the endothelium of corneal equivalents. Methods Corneal cell types were harvested from human eyes and grown separately. Stromal equivalents were constructed by seeding fibroblasts into a collagen gel on which epithelial and endothelial cells were added on each side. Alternatively, bovine endothelial cells were used. At maturity, sections of stromal equivalents were processed for Masson's trichrome or indirect...

  19. Stimulation of a calcified cartilage connecting zone by GDF-5-augmented fibrin hydrogel in a novel layered ectopic in vivo model.

    Science.gov (United States)

    Diederichs, Solvig; Renz, Yvonne; Hagmann, Sébastien; Lotz, Benedict; Seebach, Elisabeth; Richter, Wiltrud

    2017-10-25

    Tissue engineering approaches for reconstructing full-depth cartilage defects need to comprise a zone of calcified cartilage to tightly anchor cartilage constructs into the subchondral bone. Here, we investigated whether growth and differentiation factor-5-(GDF-5)-augmented fibrin hydrogel can induce a calcified cartilage-layer in vitro that seamlessly connects cartilage-relevant biomaterials with bone tissue. Human bone marrow stromal cells (BMSCs) were embedded in fibrin hydrogel and subjected to chondrogenesis with TGF-β with or without GDF-5 before constructs were implanted subcutaneously into SCID mice. A novel layered ectopic in vivo model was developed and GDF-5-augmented fibrin with BMSCs was used to glue hydrogel and collagen constructs onto bone disks to investigate formation of a calcified cartilage connecting zone. GDF-5 significantly enhanced ALP activity during in vitro chondrogenesis while ACAN and COL2A1 mRNA, proteoglycan-, collagen-type-II- and collagen-type-X-deposition remained similar to controls. Pellets pretreated with GDF-5 mineralized faster in vivo and formed more ectopic bone. In the novel layered ectopic model, GDF-5 strongly supported calcified cartilage formation that seamlessly connected with the bone. Pro-chondrogenic and pro-hypertrophic activity makes GDF-5-augmented fibrin an attractive bioactive hydrogel with high potential to stimulate a calcified cartilage connecting zone in situ that might promote integration of cartilage scaffolds with bone. Thus, GDF-5-augmented fibrin hydrogel promises to overcome poor fixation of biomaterials in cartilage defects facilitating their long-term regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017. © 2017 Wiley Periodicals, Inc.

  20. Development of a reconstructed cornea from collagen-chondroitin sulfate foams and human cell cultures.

    Science.gov (United States)

    Vrana, N Engin; Builles, Nicolas; Justin, Virginie; Bednarz, Jurgen; Pellegrini, Graziella; Ferrari, Barbara; Damour, Odile; Hulmes, David J S; Hasirci, Vasif

    2008-12-01

    To develop an artificial cornea, the ability to coculture the different cell types present in the cornea is essential. Here the goal was to develop a full-thickness artificial cornea using an optimized collagen-chondroitin sulfate foam, with a thickness close to that of human cornea, by coculturing human corneal epithelial and stromal cells and transfected human endothelial cells. Corneal extracellular matrix was simulated by a porous collagen/glycosaminoglycan-based scaffold seeded with stromal keratocytes and then, successively, epithelial and endothelial cells. Scaffolds were characterized for bulk porosity and pore size distribution. The performance of the three-dimensional construct was studied by histology, immunofluorescence, and immunohistochemistry. The scaffold had 85% porosity and an average pore size of 62.1 microm. Keratocytes populated the scaffold and produced a newly synthesized extracellular matrix as characterized by immunohistochemistry. Even though the keratocytes lost their CD34 phenotype marker, the absence of smooth muscle actin fibers showed that these cells had not differentiated into myofibroblasts. The epithelial cells formed a stratified epithelium and began basement membrane deposition. An endothelial cell monolayer beneath the foam was also apparent. These results demonstrate that collagen-chondroitin sulfate scaffolds are good substrates for artificial cornea construction with good resilience, long-term culture capability, and handling properties.

  1. Plastic compressed collagen as a novel carrier for expanded human corneal endothelial cells for transplantation.

    Directory of Open Access Journals (Sweden)

    Hannah J Levis

    Full Text Available Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.

  2. Plastic compressed collagen as a novel carrier for expanded human corneal endothelial cells for transplantation.

    Science.gov (United States)

    Levis, Hannah J; Peh, Gary S L; Toh, Kah-Peng; Poh, Rebekah; Shortt, Alex J; Drake, Rosemary A L; Mehta, Jodhbir S; Daniels, Julie T

    2012-01-01

    Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.

  3. Effects of epicatechin, a crosslinking agent, on human dental pulp cells cultured in collagen scaffolds

    Directory of Open Access Journals (Sweden)

    Eun-su Lim

    2016-02-01

    Full Text Available ABSTRACT Objective The purpose of this study was to investigate the biological effects of epicatechin (ECN, a crosslinking agent, on human dental pulp cells (hDPCs cultured in collagen scaffolds. Material and Method To evaluate the effects of ECN on the proliferation of hDPCs, cell counting was performed using optical and fluorescent microscopy. Measurements of alkaline phosphatase (ALP activity, alizarin red staining, and real-time polymerase chain reactions were performed to assess odontogenic differentiation. The compressive strength and setting time of collagen scaffolds containing ECN were measured. Differential scanning calorimetry was performed to analyze the thermal behavior of collagen in the presence of ECN. Results Epicatechin increased ALP activity, mineralized nodule formation, and the mRNA expression of dentin sialophosphoprotein (DSPP, a specific odontogenic-related marker. Furthermore, ECN upregulated the expression of DSPP in hDPCs cultured in collagen scaffolds. Epicatechin activated the extracellular signal-regulated kinase (ERK and the treatment with an ERK inhibitor (U0126 blocked the expression of DSPP. The compressive strength was increased and the setting time was shortened in a dose-dependent manner. The number of cells cultured in the ECN-treated collagen scaffolds was significantly increased compared to the cells in the untreated control group. Conclusions Our results revealed that ECN promoted the proliferation and differentiation of hDPCs. Furthermore, the differentiation was regulated by the ERK signaling pathway. Changes in mechanical properties are related to cell fate, including proliferation and differentiation. Therefore, our study suggests the ECN treatment might be desirable for dentin-pulp complex regeneration.

  4. Innervation of Tissue-Engineered Recombinant Human Collagen-Based Corneal Substitutes: A Comparative In Vivo Confocal Microscopy Study

    National Research Council Canada - National Science Library

    Lagali, Neil; Griffith, May; Fagerholm, Per; Merrett, Kimberley; Huynh, Melissa; Munger, Rejean

    2008-01-01

    ...; and the 3 Department of Ophthalmology, Linköping University Hospital, Linköping, Sweden. METHODS . Pigs received a corneal allograft or a substitute made of either recombinant human type-I or -III collagen...

  5. Inhibition of human scleral fibroblast cell attachment to collagen type I by TGFBIp.

    Science.gov (United States)

    Shelton, Lilian; Rada, Jody A Summers

    2009-08-01

    Transforming growth factor beta-induced protein (TGFBIp; 68 kDa) is a secreted extracellular matrix (ECM) protein that has been demonstrated to regulate cell attachment in a variety of cell types. The sclera synthesizes and secretes TGFBIp, which may function to facilitate scleral ECM remodeling events associated with myopia development. Here the authors report that human scleral fibroblasts (HSFs) express TGFBI and that its protein product, TGFBIp, mediates an effect on cell attachment. TGFBI/TGFBIp expression was evaluated by RT-PCR and immunoblot of HSF lysates and culture supernatants. The effect of rTGFBIp (50 microg/mL) on cell attachment to collagen type I was determined with the use of fluid-phase cell attachment assays in HSFs, human foreskin fibroblasts (HFFs), and human corneal stroma fibroblasts (HCFs). Binding assays using biotinylated rTGFBIp were used to assess TGFBIp binding to the HSF surface. Flow cytometry and immunocytochemistry were used to determine both alphavbeta3 and alphavbeta5 expression and localization to the HSF cell surface. HSFs expressed TGFBI and secreted TGFBIp (approximately 833 ng/h). rTGFBIp significantly decreased (25 microg/mL; P collagen type I, whereas rTGFBIp did not significantly affect cell attachment of HFFs (P = 0.50) or HCFs (P = 0.24) to collagen compared with BSA. Integrins alphavbeta3 and alphavbeta5 were detected on the cell surface, and both anti-alphavbeta3 and anti-alphavbeta5 functionally blocked rTGFBIp binding to HSFs. TGFBIp plays an inhibitory role in HSF attachment to collagen type I in vitro through interactions with alphavbeta3 and alphavbeta5 integrin receptors. These results suggest that TGFBIp may modulate scleral cell-matrix interactions in vivo, thereby affecting scleral viscoelasticity.

  6. Immunogold fine structural localization of extracellular matrix components in aged human cornea. I. Types I-IV collagen and laminin.

    Science.gov (United States)

    Marshall, G E; Konstas, A G; Lee, W R

    1991-01-01

    Using the immunogold technique combined with cryoultramicrotomy and London Resin white (LR white) embedding, we studied the fine structural distribution of types I-IV collagen and laminin in corneal tissue from seven enucleated human eyes (age range, 63-78 years). Type II collagen was not identified in any corneal layer. Type I and type III collagen were distributed in a similar fashion in striated collagen fibrils in Bowman's layer and in the stroma. Type IV collagen was located only in the posterior non-banded region of Descemet's membrane. Laminin was identified in subepithelial anchoring plaques and the sub-endothelial region of Descemet's membrane in accordance with its recognized adhesive function.

  7. Distinct post-translational features of type I collagen are conserved in mouse and human periodontal ligament.

    Science.gov (United States)

    Hudson, D M; Garibov, M; Dixon, D R; Popowics, T; Eyre, D R

    2017-12-01

    Specifics of the biochemical pathways that modulate collagen cross-links in the periodontal ligament (PDL) are not fully defined. Better knowledge of the collagen post-translational modifications that give PDL its distinct tissue properties is needed to understand the pathogenic mechanisms of human PDL destruction in periodontal disease. In this study, the post-translational phenotypes of human and mouse PDL type I collagen were surveyed using mass spectrometry. PDL is a highly specialized connective tissue that joins tooth cementum to alveolar bone. The main function of the PDL is to support the tooth within the alveolar bone while under occlusal load after tooth eruption. Almost half of the adult population in the USA has periodontal disease resulting from inflammatory destruction of the PDL, leading to tooth loss. Interestingly, PDL is unique from other ligamentous connective tissues as it has a high rate of turnover. Rapid turnover is believed to be an important characteristic for this specialized ligament to function within the oral-microbial environment. Like other ligaments, PDL is composed predominantly of type I collagen. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including hydroxylation, glycosylation and cross-linking. The chemistry, placement and quantity of intermolecular cross-links are believed to be important regulators of tissue-specific structural and mechanical properties of collagens. Type I collagen was isolated from several mouse and human tissues, including PDL, and analyzed by mass spectrometry for post-translational variances. The collagen telopeptide cross-linking lysines of PDL were found to be partially hydroxylated in human and mouse, as well as in other types of ligament. However, the degree of hydroxylation and glycosylation at the helical Lys87 cross-linking residue varied across species and between ligaments. These data suggest that different types of ligament collagen

  8. The architecture of cartilage: Elemental maps and scanning transmission ion microscopy/tomography

    Energy Technology Data Exchange (ETDEWEB)

    Reinert, Tilo E-mail: reinert@physik.uni-leipzig.de; Reibetanz, Uta; Schwertner, Michael; Vogt, Juergen; Butz, Tilman; Sakellariou, Arthur

    2002-04-01

    Articular cartilage is not just a jelly-like cover of the bone within the joints but a highly sophisticated architecture of hydrated macromolecules, collagen fibrils and cartilage cells. Influences on the physiological balance due to age-related or pathological changes can lead to malfunction and subsequently to degradation of the cartilage. Many activities in cartilage research are dealing with the architecture of joint cartilage but have limited access to elemental distributions. Nuclear microscopy is able to yield spatially resolved elemental concentrations, provides density information and can visualise the arrangement of the collagen fibres. The distribution of the cartilage matrix can be deduced from the elemental and density maps. The findings showed a varying content of collagen and proteoglycan between zones of different cell maturation. Zones of higher collagen content are characterised by aligned collagen fibres that can form tubular structures. Recently we focused on STIM tomography to investigate the three dimensional arrangement of the collagen structures.

  9. Quantitative (23) Na MRI of human knee cartilage using dual-tuned (1) H/(23) Na transceiver array radiofrequency coil at 7 tesla.

    Science.gov (United States)

    Moon, Chan Hong; Kim, Jung-Hwan; Zhao, Tiejun; Bae, Kyongtae Ty

    2013-11-01

    To develop quantitative dual-tuned (DT) (1) H/(23) Na MRI of human knee cartilage in vivo at 7 Tesla (T). A sensitive (23) Na transceiver array RF coil was developed at 7T. B1 fields generated by the transceiver array coil were characterized and corrected in the (23) Na images. Point spread function (PSF) of the (23) Na images was measured, and the signal decrease due to partial-volume-effect was compensated in [(23) Na] quantification of knee cartilage. SNR and [(23) Na] in anterior femoral cartilage were measured from seven healthy subjects. SNR of (23) Na image with the transceiver array coil was higher than that of birdcage coil. SNR in the cartilage at 2-mm isotropic resolution was 26.80 ± 3.69 (n = 7). B1 transmission and reception fields produced by the DT coil at 7T were similar to each other. Effective full-width-half-maximum of (23) Na image was ∼5 mm at 2-mm resolution. Mean [(23) Na] was 288.13 ± 29.50 mM (n = 7) in the anterior femoral cartilage of normal subjects. We developed a new high-sensitivity (23) Na RF coil for knee MRI at 7T. Our (1) H/(23) Na MRI allowed quantitative measurement of [(23) Na] in knee cartilage by measuring PSF and cartilage thickness from (23) Na and (1) H image, respectively. Copyright © 2013 Wiley Periodicals, Inc.

  10. Growth plate regulation and osteochondroma formation: insights from tracing proteoglycans in zebrafish models and human cartilage.

    Science.gov (United States)

    de Andrea, Carlos E; Prins, Frans A; Wiweger, Malgorzata I; Hogendoorn, Pancras C W

    2011-06-01

    Proteoglycans are secreted into the extracellular matrix of virtually all cell types and function in several cellular processes. They consist of a core protein onto which glycosaminoglycans (e.g., heparan or chondroitin sulphates), are attached. Proteoglycans are important modulators of gradient formation and signal transduction. Impaired biosynthesis of heparan sulphate glycosaminoglycans causes osteochondroma, the most common bone tumour to occur during adolescence. Cytochemical staining with positively charged dyes (e.g., polyethyleneimine-PEI) allows, visualisation of proteoglycans and provides a detailed description of how proteoglycans are distributed throughout the cartilage matrix. PEI staining was studied by electron and reflection contrast microscopy in human growth plates, osteochondromas and five different proteoglycan-deficient zebrafish mutants displaying one of the following skeletal phenotypes: dackel (dak/ext2), lacking heparan sulphate and identified as a model for human multiple osteochondromas; hi307 (β3gat3), deficient for most glycosaminoglycans; pinscher (pic/slc35b2), presenting with defective sulphation of glycosaminoglycans; hi954 (uxs1), lacking most glycosaminoglycans; and knypek (kny/gpc4), missing the protein core of the glypican-4 proteoglycan. The panel of genetically well-characterized proteoglycan-deficient zebrafish mutants serves as a convincing and comprehensive study model to investigate proteoglycan distribution and the relation of this distribution to the model mutation status. They also provide insight into the distributions and gradients that can be expected in the human homologue. Human growth plate, wild-type zebrafish and fish mutants with mild proteoglycan defects (hi307 and kny) displayed proteoglycans distributed in a gradient throughout the matrix. Although the mutants pic and hi954, which had severely impaired proteoglycan biosynthesis, showed no PEI staining, dak mutants demonstrated reduced PEI staining and no

  11. Monoclonal antibody to human cartilage cells and its reactivities to chondrocytic tumors.

    Science.gov (United States)

    Eisenbrey, A B; Chen, J C; Dyer, C A; Bernstein, J; Poulik, M D

    1991-01-01

    A murine monoclonal antibody (E10) was made against cultured cartilage cells. The E10 antibody binding is localized to the surface of cultured cartilage cells in suspension and is present in the cytoplasm in paraffin embedded sections. There is no reactivity with cartilage matrix, or with the matrix of cartilaginous tumors. Reactivity is removed by treatment with trypsin and hyaluronidase, but not by treatment with heparinase, neuraminidase, and chondroitinase. Regeneration of E10 antigen after trypsinization takes 48 hours in chondrocytes in tissue culture. SDS-polyacrylamide gel electrophoresis of an E10 immune precipitate of cultured chondrocytes results in two peaks: one at a very high molecular weight and a small fragment at approximately 250 kd. Specificity has been demonstrated by cytofluorometry, immunofluorescence, and immunohistochemistry, in both frozen and paraffin-embedded tissues. Positive reactivity was seen in cultured cartilage cells, chondrocytes in fetal and adult cartilage, chondrosarcomas, and chordomas. Minimal reactivity was found in a chondromyxoid liposarcoma. Acinar cells of salivary and sweat glands and mast cells in various tissues and tumors were also positive. There was no reactivity with other tissues and tumors, including myxoid and mucinous tumors and epithelial tissues.

  12. Leptin enhances MMP-1, MMP-3 and MMP-13 production in human osteoarthritic cartilage and correlates with MMP-1 and MMP-3 in synovial fluid from OA patients.

    Science.gov (United States)

    Koskinen, Anna; Vuolteenaho, Katriina; Nieminen, Riina; Moilanen, Teemu; Moilanen, Eeva

    2011-01-01

    In the present study, we investigated the role of adipocytokine leptin in the pathogenesis of osteoarthritis (OA) by measuring its effects on matrix metalloproteinase (MMP) production in human OA cartilage. In addition, the correlations between leptin and MMP concentrations in synovial fluid from OA patients were studied. Cartilage tissue obtained from leftover pieces of total knee replacement surgery from patients with OA was used in the experiments. Production of collagenases MMP-1, MMP-8 and MMP-13, and stromelysin-1 (MMP-3) in the cartilage was measured by immunoassay and the signalling pathways were explored by pharmacological means. In addition, synovial fluid samples were collected from 84 OA patients undergoing knee replacement surgery. The concentrations of leptin and MMPs in synovial fluid were measured by immunoassay. Leptin alone and in combination with IL-1β enhanced production of MMP-1, MMP-3, and MMP-13 in human OA cartilage, while MMP-8 concentrations remained undetectable. The effects of leptin on MMP-1, MMP-3 and MMP-13 production were mediated through transcription factor NF-κβ, and through protein kinase C and MAP kinase pathways. Interestingly, leptin concentrations in synovial fluid from OA patients correlated positively with MMP-3 (r=0.51, pleptin up-regulates MMP-1 and MMP-3 production in human OA cartilage and correlates positively to MMP-1 and MMP-3 in synovial fluid from OA patients. The findings suggest that leptin has catabolic effects in OA joints by increasing MMP production in cartilage.

  13. Tissue engineering of cartilage in space

    Science.gov (United States)

    Freed, Lisa E.; Langer, Robert; Martin, Ivan; Pellis, Neal R.; Vunjak-Novakovic, Gordana

    1997-01-01

    Tissue engineering of cartilage, i.e., the in vitro cultivation of cartilage cells on synthetic polymer scaffolds, was studied on the Mir Space Station and on Earth. Specifically, three-dimensional cell-polymer constructs consisting of bovine articular chondrocytes and polyglycolic acid scaffolds were grown in rotating bioreactors, first for 3 months on Earth and then for an additional 4 months on either Mir (10−4–10−6 g) or Earth (1 g). This mission provided a unique opportunity to study the feasibility of long-term cell culture flight experiments and to assess the effects of spaceflight on the growth and function of a model musculoskeletal tissue. Both environments yielded cartilaginous constructs, each weighing between 0.3 and 0.4 g and consisting of viable, differentiated cells that synthesized proteoglycan and type II collagen. Compared with the Earth group, Mir-grown constructs were more spherical, smaller, and mechanically inferior. The same bioreactor system can be used for a variety of controlled microgravity studies of cartilage and other tissues. These results may have implications for human spaceflight, e.g., a Mars mission, and clinical medicine, e.g., improved understanding of the effects of pseudo-weightlessness in prolonged immobilization, hydrotherapy, and intrauterine development. PMID:9391122

  14. Accelerated postero-lateral spinal fusion by collagen scaffolds modified with engineered collagen-binding human bone morphogenetic protein-2 in rats.

    Directory of Open Access Journals (Sweden)

    Xinglong Han

    Full Text Available Bone morphogenetic protein-2 (BMP-2 is a potent osteoinductive cytokine that plays a critical role in bone regeneration and repair. However, its distribution and side effects are major barriers to its success as therapeutic treatment. The improvement of therapy using collagen delivery matrices has been reported. To investigate a delivery system on postero-lateral spinal fusion, both engineered human BMP-2 with a collagen binding domain (CBD-BMP-2 and collagen scaffolds were developed and their combination was implanted into Sprague-Dawley (SD rats to study Lumbar 4-5 (L4-L5 posterolateral spine fusion. We divided SD rats into three groups, the sham group (G1, n = 20, the collagen scaffold-treated group (G2, n = 20 and the BMP-2-loaded collagen scaffolds group (G3, n = 20. 16 weeks after surgery, the spines of the rats were evaluated by X-radiographs, high-resolution micro-computed tomography (micro-CT, manual palpation and hematoxylin and eosin (H&E staining. The results showed that spine L4-L5 fusions occurred in G2(40% and G3(100% group, while results from the sham group were inconsistent. Moreover, G3 had better results than G2, including higher fusion efficiency (X score, G2 = 2.4±0.163, G3 = 3.0±0, p<0.05, higher bone mineral density (BMD, G2: 0.3337±0.0025g/cm3, G3: 0.4353±0.0234g/cm3. p<0.05 and more bone trabecular formation. The results demonstrated that with site-specific collagen binding domain, a dose of BMP-2 as low as 0.02mg CBD-BMP-2/cm3 collagen scaffold could enhance the posterolateral intertransverse process fusion in rats. It suggested that combination delivery could be an alternative in spine fusion with dramatically decreased side effects caused by high dose of BMP-2.

  15. Collagen Orientation and Crystallite Size in Human Dentin: A Small Angle X-ray Scattering Study

    Energy Technology Data Exchange (ETDEWEB)

    Pople, John A

    2001-03-29

    The mechanical properties of dentin are largely determined by the intertubular dentin matrix, which is a complex composite of type I collagen fibers and a carbonate-rich apatite mineral phase. The authors perform a small angle x-ray scattering (SAXS) study on fully mineralized human dentin to quantify this fiber/mineral composite architecture from the nanoscopic through continuum length scales. The SAXS results were consistent with nucleation and growth of the apatite phase within periodic gaps in the collagen fibers. These mineralized fibers were perpendicular to the dentinal tubules and parallel with the mineralization growth front. Within the plane of the mineralization front, the mineralized collagen fibers were isotropic near the pulp, but became mildly anisotropic in the mid-dentin. Analysis of the data also indicated that near the pulp the mineral crystallites were approximately needle-like, and progressed to a more plate-like shape near the dentino-enamel junction. The thickness of these crystallites, {approx} 5 nm, did not vary significantly with position in the tooth. These results were considered within the context of dentinogenesis and maturation.

  16. Modulation of human uterine smooth muscle cell collagen contractility by thrombin, Y-27632, TNF alpha and indomethacin

    Directory of Open Access Journals (Sweden)

    Smith Terry J

    2009-01-01

    Full Text Available Abstract Background Preterm labour occurs in approximately 10% of pregnancies and is a major cause of infant morbidity and mortality. However, the pathways involved in regulating contractility in normal and preterm labour are not fully elucidated. Our aim was to utilise a human myometrial contractility model to investigate the effect of a number of uterine specific contractility agents in this system. Therefore, we investigated the contractile response of human primary uterine smooth muscle cells or immortalised myometrial smooth muscle cells cultured within collagen lattices, to known mediators of uterine contractility, which included thrombin, the ROCK-1 inhibitor Y-27632, tumour necrosis factor alpha (TNF alpha and the non-steroidal anti-inflammatory indomethacin. Methods Cell contractility was calculated over time, with the collagen gel contraction assay, utilising human primary uterine smooth muscle cells (hUtSMCs and immortalised myometrial smooth muscle cells (hTERT-HM: a decrease in collagen gel area equated to an increase in contractility. RNA was isolated from collagen embedded cells and gene expression changes were analysed by real time fluorescence reverse transcription polymerase chain reaction. Scanning electron and fluorescence microscopy were employed to observe cell morphology and cell collagen gel interactions. Statistical analysis was performed using ANOVA followed by Tukey's post hoc tests. Results TNF alpha increased collagen contractility in comparison to the un-stimulated collagen embedded hUtSMC cells, which was inhibited by indomethacin, while indomethacin alone significantly inhibited contraction. Thrombin augmented the contractility of uterine smooth muscle cell and hTERT-HM collagen gels, this effect was inhibited by the thrombin specific inhibitor, hirudin. Y-27632 decreased both basal and thrombin-induced collagen contractility in the hTERT-HM embedded gels. mRNA expression of the thrombin receptor, F2R was up

  17. Immunofluorescent localization of collagen types I, III, IV, V, fibronectin, laminin, entactin, and heparan sulphate proteoglycan in human immature placenta.

    Science.gov (United States)

    Rukosuev, V S

    1992-03-15

    The distribution of eight components of the extracellular matrix in immature human placenta was studied by an indirect immunofluorescence method with monospecific antibodies. In the stroma of the term chorionic villi, collagen types I, III, IV, V, and fibronectin formed a mesh of fibers and conglomerates. Heparan sulphate proteoglycan formed multiple conglomerates, whereas laminin comprised small, scanty, discrete granules. Collagen type IV, laminin, entactin, and heparan sulphate proteoglycan were confined to the basement membrane of the trophoblast. Sometimes, only collagen type IV was identified in fetal vascular basement membrane.

  18. 3D collagen orientation study of the human cornea using X-ray diffraction and femtosecond laser technology.

    Science.gov (United States)

    Abahussin, Mohammad; Hayes, Sally; Knox Cartwright, Nathaniel E; Kamma-Lorger, Christina S; Khan, Yasir; Marshall, John; Meek, Keith M

    2009-11-01

    To study the distribution and predominant orientations of fibrillar collagen at different depths throughout the entire thickness of the human cornea. This information will form the basis of a full three-dimensional reconstruction of the preferred orientations of corneal lamellae. Femtosecond laser technology was used to delaminate the central zones of five human corneas into three separate layers (anterior, mid, and posterior stroma), each with predetermined thicknesses. Wide-angle x-ray diffraction was used to study the gross collagen fibril orientation and distribution within each layer. The middle and posterior parts of the human cornea demonstrated a preferential orthogonal arrangement of collagen fibrils, directed along the superior-inferior and nasal-temporal meridians, with an increase in the number of lamellae toward the periphery. However, the anterior cornea (33% of total corneal thickness) showed no systematic preferred lamellar orientation. In the posterior two thirds of the human cornea, collagen lies predominantly in the vertical and horizontal meridians (directed toward the four major rectus muscles), whereas collagen in the anterior third of the cornea is more isotropic. The predominantly orthogonal arrangement of collagen in the mid and posterior stroma may help to distribute strain in the cornea by allowing it to withstand the pull of the extraocular muscles, whereas the more isotropic arrangement in the anterior cornea may play an important role in the biomechanics of the cornea by resisting intraocular pressure while at the same time maintaining corneal curvature.

  19. Expression of BMP-receptor type 1A correlates with progress of osteoarthritis in human knee joints with focal cartilage lesions

    DEFF Research Database (Denmark)

    Schmal, Hagen; Pilz, Ingo H; Mehlhorn, Alexander T

    2012-01-01

    was significantly higher in cartilage biopsies taken in type 3 lesions with intact subchondral layer compared with type 4 defects (P ...BACKGROUND AIMS: Bone morphogenetic protein-2 (BMP-2) and its receptor type 1A (BMPR-1A) play significant roles in cartilage metabolism. The aim of this study was to evaluate a possible correlation between intra-articular expression of these proteins and the degree of osteoarthritis (OA) in human...... knees. METHODS: Biopsies of synovia and debrided cartilage were taken in 15 patients undergoing autologous chondrocyte implantation. Expression of BMP-2 and BMPR-1A was evaluated semi-quantitatively by immunohistologic staining. These data were complemented by grading of cartilage lesions according...

  20. Differences in collagen ultrastructure of human first trimester decidua basalis and parietalis: Implications for trophoblastic invasion of the placental bed

    OpenAIRE

    Sinai Talaulikar, V; Kronenberger, K; Bax, BE; Moss, R; Manyonda, I

    2014-01-01

    AIM:\\ud The human embryo-maternal interface in the first trimester of pregnancy is an area of extensive tissue remodeling. Because collagen is the most abundant constituent of the extracellular matrix of the placental bed, successful invasion must involve its rapid turnover. We compared the nature and distribution of collagen fibrils in decidua basalis and parietalis.\\ud METHODS:\\ud We used a direct-vision hysteroscopic technique to obtain biopsies of the decidua basalis and parietalis from 1...

  1. Synthesis and characterization of hyaluronic acid/human-like collagen hydrogels

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jingjing; Ma, Xiaoxuan, E-mail: xiaoxuanma@163.com; Fan, Daidi, E-mail: fandaidi@nwu.edu.cn; Zhu, Chenhui; Deng, Jianjun; Hui, Junfeng; Ma, Pei

    2014-10-01

    Injectable hydrogel plays an important role in soft tissue filling and repair. We report an injectable hydrogel based on hyaluronic acid (HA) and human-like collagen (HLC), both with favorable biocompatibility and biodegradability. These two types of biomacromolecules were crosslinked with 1,4-butanediol diglycidyl ether to form a three-dimensional network. The redundant crosslinker was removed by dialysis and distillation. An HA-based hydrogel prepared by the same method was used as a control. The cytocompatibility was studied with a Cell Counting Kit-8 (CCK-8) test. Carbazole colorimetry was used to analyze the in vitro degradation rate. The histocompatibility was evaluated by hematoxylin and eosin (H and E) staining analysis and immunohistochemical analysis. The CCK-8 assay demonstrated that the HA/HLC hydrogel was less cytotoxic than the HA-based hydrogel and could promote baby hamster kidney cell (BHK) proliferation. The cell adhesion indicated that BHK could grow well on the surface of the materials and maintain good cell viability. The in vitro degradation test showed that the HA/HLC hydrogel had a longer degradation time and an excellent antienzyme ability. In vivo injection showed that there was little inflammatory response to HA/HLC after 1, 2, and 4 weeks. Therefore, the HA/HLC hydrogel is a promising biomaterial for soft tissue filling and repair. - Highlights: • Human-like collagen was used with hyaluronic acid to prepare soft tissue filling meterials. • 1,4-Butanediol diglycidyl ether (BDDE) was introduced to treat the hydrogels. • The addition of human-like collagen could improve the biological properties of hydrogels.

  2. Cartilage Repair Using Composites of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Hyaluronic Acid Hydrogel in a Minipig Model.

    Science.gov (United States)

    Ha, Chul-Won; Park, Yong-Beom; Chung, Jun-Young; Park, Yong-Geun

    2015-09-01

    The cartilage regeneration potential of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) with a hyaluronic acid (HA) hydrogel composite has shown remarkable results in rat and rabbit models. The purpose of the present study was to confirm the consistent regenerative potential in a pig model using three different cell lines. A full-thickness chondral injury was intentionally created in the trochlear groove of each knee in 6 minipigs. Three weeks later, an osteochondral defect, 5 mm wide by 10 mm deep, was created, followed by an 8-mm-wide and 5-mm-deep reaming. A mixture (1.5 ml) of hUCB-MSCs (0.5×10(7) cells per milliliter) and 4% HA hydrogel composite was then transplanted into the defect on the right knee. Each cell line was used in two minipigs. The osteochondral defect created in the same manner on the left knee was untreated to act as the control. At 12 weeks postoperatively, the pigs were sacrificed, and the degree of subsequent cartilage regeneration was evaluated by gross and histological analysis. The transplanted knee resulted in superior and more complete hyaline cartilage regeneration compared with the control knee. The cellular characteristics (e.g., cellular proliferation and chondrogenic differentiation capacity) of the hUCB-MSCs influenced the degree of cartilage regeneration potential. This evidence of consistent cartilage regeneration using composites of hUCB-MSCs and HA hydrogel in a large animal model could be a stepping stone to a human clinical trial in the future. To date, several studies have investigated the chondrogenic potential of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs); however, the preclinical studies are still limited in numbers with various results. In parallel, in the past several years, the cartilage regeneration potential of hUCB-MSCs with a hyaluronic acid (HA) hydrogel composite have been investigated and remarkable results in rat and rabbit models have been attained. (These

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

    Science.gov (United States)

    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.

  4. In vitro effects of methotrexate on human articular cartilage and bone-derived osteoblasts

    NARCIS (Netherlands)

    van der Veen, M. J.; Scheven, B. A.; van Roy, J. L.; Damen, C. A.; Lafeber, F. P.; Bijlsma, J. W.

    1996-01-01

    Conflicting data have been published on whether low-dose methotrexate (MTX) treatment of rheumatoid arthritis (RA) is able to slow down radiological joint damage, i.e. retard the destruction of articular cartilage and (subchondral) bone. We studied the effects of MTX on proteoglycan (PG) turnover

  5. [Implantation of collagen coated hydroxyapatite particles. A clinical-histological study in humans].

    Science.gov (United States)

    Sanz, M; Bascones, A; Kessler, A; García Nuñez, J; Newman, M G; Robertson, M A; Carranza, F A

    1989-05-01

    In this study, histologic behaviour of collagen coated hydroxylapatite particles implanted in human periodontal osseous defects has been analyzed. This material was surgically implanted in four patients, and reentry and block biopsies were carried out 4 and 6 months later. The histologic results demonstrate that this material is well tolerated by surrounding tissues, not eliciting an inflammatory reaction. At four months, the hydroxylapatite particles appear encapsulated by a very cellular connective tissue and at 6 months are found in direct contact with osteoid and mature bone. This material acts as a filler material, being fully biocompatible and stimulating an osseoconductive reaction of the adjacent alveolar bone.

  6. Type I collagen synthesis and degradation in peritendinous tissue after exercise determined by microdialysis in humans

    DEFF Research Database (Denmark)

    Langberg, Henning; Skovgaard, D; Petersen, L J

    1999-01-01

    .e.m. values) for both radioactively labelled substances. 3. PICP concentration decreased in both interstitial peritendinous tissue and arterial blood immediately after exercise, but rose 3-fold from basal 72 h after exercise in the peritendinous tissue (55 +/- 10 microg l-1, mean +/- s.e.m. (rest) to 165...... as determined with microdialysis and using dialysate fibre with a very high molecular mass cut-off. This suggests an adaptation to acute physical loading also in non-bone-related collagen in humans....

  7. Growth Induction and Low-Oxygen Apoptosis Inhibition of Human CD34+ Progenitors in Collagen Gels

    Directory of Open Access Journals (Sweden)

    Daniele Avitabile

    2013-01-01

    Full Text Available Various reports have indicated low survival of injected progenitors into unfavorable environments such as the ischemic myocardium or lower limb tissues. This represents a major bottleneck in stem-cell-based cardiovascular regenerative medicine. Strategies to enhance survival of these cells in recipient tissues have been therefore sought to improve stem cell survival and ensure long-term engraftment. In the present contribution, we show that embedding human cord blood-derived CD34+ cells into a collagen I-based hydrogel containing cytokines is a suitable strategy to promote stem cell proliferation and protect these cells from anoxia-induced apoptosis.

  8. Effect of a Herbal-Leucine mix on the IL-1β-induced cartilage degradation and inflammatory gene expression in human chondrocytes

    Directory of Open Access Journals (Sweden)

    Haqqi Tariq M

    2011-08-01

    Full Text Available Abstract Background Conventional treatments for the articular diseases are often effective for symptom relief, but can also cause significant side effects and do not slow the progression of the disease. Several natural substances have been shown to be effective at relieving the symptoms of osteoarthritis (OA, and preliminary evidence suggests that some of these compounds may exert a favorable influence on the course of the disease. The objective of this study was to investigate the anti-inflammatory/chondroprotective potential of a Herbal and amino acid mixture containing extract of the Uncaria tomentosa, Boswellia spp., Lepidium meyenii and L-Leucine on the IL-1β-induced production of nitric oxide (NO, glycosaminoglycan (GAG, matrix metalloproteinases (MMPs, aggrecan (ACAN and type II collagen (COL2A1 in human OA chondrocytes and OA cartilage explants. Methods Primary OA chondrocytes or OA cartilage explants were pretreated with Herbal-Leucine mixture (HLM, 1-10 μg/ml and then stimulated with IL-1β (5 ng/ml. Effect of HLM on IL-1β-induced gene expression of iNOS, MMP-9, MMP-13, ACAN and COL2A1 was verified by real time-PCR. Estimation of NO and GAG release in culture supernatant was done using commercially available kits. Results HLM tested in these in vitro studies was found to be an effective anti-inflammatory agent, as evidenced by strong inhibition of iNOS, MMP-9 and MMP-13 expression and NO production in IL-1β-stimulated OA chondrocytes (p Leucine mixture (HLM up-regulation of ACAN and COL2A1 expression in IL-1β-stimulated OA chondrocytes was also noted (p Conclusion Our data suggests that HLM could be chondroprotective and anti-inflammatory agent in arthritis, switching chondrocyte gene expression from catabolic direction towards anabolic and regenerative, and consequently this approach may be potentially useful as a new adjunct therapeutic/preventive agent for OA or injury recovery.

  9. Collagen and mature elastic fibre organisation as a function of depth in the human cornea and limbus.

    Science.gov (United States)

    Kamma-Lorger, Christina S; Boote, Craig; Hayes, Sally; Moger, Julian; Burghammer, Manfred; Knupp, Carlo; Quantock, Andrew J; Sorensen, Thomas; Di Cola, Emanuela; White, Nick; Young, Robert D; Meek, Keith M

    2010-03-01

    A network of circumferentially oriented collagen fibrils exists in the periphery of the human cornea, and is thought to be pivotal in maintaining corneal biomechanical stability and curvature. However, it is unknown whether or not this key structural arrangement predominates throughout the entire corneal thickness or exists as a discrete feature at a particular tissue depth; or if it incorporates any elastic fibres and how, with respect to tissue depth, the circumcorneal annulus integrates with the orthogonally arranged collagen of the central cornea. To address these issues we performed a three-dimensional investigation of fibrous collagen and elastin architecture in the peripheral and central human cornea using synchrotron X-ray scattering and non-linear microscopy. This showed that the network of collagen fibrils circumscribing the human cornea is located in the posterior one-third of the tissue and is interlaced with significant numbers of mature elastic fibres which mirror the alignment of the collagen. The orthogonal arrangement of collagen in the central cornea is also mainly restricted to the posterior stromal layers. This information will aid the development of corneal biomechanical models aimed at explaining how normal corneal curvature is sustained and further predicting the outcome of surgical procedures. (c) 2009 Elsevier Inc. All rights reserved.

  10. {mu}-PIXE and SAXS studies at the bone-cartilage interface

    Energy Technology Data Exchange (ETDEWEB)

    Kaabar, W. [Department of Physics, University of Surrey, Guildford GU2 7XH (United Kingdom)], E-mail: w.kaabar@surrey.ac.uk; Gundogdu, O. [Umuttepe Campus, University of Kocaeli, 41380, Kocaeli (Turkey); Laklouk, A. [Food Science Department, Al-Fateh Unversity, Tripoli (Libyan Arab Jamahiriya); Bunk, O. [Swiss Light Source, Paul Scherrer Institute, 5232 Villigen (Switzerland); Pfeiffer, F. [Swiss Light Source, Paul Scherrer Institute, 5232 Villigen (Switzerland); Ecole Polytechnique Federale de Lausanne, 1015 Lausanne (Switzerland); Farquharson, M.J. [Department of Radiography, City University, London EC1V OHB (United Kingdom); Bradley, D.A. [Department of Physics, University of Surrey, Guildford GU2 7XH (United Kingdom)

    2010-04-15

    Micro Proton Induced X-ray Emission ({mu}-PIXE) analysis has been employed herein in investigating and quantifying the distribution of a number of essential elements in thin human diseased articular cartilage sections affected by osteoarthritis (OA). Various cations Ca, P and Zn have been reported to play an important role both in the normal growth and remodelling of articular cartilage and subchondral bone as well as in the degenerative and inflammatory processes associated with the disease; they act as co-factors of a class of enzymes known as metalloproteinases which are believed to be active during the initiation, progress and remodelling processes associated with osteoarthritis. Other important enzymes such as alkaline phosphatase are associated with cartilage mineralization. Synchrotron radiation X-ray fluorescence (SR-XRF) for mapping of elemental distributions in bone and cartilage has also been employed by the present group and others. In the current investigations using the cSAXS beamline at the Swiss light source, Small-Angle X-ray Scattering (SAXS) was carried out on decalcified human articular cartilage to explore the structural and organizational changes of collagen networks in diseased articular cartilage.

  11. Cinnamon extract promotes type I collagen biosynthesis via activation of IGF-I signaling in human dermal fibroblasts.

    Science.gov (United States)

    Takasao, Naoko; Tsuji-Naito, Kentaro; Ishikura, Seiko; Tamura, Azusa; Akagawa, Mitsugu

    2012-02-08

    The breakdown of collagenous networks with aging results in hypoactive changes in the skin. Accordingly, reviving stagnant collagen synthesis can help protect dermal homeostasis against aging. We searched for type I collagen biosynthesis-inducing substances in various foods using human dermal fibroblasts and found that cinnamon extract facilitates collagen biosynthesis. Cinnamon extract potently up-regulated both mRNA and protein expression levels of type I collagen without cytotoxicity. We identified cinnamaldehyde as a major active component promoting the expression of collagen by HPLC and NMR analysis. Since insulin-like growth factor-I (IGF-I) is the most potent stimulator of collagen biosynthesis in fibroblasts, we examined the effect of cinnamaldehyde on IGF-I signaling. Treatment with cinnamaldehyde significantly increased the phosphorylation levels of the IGF-I receptor and its downstream signaling molecules such as insulin receptor substrate-1 and Erk1/2 in an IGF-I-independent manner. These results suggested that cinnamon extract is useful in antiaging treatment of skin.

  12. Biofabricated soft network composites for cartilage tissue engineering.

    Science.gov (United States)

    Bas, Onur; De-Juan-Pardo, Elena M; Meinert, Christoph; D'Angella, Davide; Baldwin, Jeremy G; Bray, Laura J; Wellard, R Mark; Kollmannsberger, Stefan; Rank, Ernst; Werner, Carsten; Klein, Travis J; Catelas, Isabelle; Hutmacher, Dietmar W

    2017-05-12

    Articular cartilage from a material science point of view is a soft network composite that plays a critical role in load-bearing joints during dynamic loading. Its composite structure, consisting of a collagen fiber network and a hydrated proteoglycan matrix, gives rise to the complex mechanical properties of the tissue including viscoelasticity and stress relaxation. Melt electrospinning writing allows the design and fabrication of medical grade polycaprolactone (mPCL) fibrous networks for the reinforcement of soft hydrogel matrices for cartilage tissue engineering. However, these fiber-reinforced constructs underperformed under dynamic and prolonged loading conditions, suggesting that more targeted design approaches and material selection are required to fully exploit the potential of fibers as reinforcing agents for cartilage tissue engineering. In the present study, we emulated the proteoglycan matrix of articular cartilage by using highly negatively charged star-shaped poly(ethylene glycol)/heparin hydrogel (sPEG/Hep) as the soft matrix. These soft hydrogels combined with mPCL melt electrospun fibrous networks exhibited mechanical anisotropy, nonlinearity, viscoelasticity and morphology analogous to those of their native counterpart, and provided a suitable microenvironment for in vitro human chondrocyte culture and neocartilage formation. In addition, a numerical model using the p-version of the finite element method (p-FEM) was developed in order to gain further insights into the deformation mechanisms of the constructs in silico, as well as to predict compressive moduli. To our knowledge, this is the first study presenting cartilage tissue-engineered constructs that capture the overall transient, equilibrium and dynamic biomechanical properties of human articular cartilage.

  13. No donor age effect of human serum on collagen synthesis signaling and cell proliferation of human tendon fibroblasts

    DEFF Research Database (Denmark)

    Bayer, Monika L; Schjerling, Peter; Biskup, Edyta

    2012-01-01

    The aging process of tendon tissue is associated with decreased collagen content and increased risk for injuries. An essential factor in tendon physiology is transforming growth factor-ß1 (TGF-ß1), which is presumed to be reduced systemically with advanced age. The aim of this study was to invest......The aging process of tendon tissue is associated with decreased collagen content and increased risk for injuries. An essential factor in tendon physiology is transforming growth factor-ß1 (TGF-ß1), which is presumed to be reduced systemically with advanced age. The aim of this study...... and elderly donors, and we found no difference in collagen expression when cells were subjected to human serum from elderly versus young donors. In addition, tendon cell proliferation was similar when culture medium was supplemented with serum of different donor age. These findings suggest that factors...... such as the cell intrinsic capacity or the tissue-specific environment rather than systemic circulating factors are important for functional capacity throughout life in human tendon cells....

  14. Introduction of the human pro. cap alpha. 1(I) collagen gene into pro. cap alpha. 1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen

    Energy Technology Data Exchange (ETDEWEB)

    Schnieke, A.; Dziadek, M.; Bateman, J.; Mascara, T.; Harbers, K.; Gelinas, R.; Jaenisch, R.

    1987-02-01

    The Mov-13 mouse strain carries a retroviral insertion in the pro..cap alpha..1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of pro..cap alpha..2 mRNA are synthesized. The authors have introduced genomic clones of either the human or mouse pro..cap alpha..1(I) collagen gene into homozygous cell lines to assess whether the human or mouse pro..cap alpha..1(I) chains can associate with the endogenous mouse pro..cap alpha..2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human ..cap alpha..1 chains and one mouse ..cap alpha..2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both ..cap alpha..1(I) and ..cap alpha..2(I) chains in the human-mouse hybrid molecules were retarded, compared to the ..cap alpha..(I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse ..cap alpha..1 and ..cap alpha..2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human ..cap alpha.. chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow the authors to study the effect specific mutations introduced in transfected pro..cap alpha..1(I) genes have on the synthesis, assembly, and function of collagen I.

  15. Immunohistochemical and biochemical studies on the collagenous proteins of human osteosarcomas.

    Science.gov (United States)

    Ueda, Y; Nakanishi, I

    1989-01-01

    The distribution of type I, II, III, IV, V and VI collagens in 20 cases of osteosarcoma was demonstrated immunohistochemically using monospecific antibodies to different collagen types. In addition, biochemical analysis was made on collagenous proteins synthesized by tumor cells in short-term cultures obtained from seven representative cases and compared with dermal fibroblasts. In osteoblastic areas, most of the tumor osteoid consisted exclusively of type I collagen. Type V collagen was associated in some of them. Type III and type VI collagens were mainly localized in the perivascular fibrous stroma. Cultured tumor cells from osteoblastic osteosarcomas produced type I collagen exclusively and small amount of type V collagen constantly, while the synthetic activity of type III collagen was extremely low. In contrast, fibroblastic areas were characterized by the codistribution of type I, III, VI collagens and chondroblastic areas by type I, V, VI collagens as well as type II. Furthermore, type IV collagen was demonstrated in the stroma, other than the basement membrane region of blood vessels, in fibroblastic, intramedullary well-differentiated and telangiectatic osteosarcomas. In vitro, the production of variable amounts of type IV collagen, which was not detected in cultured dermal fibroblasts, was also recognized in the osteoblastic, fibroblastic, undifferentiated and intramedullary well-differentiated osteosarcomas examined. These findings suggest that the immunohistochemical approach using monospecific antibodies to different collagen types is useful not only in identifying some specific organoid components, such as tumor osteoid, but also in disclosing the biological properties of osteosarcoma cells with diverse differentiation.

  16. Structural response of human corneal and scleral tissues to collagen cross-linking treatment with riboflavin and ultraviolet A light.

    Science.gov (United States)

    Choi, Samjin; Lee, Seung-Chan; Lee, Hui-Jae; Cheong, Youjin; Jung, Gyeong-Bok; Jin, Kyung-Hyun; Park, Hun-Kuk

    2013-09-01

    High success rates in clinical trials on keratoconic corneas suggest the possibility of efficient treatment against myopic progression. This study quantitatively investigated the in vitro ultrastructural effects of a photooxidative collagen cross-linking treatment with photosensitizer riboflavin and UVA light in human corneo-scleral collagen fibrils. A total of 30.8 × 2 mm corneo-scleral strips from donor tissue were sagittally dissected using a scalpel. The five analytic parameters namely fibril density, fibril area, corneo-scleral thickness, fibril diameter, and fibril arrangement were investigated before and after riboflavin-UVA-catalyzed collagen cross-linking treatment. Collagen cross-linking effects were measured at the corneo-scleral stroma and were based on clinical corneal cross-linking procedures. The structural response levels were assessed by histology, digital mechanical caliper measurement, scanning electron microscopy, and atomic force microscopy. Riboflavin-UVA-catalyzed collagen cross-linking treatment led to an increase in the area, density, and diameters of both corneal (110, 112, and 103 %) and scleral (133, 133, and 127 %) stromal collagens. It also led to increases in corneal (107 %) and scleral (105 %) thickness. Collagen cross-linking treatment through riboflavin-sensitized photoreaction may cause structural property changes in the collagen fibril network of the cornea and sclera due to stromal edema and interfibrillar spacing narrowing. These changes were particularly prominent in the sclera. This technique can be used to treat progressive keratoconus in the cornea as well as progressive myopia in the sclera. Long-term collagen cross-linking treatment of keratoconic and myopic progression dramatically improves weakened corneo-scleral tissues.

  17. Endocytic collagen degradation

    DEFF Research Database (Denmark)

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

    2012-01-01

    it crucially important to understand both the collagen synthesis and turnover mechanisms in this condition. Here we show that the endocytic collagen receptor, uPARAP/Endo180, is a major determinant in governing the balance between collagen deposition and degradation. Cirrhotic human livers displayed a marked...

  18. Morphometric computerized analysis on the dentinal tubules and the collagen fibers in the dentine of human permanent teeth.

    Science.gov (United States)

    Marchetti, C; Piacentini, C; Menghini, P

    1992-01-01

    A morphometric analysis has been performed on important components of human dentine using an image computerized analyzer. The dentinal tubule diameter and their area percentage were calculated. Moreover the area percentage of the collagen fibers in the dentinal matrix was measured. These parameters have been evaluated in different areas of the coronal and the radicular dentine in permanent teeth. Measurements have been performed on undecalcified and decalcified teeth and on teeth treated with enzymatic digestion to remove the organic non collagen matrix and to evidentiate the collagen fiber network. The values obtained in different areas of the tooth and in samples submitted to different treatments were evaluated by statistical analysis. Dentinal tubule diameter and area percentage significatively decrease from the inner to the peripheral dentine both in the undecalcified teeth as in the decalcified ones and in the samples undergone to enzymatic digestion. The collagen fiber percentage in the organic matrix is significatively lower in the mantle dentine.

  19. Growth hormone stimulates the collagen synthesis in human tendon and skeletal muscle without affecting myofibrillar protein synthesis

    DEFF Research Database (Denmark)

    Doessing, Simon; Heinemeier, Katja; Holm, Lars

    2010-01-01

    matrix collagen synthesis in skeletal muscle and tendon, but without any effect upon myofibrillar protein synthesis. The results suggest that GH is more important in strengthening the matrix tissue than for muscle cell hypertrophy in adult human musculotendinous tissue.......In skeletal muscle and tendon the extracellular matrix confers important tensile properties and is crucially important for tissue regeneration after injury. Musculoskeletal tissue adaptation is influenced by mechanical loading, which modulates the availability of growth factors, including growth...... young individuals. rhGH administration caused an increase in serum GH, serum IGF-I, and IGF-I mRNA expression in tendon and muscle. Tendon collagen I mRNA expression and tendon collagen protein synthesis increased by 3.9-fold and 1.3-fold, respectively (P muscle collagen I m...

  20. Ascorbic acid enhances the expression of type 1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts.

    Science.gov (United States)

    Kishimoto, Yuki; Saito, Norikatsu; Kurita, Katsumi; Shimokado, Kentaro; Maruyama, Naoki; Ishigami, Akihito

    2013-01-11

    Ascorbic acid (AA) is essential for collagen biosynthesis as a cofactor for prolyl and lysyl hydroxylase and as a stimulus for collagen gene expression. Many studies have evaluated the relationship between AA and collagen expression in short- and long-term effects on cells after a single administration of AA into the culture medium. However, no such study has monitored in detail the stability of AA in medium or the alterations of intracellular AA levels during a protracted interval. Therefore, we examined here intracellular AA levels and stability throughout its exposure to human skin fibroblasts in vitro. Moreover, we determined the effects on type 1 and type 4 collagen and sodium-dependent vitamin C transporter (SVCT) gene expression when medium containing 100 μM AA was replaced every 24h for 5 days to avoid depletion of AA. Throughout this long-term culture, intracellular AA levels remained constant; the expression of type 1 and type 4 collagens and SVCT2 mRNA was enhanced, and type 1 procollagen synthesis increased. Thus, these results indicate that human skin fibroblasts exposed to AA over time had rising levels of type 1/type 4 collagens and SVCT2 mRNA expression and type 1 procollagen synthesis. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  1. The collagen matrix of the human trabecular meshwork is an extension of the novel pre-Descemet's layer (Dua's layer).

    Science.gov (United States)

    Dua, Harminder S; Faraj, Lana A; Branch, Matthew J; Yeung, Aaron M; Elalfy, Mohamed S; Said, Dalia G; Gray, Trevor; Lowe, James

    2014-05-01

    The trabecular meshwork (TM) located at the angle of the anterior chamber of the eye contributes to aqueous drainage. A novel layer in the posterior part of the human cornea has recently been reported (the pre-Descemet's layer (Dua's layer (PDL)). We examined the peripheral part of this layer in relation to the origin of the TM. The PDL and TM of 19 human donor eyes and one exenterated sample were studied. Samples were examined by light and electron microscopy (EM) for tissue architecture and by immunohistology for four matricellular proteins, five collagen types and CD34. EM revealed that beams of collagen emerged from the periphery of PDL on the anterior surface of the Descemet's membrane and divided and subdivided to continue as the beams of the TM. Long-spacing collagen was seen in the PDL and TM. Trabecular cells (CD34-ve) associated with basement membrane were seen in the peripheral part of the PDL and corresponded to the start of the separation of the collagen lamellae of PDL. Collagen VI was present continuously in PDL and extended into the TM. Matricellular proteins were seen predominantly in the TM with only laminin extending into the periphery of PDL. This study provides an insight into the origins of the collagen core of the TM as an extension of the PDL of the cornea. This finding adds to the knowledge base of the TM and cornea and has the potential to impact future research into the TM and glaucoma.

  2. Aging is associated with increased collagen type IV accumulation in the basal lamina of human cerebral microvessels

    Directory of Open Access Journals (Sweden)

    Danek Adrian

    2004-09-01

    Full Text Available Abstract Background Microvascular alterations contribute to the development of stroke and vascular dementia. The goal of this study was to evaluate age and hypertension related changes of the basal lamina in cerebral microvessels of individuals, who died from non-cerebral causes. Results We examined 27 human brains: 11 young and 16 old patients. Old patients were divided into two subgroups, those with hypertension (n = 8 and those without hypertension (n = 8. Basal lamina changes of the cerebral microvessels were determined in the putamen using antibodies against collagen type IV and by quantitative analysis of vessel number, total stained area of collagen, thickness of the vessel wall and lumen, and relative staining intensity using immunofluorescence. The total number of collagen positive vessels per microscopic field was reduced in old compared to young subjects (12.0+/-0.6 vs. 15.1+/-1.2, p = 0.02. The relative collagen content per vessel (1.01+/-0.06 vs. 0.76+/-0.05, p = 0.01 and the relative collagen intensity (233.1+/-4.5 vs. 167.8+/-10.6, p Conclusions The present data show age-related changes of the cerebral microvessels in sections of human putamen for the first time. Due to the accumulation of collagen, microvessels thicken and show a reduction in their lumen. Besides this, the number of vessels decreases. These findings might represent a precondition for the development of vascular cognitive impairment. However, hypertension was not proven to modulate these changes.

  3. Aging is associated with increased collagen type IV accumulation in the basal lamina of human cerebral microvessels.

    Science.gov (United States)

    Uspenskaia, Olga; Liebetrau, Martin; Herms, Jochen; Danek, Adrian; Hamann, Gerhard F

    2004-09-24

    Microvascular alterations contribute to the development of stroke and vascular dementia. The goal of this study was to evaluate age and hypertension related changes of the basal lamina in cerebral microvessels of individuals, who died from non-cerebral causes. We examined 27 human brains: 11 young and 16 old patients. Old patients were divided into two subgroups, those with hypertension (n = 8) and those without hypertension (n = 8). Basal lamina changes of the cerebral microvessels were determined in the putamen using antibodies against collagen type IV and by quantitative analysis of vessel number, total stained area of collagen, thickness of the vessel wall and lumen, and relative staining intensity using immunofluorescence. The total number of collagen positive vessels per microscopic field was reduced in old compared to young subjects (12.0+/-0.6 vs. 15.1+/-1.2, p = 0.02). The relative collagen content per vessel (1.01+/-0.06 vs. 0.76+/-0.05, p = 0.01) and the relative collagen intensity (233.1+/-4.5 vs. 167.8+/-10.6, p hypertensive and non-hypertensive patients. The present data show age-related changes of the cerebral microvessels in sections of human putamen for the first time. Due to the accumulation of collagen, microvessels thicken and show a reduction in their lumen. Besides this, the number of vessels decreases. These findings might represent a precondition for the development of vascular cognitive impairment. However, hypertension was not proven to modulate these changes.

  4. STAT6-Dependent Collagen Synthesis in Human Fibroblasts Is Induced by Bovine Milk.

    Directory of Open Access Journals (Sweden)

    Stefan Kippenberger

    Full Text Available Since the domestication of the urus, 10.000 years ago, mankind utilizes bovine milk for different purposes. Besides usage as a nutrient also the external application of milk on skin has a long tradition going back to at least the ancient Aegypt with Cleopatra VII as a great exponent. In order to test whether milk has impact on skin physiology, cultures of human skin fibroblasts were exposed to commercial bovine milk. Our data show significant induction of proliferation by milk (max. 2,3-fold, EC50: 2,5% milk without toxic effects. Surprisingly, bovine milk was identified as strong inducer of collagen 1A1 synthesis at both, the protein (4-fold, EC50: 0,09% milk and promoter level. Regarding the underlying molecular pathways, we show functional activation of STAT6 in a p44/42 and p38-dependent manner. More upstream, we identified IGF-1 and insulin as key factors responsible for milk-induced collagen synthesis. These findings show that bovine milk contains bioactive molecules that act on human skin cells. Therefore, it is tempting to test the herein introduced concept in treatment of atrophic skin conditions induced e.g. by UV light or corticosteroids.

  5. STAT6-Dependent Collagen Synthesis in Human Fibroblasts Is Induced by Bovine Milk.

    Science.gov (United States)

    Kippenberger, Stefan; Zöller, Nadja; Kleemann, Johannes; Müller, Jutta; Kaufmann, Roland; Hofmann, Matthias; Bernd, August; Meissner, Markus; Valesky, Eva

    2015-01-01

    Since the domestication of the urus, 10.000 years ago, mankind utilizes bovine milk for different purposes. Besides usage as a nutrient also the external application of milk on skin has a long tradition going back to at least the ancient Aegypt with Cleopatra VII as a great exponent. In order to test whether milk has impact on skin physiology, cultures of human skin fibroblasts were exposed to commercial bovine milk. Our data show significant induction of proliferation by milk (max. 2,3-fold, EC50: 2,5% milk) without toxic effects. Surprisingly, bovine milk was identified as strong inducer of collagen 1A1 synthesis at both, the protein (4-fold, EC50: 0,09% milk) and promoter level. Regarding the underlying molecular pathways, we show functional activation of STAT6 in a p44/42 and p38-dependent manner. More upstream, we identified IGF-1 and insulin as key factors responsible for milk-induced collagen synthesis. These findings show that bovine milk contains bioactive molecules that act on human skin cells. Therefore, it is tempting to test the herein introduced concept in treatment of atrophic skin conditions induced e.g. by UV light or corticosteroids.

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

    Science.gov (United States)

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

    2017-08-01

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

  7. Adherens junction proteins are expressed in collagen corneal equivalents produced in vitro with human cells.

    Science.gov (United States)

    Giasson, Claude J; Deschambeault, Alexandre; Carrier, Patrick; Germain, Lucie

    2014-01-01

    To test whether adherens junction proteins are present in the epithelium and the endothelium of corneal equivalents. Corneal cell types were harvested from human eyes and grown separately. Stromal equivalents were constructed by seeding fibroblasts into a collagen gel on which epithelial and endothelial cells were added on each side. Alternatively, bovine endothelial cells were used. At maturity, sections of stromal equivalents were processed for Masson's trichrome or indirect immunofluorescence using antibodies against pan-, N-, or E-cadherins or α- or β-catenins. Alternatively, stromal equivalents were dissected, to separate the proteins from the epithelium, endothelium, and stroma with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots of the transferred proteins exposed to these primary antibodies were detected with chemiluminescence. Native corneas were processed similarly. Three or four layers of epithelial cells reminiscent of the native cornea (basal cuboidal and superficial flatter cells) lay over a stromal construct containing fibroblastic cells under which an endothelium is present. Western blots and indirect immunofluorescence revealed that, similarly to the native cornea, the epithelium reacted positively to antibodies against catenins (α and β) and E-cadherin. The endothelium of corneal constructs, whether of human or bovine origin, reacted mildly to catenins and N-cadherin. This collagen-based corneal equivalent simulated the native cornea. Cells from the epithelial and endothelial layers expressed adherens junction proteins, indicating the presence of cell-cell contacts and the existence of polarized morphology of these layers over corneal equivalents.

  8. Lysylhydroxylation and non-reducible crosslinking of human supraspinatus tendon collagen: changes with age and in chronic rotator cuff tendinitis.

    Science.gov (United States)

    Bank, R A; TeKoppele, J M; Oostingh, G; Hazleman, B L; Riley, G P

    1999-01-01

    To investigate age related and site specific variations in turnover and chemistry of the collagen network in healthy tendons as well as the role of collagen remodelling in the degeneration of the supraspinatus tendon (ST-D) in rotator cuff tendinitis. Collagen content and the amount of hydroxylysine (Hyl), hydroxy-lysylpyridinoline (HP), lysylpyridinoline (LP), and the degree of non-enzymatic glycation (pentosidine) were investigated in ST-D and in normal human supraspinatus (ST-N) and biceps brachii tendons (BT-N) by high-performance liquid chromatography. In BT-N, tendons that served as control tissue as it shows rarely matrix abnormalities, pentosidine levels rise linearly with age (20-90 years), indicating little tissue remodelling (resulting in an undisturbed accumulation of pentosidine). A similar accumulation was observed in ST-N up to 50 years. At older ages, little pentosidine accumulation was observed and pentosidine levels showed large interindividual variability. This was interpreted as remodelling of collagen in normal ST after age 50 years because of microruptures (thus diluting old collagen with newly synthesised collagen). All degenerate ST samples showed decreased pentosidine levels compared with age matched controls, indicating extensive remodelling in an attempt to repair the tendon defect. Collagen content and the amount of Hyl, HP, and LP of ST-N and BT-N did not change with age. With the exception of collagen content, which did not differ, all parameters were significantly (p collagen content and had higher Hyl, HP, and LP levels than ST-N (p collagen. On the other hand, the clearly different profile of post-translational modifications in ST-D indicates that the newly deposited collagen network in degenerated tendons is qualitatively different. It is concluded that in ST-D the previously functional and carefully constructed matrix is replaced by aberrant collagen. This may result in a mechanically less stable tendon; as the supraspinatus is

  9. Computer-aided diagnosis for phase-contrast X-ray computed tomography: quantitative characterization of human patellar cartilage with high-dimensional geometric features.

    Science.gov (United States)

    Nagarajan, Mahesh B; Coan, Paola; Huber, Markus B; Diemoz, Paul C; Glaser, Christian; Wismüller, Axel

    2014-02-01

    Phase-contrast computed tomography (PCI-CT) has shown tremendous potential as an imaging modality for visualizing human cartilage with high spatial resolution. Previous studies have demonstrated the ability of PCI-CT to visualize (1) structural details of the human patellar cartilage matrix and (2) changes to chondrocyte organization induced by osteoarthritis. This study investigates the use of high-dimensional geometric features in characterizing such chondrocyte patterns in the presence or absence of osteoarthritic damage. Geometrical features derived from the scaling index method (SIM) and statistical features derived from gray-level co-occurrence matrices were extracted from 842 regions of interest (ROI) annotated on PCI-CT images of ex vivo human patellar cartilage specimens. These features were subsequently used in a machine learning task with support vector regression to classify ROIs as healthy or osteoarthritic; classification performance was evaluated using the area under the receiver-operating characteristic curve (AUC). SIM-derived geometrical features exhibited the best classification performance (AUC, 0.95 ± 0.06) and were most robust to changes in ROI size. These results suggest that such geometrical features can provide a detailed characterization of the chondrocyte organization in the cartilage matrix in an automated and non-subjective manner, while also enabling classification of cartilage as healthy or osteoarthritic with high accuracy. Such features could potentially serve as imaging markers for evaluating osteoarthritis progression and its response to different therapeutic intervention strategies.

  10. Microwave treatment of xenogeneic cartilage transplants

    NARCIS (Netherlands)

    Visser, C. E.; Boon, M. E.; Visser, P. E.; Kok, L. P.

    1989-01-01

    Human rib cartilage was irradiated with microwaves according to six different methods and transplanted into rabbits. Untreated rib cartilage preserved in Cialit served as a control. After 12 and 40 wk of implantation, the microscopic appearance of these xenogeneic cartilage transplants was given a

  11. Type I collagen as an extracellular matrix for the in vitro growth of human small intestinal epithelium.

    Directory of Open Access Journals (Sweden)

    Ziyad Jabaji

    Full Text Available We previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells.Human small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry.Collagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages.Type I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet

  12. Changes in the epigenetic status of the SOX-9 promoter in human osteoarthritic cartilage.

    Science.gov (United States)

    Kim, Kyung-Il; Park, Youn-Soo; Im, Gun-Il

    2013-05-01

    Whether osteoarthritis (OA) is associated with alterations in the epigenetic status of anabolic factors is largely unknown. To answer the question, we investigated the DNA methylation and histone modification of SOX-9 gene promoter, a typical anabolic gene, in the articular cartilage from nine patients with femoral neck fractures without OA and from nine hip OA patients. Methylation-specific PCR (MSP) and bisulfite sequencing analysis (BSQ) showed that the methylation of SOX-9 promoter was increased in OA cartilage compared to normal cartilage. The decreased SOX-9 gene and protein expression in OA chondrocytes was reversed by the treatment of 5-azacytidine (5-AzaC), the demethylating agent. Methylation of SOX-9 proximal promoters reduced the binding affinity of transcription factors CCAAT-binding factor/nuclear factor-Y and cyclic adenosine monophosphate (cAMP) response element-binding. There was a significant increase in H3K9 and H3K27 trimethylation and a significant decrease in the acetylation of H3K9, 15, 18, 23, and 27 at SOX-9 promoters in OA chondrocytes. These findings suggest that hip OA is associated with a change in the epigenetic status of SOX-9 promoter, including increased DNA methylation and altered histone modification. Copyright © 2013 American Society for Bone and Mineral Research.

  13. Asiaticoside induces cell proliferation and collagen synthesis in human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Linda Yuliati

    2015-12-01

    Asiaticoside induces HDF proliferation and type I and III collagen synthesis in a time- and dose-dependent pattern. Asiaticoside has a similar effect as retinoic acid on type I and type III collagen synthesis.

  14. The extracellular matrix is an integrated unit: ultrastructural localization of collagen types I, III, IV, V, VI, fibronectin, and laminin in human term placenta.

    Science.gov (United States)

    Amenta, P S; Gay, S; Vaheri, A; Martinez-Hernandez, A

    1986-06-01

    The human term placenta is used extensively as a source of extracellular matrix components. To elucidate the tissue distribution and interrelationships of seven of these components, monospecific antibodies directed against collagen types I, III, IV, V, VI, fibronectin, and laminin were reacted with human term placenta and studied by light and electron immunohistochemistry. Type I collagen was the basic structural unit of human term placenta, present as 30-35 nm, cross-banded fibers, often in the form of large fiber bundles. Type III collagen was present as thin 10-15 nm, beaded fibers often forming a meshwork which encased type I collagen fibers. Types V and VI collagen were present as 6-10 nm filaments, often closely associated with types I and III collagen. Type VI collagen also coated collagen fibers of all diameters, enhancing their periodicity, providing a staining pattern often similar to that observed with anti-fibronectin antibodies. Fibronectin was present in both maternal and fetal plasma and throughout the stroma of the chorionic villus, as both free filaments and coating collagen fibers. Basement membranes contained laminin and type IV collagen, but no fibronectin. In summary, the non-basement membrane proteins studied often codistributed with type I collagen, between and apparently attached to fibers, suggesting that they may act as binding proteins, linking type I fibers and bundles, to themselves and to other structures.

  15. Regeneration of Cartilage in Human Knee Osteoarthritis with Autologous Adipose Tissue-Derived Stem Cells and Autologous Extracellular Matrix

    Directory of Open Access Journals (Sweden)

    Jaewoo Pak

    2016-08-01

    Full Text Available This clinical case series demonstrates that percutaneous injections of autologous adipose tissue-derived stem cells (ADSCs and homogenized extracellular matrix (ECM in the form of adipose stromal vascular fraction (SVF, along with hyaluronic acid (HA and platelet-rich plasma (PRP activated by calcium chloride, could regenerate cartilage-like tissue in human knee osteoarthritis (OA patients. Autologous lipoaspirates were obtained from adipose tissue of the abdominal origin. Afterward, the lipoaspirates were minced to homogenize the ECM. These homogenized lipoaspirates were then mixed with collagenase and incubated. The resulting mixture of ADSCs and ECM in the form of SVF was injected, along with HA and PRP activated by calcium chloride, into knees of three Korean patients with OA. The same affected knees were reinjected weekly with additional PRP activated by calcium chloride for 3 weeks. Pretreatment and post-treatment magnetic resonance imaging (MRI data, functional rating index, range of motion (ROM, and pain score data were then analyzed. All patients' MRI data showed cartilage-like tissue regeneration. Along with MRI evidence, the measured physical therapy outcomes in terms of ROM, subjective pain, and functional status were all improved. This study demonstrates that percutaneous injection of ADSCs with ECM contained in autologous adipose SVF, in conjunction with HA and PRP activated by calcium chloride, is a safe and potentially effective minimally invasive therapy for OA of human knees.

  16. Exercise-dependent IGF-I, IGFBPs, and type I collagen changes in human peritendinous connective tissue determined by microdialysis

    DEFF Research Database (Denmark)

    Olesen, Jens L; Heinemeier, Katja M; Gemmer, Carsten

    2007-01-01

    and exercise groups after 48 h (P human peritendinous tissue in response to prolonged mechanical loading with part of the increase due to trauma from the sampling......Microdialysis studies indicate that mechanical loading of human tendon during exercise elevates type I collagen production in tendon. However, the possibility that the insertion of microdialysis fibers per se may increase the local collagen production due to trauma has not been explored. Insulin......-terminal propeptide (PICP) and COOH-terminal telopeptide of type I collagen] were measured by microdialysis in peritendinous tissue of the human Achilles tendon in an exercise group (performing a 36-km run, n = 6) and a control group (no intervention, n = 6). An increase in local PICP concentration was seen in both...

  17. Quantifying Baseline Fixed Charge Density in Healthy Human Cartilage Endplate: A Two-point Electrical Conductivity Method.

    Science.gov (United States)

    Wu, Yongren; Cisewski, Sarah E; Sun, Yi; Damon, Brooke J; Sachs, Barton L; Pellegrini, Vincent D; Slate, Elizabeth H; Yao, Hai

    2017-09-01

    Regional measurements of fixed charge densities (FCDs) of healthy human cartilage endplate (CEP) using a two-point electrical conductivity approach. The aim of this study was to determine the FCDs at four different regions (central, lateral, anterior, and posterior) of human CEP, and correlate the FCDs with tissue biochemical composition. The CEP, a thin layer of hyaline cartilage on the cranial and caudal surfaces of the intervertebral disc, plays an irreplaceable role in maintaining the unique physiological mechano-electrochemical environment inside the disc. FCD, arising from the carboxyl and sulfate groups of the glycosaminoglycans (GAG) in the extracellular matrix of the disc, is a key regulator of the disc ionic and osmotic environment through physicochemical and electrokinetic effects. Although FCDs in the annulus fibrosus (AF) and nucleus pulposus (NP) have been reported, quantitative baseline FCD in healthy human CEP has not been reported. CEP specimens were regionally isolated from human lumbar spines. FCD and ion diffusivity were concurrently investigated using a two-point electrical conductivity method. Biochemical assays were used to quantify regional GAG and water content. FCD in healthy human CEP was region-dependent, with FCD lowest in the lateral region (P = 0.044). Cross-region FCD was 30% to 60% smaller than FCD in NP, but similar to the AF and articular cartilage (AC). CEP FCD (average: 0.12 ± 0.03 mEq/g wet tissue) was correlated with GAG content (average: 31.24 ± 5.06 μg/mg wet tissue) (P = 0.005). In addition, the cross-region ion diffusivity in healthy CEP (2.97 ± 1.00 × 10 cm/s) was much smaller than the AF and NP. Healthy human CEP acts as a biomechanical interface, distributing loads between the bony vertebral body and soft disc tissues and as a gateway impeding rapid solute diffusion through the disc. N/A.

  18. Type XV collagen exhibits a widespread distribution in human tissues but a distinct localization in basement membrane zones.

    Science.gov (United States)

    Myers, J C; Dion, A S; Abraham, V; Amenta, P S

    1996-12-01

    The collagen family of proteins consists of 19 types encoded by 33 genes. One of the more recently discovered collagens is the alpha1 chain of type XV. Type XV collagen is comprised of a 577-amino-acid, highly interrupted, triple-helical region that is flanked by amino and carboxy noncollagenous domains of 555 and 256 residues, respectively. To address questions of where this collagen is localized and what its function may entail, we produced a bacteria-expressed recombinant protein representing the first half of the type XV collagen carboxy-terminal domain in order to generate highly specific polyclonal antisera. Immunoscreening of an expression library with the affinity-purified antibody revealed three clones coding for part of the type XV triple-helical region and the entire noncollagenous carboxy-terminus. Western blot analysis of human tissue homogenates identified a 116-kDa collagenase-sensitive protein and a 27-kDa collagenase-resistant fragment, whose electrophoretic mobilities were unchanged in the presence and absence of reductant. Northern blot hybridization to human tissue RNAs indicated that type XV has a prevalent and widespread distribution. To determine the precise localization of type XV collagen, immunohistochemical analyses at the light- and electron-microscopic levels were performed. Type XV exhibited a surprisingly restricted and uniform presence in many human tissues as evidenced by a strong association with vascular, neuronal, mesenchymal, and some epithelial basement membrane zones. These data suggest that type XV collagen may function in some manner to adhere basement membrane to the underlying connective tissue stroma.

  19. Human platelet lysate improves human cord blood derived ECFC survival and vasculogenesis in three dimensional (3D) collagen matrices.

    Science.gov (United States)

    Kim, Hyojin; Prasain, Nutan; Vemula, Sasidhar; Ferkowicz, Michael J; Yoshimoto, Momoko; Voytik-Harbin, Sherry L; Yoder, Mervin C

    2015-09-01

    Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Since diminished ECFC survival is known to dampen the vasculogenic response in vivo, we tested how long implanted ECFC survive and generate vessels in three-dimensional (3D) type I collagen matrices in vitro and in vivo. We hypothesized that human platelet lysate (HPL) would promote cell survival and enhance vasculogenesis in the 3D collagen matrices. We report that the percentage of ECFC co-cultured with HPL that were alive was significantly enhanced on days 1 and 3 post-matrix formation, compared to ECFC alone containing matrices. Also, co-culture of ECFC with HPL displayed significantly more vasculogenic activity compared to ECFC alone and expressed significantly more pro-survival molecules (pAkt, p-Bad and Bcl-xL) in the 3D collagen matrices in vitro. Treatment with Akt1 inhibitor (A-674563), Akt2 inhibitor (CCT128930) and Bcl-xL inhibitor (ABT-263/Navitoclax) significantly decreased the cell survival and vasculogenesis of ECFC co-cultured with or without HPL and implicated activation of the Akt1 pathway as the critical mediator of the HPL effect on ECFC in vitro. A significantly greater average vessel number and total vascular area of human CD31(+) vessels were present in implants containing ECFC and HPL, compared to the ECFC alone implants in vivo. We conclude that implantation of ECFC with HPL in vivo promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro.

    Science.gov (United States)

    McKay, Tina B; Hjortdal, Jesper; Priyadarsini, Shrestha; Karamichos, Dimitrios

    2017-01-01

    Keratoconus (KC) is a progressive corneal ectasia linked to thinning of the central cornea. Hard contact lenses, rigid gas permeable lenses, and scleral lenses are the primary treatment modalities for early to mid- stages of KC to correct refractive error and astigmatism that develops as a result of an irregular corneal structure. These treatments are associated with significant drawbacks, including reduced availability of the tear film and oxygen to the corneal epithelium and stroma. However, it remains unknown whether hypoxia affects corneal integrity in the KC pathobiology. A number of studies have associated elevated oxidative stress with KC both in vitro and ex vivo. We hypothesized that KC-derived corneal fibroblasts are more susceptible to hypoxia-induced oxidative stress compared to healthy controls leading to exacerbation of corneal thinning in KC. This study investigated the effects of hypoxia on ECM secretion, assembly, and matrix metalloproteinase (MMP) expression in human corneal fibroblasts from healthy controls (HCFs) and KC patients (HKCs) in vitro. HCFs and HKCs were cultured in 3D constructs for 3 weeks and maintained or transferred to normoxic (21% O2) or hypoxic (2% O2) conditions, respectively, for 1 additional week. At the 4 week time-point, constructs were isolated and probed for Collagen I, III, and V, keratocan and MMP-1, -2, -3, -9, and -13, as well as hypoxia markers, hypoxia inducible factor-1α and lactoferrin. Conditioned media was also collected and probed for Collagen I, III, and V by Western blot. Thickness of the ECM assembled by HCFs and HKCs was measured using immunofluorescence microscopy. Results showed that hypoxia significantly reduced Collagen I secretion in HKCs, as well as upregulated the expression of MMP-1 and -2 with no significant effects on MMP-3, -9, or -13. ECM thickness was reduced in both cell types following 1 week in a low oxygen environment. Our study shows that hypoxia influences collagen and MMP expression by

  1. Collagen mineralization in human aortic valve stenosis: a field emission scanning electron microscopy and energy dispersive spectroscopy analysis.

    Science.gov (United States)

    Perrotta, Ida; Davoli, Mariano

    2014-08-01

    Abstract Calcific aortic stenosis is a slowly progressive disorder characterized by an important extracellular matrix remodeling with fibrosis and massive deposition of minerals (primarily calcium) in the valve leaflet. The main structural components of human aortic valve are the large, thick collagen bundles that withstand the diastolic loading. Collagen has been studied in a number of reports that aim to clarify the mechanisms underlying the structural deterioration of heart valve substitutes, however to date, little is known regarding the morphological interaction between collagen and mineral crystals in the calcifying tissue of native aortic valve. Here, we have analyzed a total of 12 calcified native aortic valves by using scanning electron microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX) to depict the morphological appearance of mineralized collagen and to determine the location of calcium phosphate minerals in the collagen matrix of the valve cusp. Our results demonstrate that crystals probably nucleate and grow in the interior of the collagen fibers in the absence of surface events.

  2. Cartilage tissue engineering using electrospun PCL nanofiber meshes and MSCs.

    Science.gov (United States)

    Alves da Silva, M L; Martins, A; Costa-Pinto, A R; Costa, P; Faria, S; Gomes, M; Reis, R L; Neves, N M

    2010-12-13

    Mesenchymal stem cells (MSCs) have been recognized for their ability to differentiate into cells of different tissues such as bone, cartilage, or adipose tissue, and therefore are of great interest for potential therapeutic strategies. Adherent, colony-forming, fibroblastic cells were isolated from human bone marrow aspirates, from patients undergoing knee arthroplasties, and the MSCs phenotype characterized by flow cytometry. Afterward, cells were seeded onto electrospun polycaprolactone nanofiber meshes and cultured in a multichamber flow perfusion bioreactor to determine their ability to produce cartilagineous extracellular matrix. Results indicate that the flow perfusion bioreactor increased the chondrogenic differentiation of hBM-MSCs, as confirmed either by morphological and RT-PCR analysis. Cartilage-related genes such as aggrecan, collagen type II, and Sox9 were expressed. ECM deposition was also detected by histological procedures. Collagen type II was present in the samples, as well as collagen type I. Despite no statistically significant values being obtained for gene expression, the other results support the choice of the bioreactor for this type of culture.

  3. Alignment and cell-matrix interactions of human corneal endothelial cells on nanostructured collagen type I matrices.

    Science.gov (United States)

    Gruschwitz, Rita; Friedrichs, Jens; Valtink, Monika; Franz, Clemens M; Müller, Daniel J; Funk, Richard H W; Engelmann, Katrin

    2010-12-01

    To use nanoscopically defined, two-dimensional matrices assembled from aligned collagen type I fibrils as a sheet substratum for in vitro cultivation of human corneal endothelial cells (HCECs). To assess the effect of matrix architecture on HCEC morphology and to characterize integrin-mediated HCEC-matrix interaction. Cell alignment and cell-matrix interactions of primary HCECs and three different immortalized HCEC populations on native and UV-cross-linked collagen type I matrices were examined by time-lapse microscopy. Specific integrin α(2)β(1) binding to the collagen matrix was demonstrated using a function-blocking α(2) antibody. Integrin α(2) subunit expression levels of the four HCEC populations were analyzed by Western blot analysis. All HCEC populations aligned along the oriented collagen fibrils. Primary HCECs and, to a lesser extent, the other tested HCEC populations exerted high traction forces, leading to progressive matrix destruction. Cross-linking of the collagen matrices considerably increased matrix stability. Integrin subunit α(2) expression levels of the four cell types correlated with the degree of cell alignment and exertion of traction forces. In turn, blocking integrin subunit α(2) reduced cell alignment and prevented matrix destruction. HCECs align directionally along parallel arrays of collagen type I fibrils. The interactions of HCECs with collagen type I are primarily mediated by integrin α(2)β(1). Integrin subunit α(2) levels correlate with matrix contraction and subsequent destruction. Sustained cultivation of HCECs on ultrathin collagen matrices thus requires matrix cross-linking and moderate integrin α(2)β(1) expression levels.

  4. Age-dependent changes of the immunohistochemical distribution of various collagen types and structural glycoproteins in the human uterine tube.

    Science.gov (United States)

    Schultka, R; Göpel, C; Schuppan, D; Schmidt, T

    1993-12-01

    This immunohistochemical investigation deals with the age-dependent localization and distribution of types I, III, IV, V, and VI collagen and the structural glycoproteins undulin, fibronectin, laminin, tenascin, and vitronectin in the connective tissue of the human uterine tube. The stroma of this oviductal region consisted of all collagen types. Collagen types I and VI were distributed throughout the connective tissue of the mucosa reaching the basal membrane. The findings suggest that the amount of these collagen types and type III collagen increases in relation to age, since the coarser fibres of the mucosal stroma in the uterine tubes of older women were strongly labelled by immunohistochemistry. The pattern of undulin reactivity was similar to that of types I and VI collagen. The exact quantitative proportions of age-related oviductal changes for types I, III, and VI as well as of undulin are still unknown. Type V collagen was associated with a fine fibre meshwork in the mucosal stroma. The fibres reached the subepithelial zone which appeared membrane-like. The location of type V collagen-associated fibres and aldehyde fuchsin-positive fibres characterized in our previous studies appears to be identical. Moreover, the structural glycoproteins undulin, fibronectin, laminin, tenascin, and vitronectin were detected in the mucosal stroma. The staining of fibronectin was less pronounced than that of undulin. Laminin was located in the zone of the basal membrane, whereas tenascin was mainly found in the mucosal vessels. Contrary to these findings, tenascin showed a unique distribution in the region near the basis of the mucosal folds in the isthmic part. Vitronectin could be observed in the same region of the isthmic part of uterine tubes obtained from younger women. However, the zonal localization of vitronectin reactivity was absent in the isthmic part of older women.

  5. The Role of Two Different Collagen Membranes for Dehiscence Defect Around Implants in Humans.

    Science.gov (United States)

    Lee, Dong-Woon; Kim, Kyeong-Taek; Joo, Yon-Soo; Yoo, Mi-Kyung; Yu, Jeoung-A; Ryu, Jae-Jun

    2015-08-01

    The aim of this study was to elucidate the role of 2 types of collagen membranes (cross-linked vs noncross-linked) used in conjunction with autogenous or allogenic bone followed by xenogeneic bone particles for dehiscence defect around implants in humans. Experimental groups were divided into 2 groups: Group CL (cross-linked, Ossix Plus, n = 24 implants, 16 patients) and Group NCL (noncross-linked, Bio-Gide, n = 25 implants, 18 patients). At the time of implant insertion and uncovery surgery, measurements of the dehiscence bony height, width, and surface area were made. Before applying the membrane to defects, guided bone regeneration was performed. Because it is difficult to measure the degree of exposure, early exposed cases were excluded from the result analysis. The mean percentage gain of the dehiscence defect and the mean marginal bone reduction value of follow-up radiograph did not show statistically significant differences between the 2 groups. Both membranes exhibited satisfactory results on dehiscence defects. As a result, our authors concluded the success of guided bone regeneration was performed simultaneously for dehiscence defects around the implant, regardless whether collagen membranes were cross-linked or noncross-linked.

  6. Collagen-Hydroxyapatite Scaffolds Induce Human Adipose Derived Stem Cells Osteogenic Differentiation In Vitro.

    Directory of Open Access Journals (Sweden)

    Giovanna Calabrese

    Full Text Available Mesenchymal stem cells (MSCs play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I, osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects.

  7. Hypoxia-Induced Collagen Synthesis of Human Lung Fibroblasts by Activating the Angiotensin System

    Directory of Open Access Journals (Sweden)

    Shan-Shan Liu

    2013-12-01

    Full Text Available The exact molecular mechanism that mediates hypoxia-induced pulmonary fibrosis needs to be further clarified. The aim of this study was to explore the effect and underlying mechanism of angiotensin II (Ang II on collagen synthesis in hypoxic human lung fibroblast (HLF cells. The HLF-1 cell line was used for in vitro studies. Angiotensinogen (AGT, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AT1R and angiotensin II type 2 receptor (AT2R expression levels in human lung fibroblasts were analysed using real-time polymerase chain reaction (RT-PCR after hypoxic treatment. Additionally, the collagen type I (Col-I, AT1R and nuclear factor κappaB (NF-κB protein expression levels were detected using Western blot analysis, and NF-κB nuclear translocation was measured using immunofluorescence localization analysis. Ang II levels in HLF-1 cells were measured with an enzyme-linked immunosorbent assay (ELISA. We found that hypoxia increased Col-I mRNA and protein expression in HLF-1 cells, and this effect could be inhibited by an AT1R or AT2R inhibitor. The levels of NF-κB, RAS components and Ang II production in HLF-1 cells were significantly increased after the hypoxia exposure. Hypoxia or Ang II increased NF-κB-p50 protein expression in HLF-1 cells, and the special effect could be inhibited by telmisartan (TST, an AT1R inhibitor, and partially inhibited by PD123319, an AT2R inhibitor. Importantly, hypoxia-induced NF-κB nuclear translocation could be nearly completely inhibited by an AT1R or AT2R inhibitor. Furthermore pyrrolidine dithiocarbamate (PDTC, a NF-κB blocker, abolished the expression of hypoxia-induced AT1R and Col-I in HLF-1 cells. Our results indicate that Ang II-mediated NF-κB signalling via ATR is involved in hypoxia-induced collagen synthesis in human lung fibroblasts.

  8. Quantitative mapping of collagen fiber orientation in non-glaucoma and glaucoma posterior human sclerae.

    Science.gov (United States)

    Pijanka, Jacek K; Coudrillier, Baptiste; Ziegler, Kimberly; Sorensen, Thomas; Meek, Keith M; Nguyen, Thao D; Quigley, Harry A; Boote, Craig

    2012-08-07

    The posterior sclera has a major biomechanical influence on the optic nerve head, and may therefore be important in glaucoma. Scleral material properties are influenced significantly by collagen fiber architecture. Here we quantitatively map fiber orientation in non-glaucoma and glaucoma posterior human sclerae. Wide-angle x-ray scattering quantified fiber orientation at 0.5-mm intervals across seven non-glaucoma post-mortem human sclerae, and five sclerae with glaucoma history and confirmed axon loss. Multiphoton microscopy provided semiquantitative depth-profiling in the peripapillary sclera. Midposterior fiber orientation was either uniaxial (one preferred direction) or biaxial (two directions). The peripapillary sclera was characterized by a ring of fibers located mainly in the mid-/outer stromal depth and encompassing ∼50% of the total tissue thickness. Fiber anisotropy was 37% higher in the peripapillary sclera compared with midposterior, varied up to 4-fold with position around the scleral canal, and was consistently lowest in the superior-nasal quadrant. Mean fiber anisotropy was significantly lower in the superior-temporal (P glaucoma compared with non-glaucoma eyes. The collagen fiber architecture of the posterior human sclera is highly anisotropic and inhomogeneous. Regional differences in peripapillary fiber anisotropy between non-glaucoma and glaucoma eyes may represent adaptive changes in response to elevated IOP and/or glaucoma, or baseline structural properties that associate with predisposition to glaucomatous axon damage. Quantitative fiber orientation data will benefit numerical eye models aimed at predicting the sclera's influence on nerve head biomechanics, and thereby its possible role in glaucoma.

  9. Global Gene Expression Profiling and Alternative Splicing Events during the Chondrogenic Differentiation of Human Cartilage Endplate-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Jin Shang

    2015-01-01

    Full Text Available Low back pain (LBP is a very prevalent disease and degenerative disc diseases (DDDs usually account for the LBP. However, the pathogenesis of DDDs is complicated and difficult to elucidate. Alternative splicing is a sophisticated regulatory process which greatly increases cellular complexity and phenotypic diversity of eukaryotic organisms. In addition, the cartilage endplate-derived stem cells have been discovered and identified by our research group. In this paper, we continue to investigate gene expression profiling and alternative splicing events during chondrogenic differentiation of cartilage endplate-derived stem cells. We adopted Affymetrix Human Transcriptome Array 2.0 (HTA 2.0 to compare the transcriptional and splicing changes between the control and differentiated samples. RT-PCR and quantitative PCR are used to validate the microarray results. The GO and KEGG pathway analysis was also performed. After bioinformatics analysis of the data, we detected 1953 differentially expressed genes. In terms of alternative splicing, the Splicing Index algorithm was used to select alternatively spliced genes. We detected 4411 alternatively spliced genes. GO and KEGG pathway analysis also revealed several functionally involved biological processes and signaling pathways. To our knowledge, this is the first study to investigate the alternative splicing mechanisms in chondrogenic differentiation of stem cells on a genome-wide scale.

  10. Dynamic Culturing of Cartilage Tissue: The Significance of Hydrostatic Pressure

    Science.gov (United States)

    Pereira, Ana L.; Duarte, Ana R.C.; Frias, Ana M.; Pedro, Adriano J.; Oliveira, João T.; Sousa, Rui A.; Reis, Rui L.

    2012-01-01

    Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10×106 cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4 MPa Pulsatile HP; (2) 0.4 MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASCs (10×106 cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4 MPa Pulsatile HP; (2) 5 MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and

  11. Application of an acoustofluidic perfusion bioreactor for cartilage tissue engineering

    OpenAIRE

    Li, Siwei; Glynne-Jones, Peter; Andriotis, Orestis G.; Ching, Kuan Y.; Jonnalagadda, Umesh S.; Oreffo, Richard O.C.; Hill, Martyn; Tare, Rahul S.

    2014-01-01

    Cartilage grafts generated using conventional static tissue engineering strategies are characterised by low cell viability, suboptimal hyaline cartilage formation and, critically, inferior mechanical competency, which limit their application for resurfacing articular cartilage defects. To address the limitations of conventional static cartilage bioengineering strategies and generate robust, scaffold-free neocartilage grafts of human articular chondrocytes, the present study utilised custom-bu...

  12. Reliability of cut mark analysis in human costal cartilage: the effects of blade penetration angle and intra- and inter-individual differences.

    Science.gov (United States)

    Puentes, K; Cardoso, H F V

    2013-09-10

    Identification of tool class characteristics from cut marks in either bone or cartilage is a valuable source of data for the forensic scientist. Various animal models have been used in experimental studies for the analysis of individual and class characteristics. However, human tissue has seldom been used and it is likely to differ from that of non-humans in key aspects. This study wishes to assess how the knife's blade angle, and both intra- and inter-individual variation in cartilage samples affect the ability of costal cartilage to retain the original class characteristics of the knife, as measured microscopically by the distance between consecutive striations. The 120 cartilaginous samples used in this study originated from the ribcage of 6 male cadavers which were submitted to autopsy at the North Branch of the National Institute of Legal Medicine, in Portugal. Three different serrated knives were purchased from a large department store, and were used in the experimental cuts. Samples of costal cartilage from 2 individuals were assigned to each knife. Each individual provided 20 cartilage samples. Cartilage samples were manually cut using each of the three knives, following two motions: one straight up-and-down cutting motion and parallel and one perpendicular to the blade's teeth long axis forward cutting motion. Casts of the samples were made with Mikrosil(®). Image capture and processing were performed with an Olympus stereomicroscope and its software. The blade's penetration angle and inter-individual variation were shown to affect the identification of the tool class characteristics from the striation pattern observed in a kerf wall, although this seems to be related only to the degree of calcification of the costal cartilage. Intra-individual variation does not seem to significantly affect the identification of the tool class characteristics from the striation pattern observed in a kerf wall, for the same knife following the same motion. Although this

  13. Ex Vivo Transepithelial Collagen Cross-linking in Porcine and Human Corneas Using Human Decorin Core Protein.

    Science.gov (United States)

    Metzler, Kimberly M; Roberts, Cynthia J; Mahmoud, Ashraf M; Agarwal, Gunjan; Liu, Jun

    2016-06-01

    To investigate changes in corneal biomechanics after cross-linking with human decorin core protein (decoron), which is a small, naturally occurring proteoglycan that bridges collagen fibrils, organizing and stabilizing lamellar collagen architecture. Five human donor pairs (10 eyes) and 5 porcine pairs (10 eyes) had one random eye treated transepithelially with decoron, with the untreated fellow eye serving as control. Pretreatment (45 to 60 seconds) and penetration enhancer (45 to 60 seconds) preceded instillation of decoron (45 to 60 seconds). Total treatment time was less than 4 minutes per eye. Human donor eyes were evaluated using the CorVis ST (Oculus Optikgeräte GmbH, Wetzlar, Germany) at 15, 20, 30, 40, and 50 mm Hg of intraocular pressure. Elastic modulus was calculated for human corneas, using parameters derived from Scheimpflug images. Analysis of variance was performed. Porcine corneas underwent uniaxial tensile testing with a Rheometrics Systems Analyzer (RSA III; TA Instruments, New Castle, DE). Secant modulus was calculated and paired t tests were performed between treated and control groups. One human eye pair was excluded based on initial corneal thickness greater than 850 µm. Analysis of variance of the included four pairs demonstrated a significant treatment effect (P cross-linking. Elastic modulus demonstrated a significant treatment effect with a higher elastic modulus in the treatment group. In porcine eye pairs, the secant modulus was significantly higher in the treated than the untreated corneas at 4%, 5%, and 6% strain (P biomechanical behavior and higher elastic modulus in both human and porcine corneas in this preliminary ex vivo study. Further studies are needed to evaluate clinical safety, efficacy, and long-term stability. [J Refract Surg. 2016;32(6):410-417.]. Copyright 2016, SLACK Incorporated.

  14. From mechanical loading to collagen synthesis, structural changes and function in human tendon

    DEFF Research Database (Denmark)

    Kjær, Michael; Langberg, Henning; Heinemeier, Katja

    2009-01-01

    a similar response whether the tendon was stimulated by concentric, isometric or eccentric muscle contraction, suggesting that strain rather that stress/torque determines the collagen-synthesis stimulating response seen with exercise. The adaptation time to chronic loading is longer in tendon tissue...... of TGF-beta, PGE2, IGF-I plus its binding proteins and interleukin-6 takes place after exercise. The increase in IGF-I expression in tendon includes the isoform that has so far been thought only to exist in skeletal muscle (mechano growth factor). The increase in IGF-I and procollagen expression showed...... compared with contractile elements of skeletal muscle or the heart, and only with very prolonged loading are significant changes in gross dimensions of the tendon observed, suggesting that habitual loading is associated with a robust change in the size and mechanical properties of human tendons...

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

    Science.gov (United States)

    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.

  16. Human Bone Derived Collagen for the Development of an Artificial Corneal Endothelial Graft. In Vivo Results in a Rabbit Model.

    Directory of Open Access Journals (Sweden)

    Natalia Vázquez

    Full Text Available Corneal keratoplasty (penetrating or lamellar using cadaveric human tissue, is nowadays the main treatment for corneal endotelial dysfunctions. However, there is a worldwide shortage of donor corneas available for transplantation and about 53% of the world's population have no access to corneal transplantation. Generating a complete cornea by tissue engineering is still a tough goal, but an endothelial lamellar graft might be an easier task. In this study, we developed a tissue engineered corneal endothelium by culturing human corneal endothelial cells on a human purified type I collagen membrane. Human corneal endothelial cells were cultured from corneal rims after corneal penetrating keratoplasty and type I collagen was isolated from remnant cancellous bone chips. Isolated type I collagen was analyzed by western blot, liquid chromatography -mass spectrometry and quantified using the exponentially modified protein abundance index. Later on, collagen solution was casted at room temperature obtaining an optically transparent and mechanically manageable membrane that supports the growth of human and rabbit corneal endothelial cells which expressed characteristic markers of corneal endothelium: zonula ocluddens-1 and Na+/K+ ATPase. To evaluate the therapeutic efficiency of our artificial endothelial grafts, human purified type I collagen membranes cultured with rabbit corneal endothelial cells were transplanted in New Zealand white rabbits that were kept under a minimal immunosuppression regimen. Transplanted corneas maintained transparency for as long as 6 weeks without obvious edema or immune rejection and maintaining the same endothelial markers that in a healthy cornea. In conclusion, it is possible to develop an artificial human corneal endothelial graft using remnant tissues that are not employed in transplant procedures. This artificial endothelial graft can restore the integrality of corneal endothelium in an experimental model of

  17. Functional fabrication of recombinant human collagen-phosphorylcholine hydrogels for regenerative medicine applications.

    Science.gov (United States)

    Mirazul Islam, M; Cėpla, Vytautas; He, Chaoliang; Edin, Joel; Rakickas, Tomas; Kobuch, Karin; Ruželė, Živilė; Jackson, W Bruce; Rafat, Mehrdad; Lohmann, Chris P; Valiokas, Ramūnas; Griffith, May

    2015-01-01

    The implant-host interface is a critical element in guiding tissue or organ regeneration. We previously developed hydrogels comprising interpenetrating networks of recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) as substitutes for the corneal extracellular matrix that promote endogenous regeneration of corneal tissue. To render them functional for clinical application, we have now optimized their composition and thereby enhanced their mechanical properties. We have demonstrated that such optimized RHCIII-MPC hydrogels are suitable for precision femtosecond laser cutting to produce complementing implants and host surgical beds for subsequent tissue welding. This avoids the tissue damage and inflammation associated with manual surgical techniques, thereby leading to more efficient healing. Although we previously demonstrated in clinical testing that RHCIII-based implants stimulated cornea regeneration in patients, the rate of epithelial cell coverage of the implants needs improvement, e.g. modification of the implant surface. We now show that our 500μm thick RHCIII-MPC constructs comprising over 85% water are suitable for microcontact printing with fibronectin. The resulting fibronectin micropatterns promote cell adhesion, unlike the bare RHCIII-MPC hydrogel. Interestingly, a pattern of 30μm wide fibronectin stripes enhanced cell attachment and showed the highest mitotic rates, an effect that potentially can be utilized for faster integration of the implant. We have therefore shown that laboratory-produced mimics of naturally occurring collagen and phospholipids can be fabricated into robust hydrogels that can be laser profiled and patterned to enhance their potential function as artificial substitutes of donor human corneas. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  18. Interlacing of collagen lamellae in the midstroma of the human cornea.

    Science.gov (United States)

    Radner, Wolfgang; Mallinger, Rudolf

    2002-08-01

    To investigate by means of scanning electron microscopy (SEM) the interlacing of collagen lamellae in the midstroma of the human cornea after opening the interlamellar spaces. For SEM, cells and noncollagenous extracellular matrix were removed with 10% sodium hydroxide. Specimens were dehydrated in a series of graded tertiary butanols, frozen at -24 degrees C and dried in an exsiccator by sublimation of the frozen butanol. Dried corneas were cut vertically with a razor blade, and the interlamellar spaces were exposed by stretching the stroma along its anterior-posterior axis by pulling apart the inner (endothelial) and outer (epithelial) edges. Specimens were sputtered with gold and examined with a Cambridge Stereoscan 90 microscope. The opened interlamellar spaces gave the stroma at the cutting edge the appearance of a polymorphic honeycomb. The stromal openings differed in size, from approximately 10 microm to over 150 microm length and up to 80 microm in height. Adjacent lamellae remained connected at several interconnecting regions, either through an exchange of short merging sublamella or single fibrils. Interlacing lamellae crossed through fissures between the branches of splitting lamellae. Others crossed clefts of neighboring lamellae, and other lamellae tunneled crosswise through a horizontally split lamellae hanging in the inferior branch as in a hammock. Large interweaving zones in which a mixture of several types of interlacing was localized close together could also be found. The current study indicates that interlacing is a distinct and important feature of the human cornea, and it gives new insights into the stromal morphology by demonstrating various types of interlacing that occur between collagen lamellae.

  19. Differential induction of collagens by mechanical stress in human periodontal ligament cells.

    Science.gov (United States)

    Nemoto, Tetsuomi; Kajiya, Hiroshi; Tsuzuki, Takashi; Takahashi, Yutaka; Okabe, Koji

    2010-12-01

    Excessive mechanical stress (MS) during hyperocclusion is known to result in destruction of periodontal tissues and alveolar bone, leading to occlusal trauma. Collagens are extracellular matrix components that are encoded by more than 30 different genes. They are classified into three types: fibril-forming, fibril-associated with interrupted triple helices (FACIT), and non-fibril forming collagens. Although MS is known to affect COL I, little is known about its effects on other types of collagens in the periodontal ligament (PDL). We hypothesised that MS could induce expression of the three different types of collagens, thus protecting against occlusal trauma. The aim of this study was to investigate intermittent uniaxial stretching-induced collagen expression in PDL cells using DNA microarray, polymerase chain reaction, and western blotting analysis. We compared changes in collagen expression caused by MS stimulation and osteogenic stimulation, and examined relationships between expression of collagen and their digestive enzymes, matrix metalloproteases (MMPs). Expression of both fibril-forming and FACIT collagens was transiently decreased in the initial phase after MS, while the expression of non-fibril-forming collagens was gradually increased. MS for 3-7 days resulted in gradual upregulation of all three types of collagen. Furthermore, the expression of fibril- and non-fibril-forming collagens was reciprocally related to expression of MMPs. In contrast, expression of all three types of collagen was slightly upregulated during osteogenesis. The MS-induced expression patterns of fibril-forming and FACIT collagens suggest changes in the composition of the extracellular matrix to increase the resistance of PDL cells to hyperocclusal force. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. [Bionic design of articular cartilage].

    Science.gov (United States)

    Qin, Jun; Zhang, Wenguang; Wu, Gang; Wang, Chengtao

    2008-02-01

    Natural articular cartilage is well known as a special connective tissue with multiple effects and functions, which are important and irreplaceable, in human synovial joints. Biomedical, histological and pathological characteristics of articular cartilage, as well as biomaterial, biomechanical and bio-tribological properties thereof, are summarized from a novel aspect of bionics. Bionic design of articualr cartilage at macro-level and micro-level is carried out from three aspects, i.e., structure, material, and function; and a bionic design model of articular cartilage is set up. As a result, this basic research would be helpful to providing theoretical and practical basis for innovational design and manufacturing of new-style artificial joint with "soft-cushion bearing", and of bionic artificial cartilage.

  1. Collagen: A review on its sources and potential cosmetic applications.

    Science.gov (United States)

    Avila Rodríguez, María Isabela; Rodríguez Barroso, Laura G; Sánchez, Mirna Lorena

    2018-02-01

    Collagen is a fibrillar protein that conforms the conjunctive and connective tissues in the human body, essentially skin, joints, and bones. This molecule is one of the most abundant in many of the living organisms due to its connective role in biological structures. Due to its abundance, strength and its directly proportional relation with skin aging, collagen has gained great interest in the cosmetic industry. It has been established that the collagen fibers are damaged with the pass of time, losing thickness and strength which has been strongly related with skin aging phenomena [Colágeno para todo. 60 y más. 2016. http://www.revista60ymas.es/InterPresent1/groups/revistas/documents/binario/ses330informe.pdf.]. As a solution, the cosmetic industry incorporated collagen as an ingredient of different treatments to enhance the user youth and well-being, and some common presentations are creams, nutritional supplement for bone and cartilage regeneration, vascular and cardiac reconstruction, skin replacement, and augmentation of soft skin among others [J App Pharm Sci. 2015;5:123-127]. Nowadays, the biomolecule can be obtained by extraction from natural sources such as plants and animals or by recombinant protein production systems including yeast, bacteria, mammalian cells, insects or plants, or artificial fibrils that mimic collagen characteristics like the artificial polymer commercially named as KOD. Because of its increased use, its market size is valued over USD 6.63 billion by 2025 [Collagen Market By Source (Bovine, Porcine, Poultry, Marine), Product (Gelatin, Hydrolyzed Collagen), Application (Food & Beverages, Healthcare, Cosmetics), By Region, And Segment Forecasts, 2014 - 2025. Grand View Research. http://www.grandviewresearch.com/industry-analysis/collagen-market. Published 2017.]. Nevertheless, there has been little effort on identifying which collagen types are the most suitable for cosmetic purposes, for which the present review will try to enlighten

  2. Cytocompatibility and biologic characteristics of synthetic scaffold materials of rabbit acellular vascular matrix combining with human-like collagen I.

    Science.gov (United States)

    Liu, Xuqian; Wang, Jie; Dong, Fusheng; Song, Peng; Tian, Songbo; Li, Hexiang; Hou, Yali

    2017-10-01

    Scaffold material provides a three-dimensional growing environment for seed cells in the research field of tissue engineering. In the present study, rabbit arterial blood vessel cells were chemically removed with trypsin and Triton X-100 to prepare rabbit acellular vascular matrix scaffold material. Observation by He&Masson staining revealed that no cellular components or nuclei existed in the vascular intima and media after decellularization. Human-like collagen I was combined with acellular vascular matrix by freeze-drying to prepare an acellular vascular matrix-0.25% human-like collagen I scaffold to compensate for the extracellular matrix loss during the decellularization process. We next performed a series of experiments to test the water absorbing quality, biomechanics, pressure resistance, cytotoxicity, and ultra-micro structure of the acellular vascular matrix composite material and natural rabbit artery and found that the acellular vascular matrix-0.25% human-like collagen I material behaved similarly to natural rabbit artery. In conclusion, the acellular vascular matrix-0.25% human-like collagen I composite material provides a new approach and lays the foundation for novel scaffold material research into tissue engineering of blood vessels.

  3. Collagen turnover in normal and degenerate human intervertebral discs as determined by the racemization of aspartic acid

    NARCIS (Netherlands)

    Sivan, S.-S.; Wachtel, E.; Tsitron, E.; Sakkee, N.; Ham, F. van der; Groot, J.de; Roberts, S.; Maroudas, A.

    2008-01-01

    Knowledge of rates of protein turnover is important for a quantitative understanding of tissue synthesis and catabolism. In this work, we have used the racemization of aspartic acid as a marker for the turnover of collagen obtained from healthy and pathological human intervertebral disc matrices. We

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

    DEFF Research Database (Denmark)

    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E

    2010-01-01

    Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts t...

  5. Changes in the Chondrocyte and Extracellular Matrix Proteome during Post-natal Mouse Cartilage Development*

    Science.gov (United States)

    Wilson, Richard; Norris, Emma L.; Brachvogel, Bent; Angelucci, Constanza; Zivkovic, Snezana; Gordon, Lavinia; Bernardo, Bianca C.; Stermann, Jacek; Sekiguchi, Kiyotoshi; Gorman, Jeffrey J.; Bateman, John F.

    2012-01-01

    Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296–1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1

  6. Neurotrophin 3 upregulates proliferation and collagen production in human aortic valve interstitial cells: a potential role in aortic valve sclerosis.

    Science.gov (United States)

    Yao, Qingzhou; Song, Rui; Ao, Lihua; Cleveland, Joseph C; Fullerton, David A; Meng, Xianzhong

    2017-06-01

    Calcific aortic valve disease (CAVD) is a leading cardiovascular disorder in the elderly. Diseased aortic valves are characterized by sclerosis (fibrosis) and nodular calcification. Sclerosis, an early pathological change, is caused by aortic valve interstitial cell (AVIC) proliferation and overproduction of extracellular matrix (ECM) proteins. However, the mechanism of aortic valve sclerosis remains unclear. Recently, we observed that diseased human aortic valves overexpress growth factor neurotrophin 3 (NT3). In the present study, we tested the hypothesis that NT3 is a profibrogenic factor to human AVICs. AVICs isolated from normal human aortic valves were cultured in M199 growth medium and treated with recombinant human NT3 (0.10 µg/ml). An exposure to NT3 induced AVIC proliferation, upregulated the production of collagen and matrix metalloproteinase (MMP), and augmented collagen deposition. These changes were abolished by inhibition of the Trk receptors. NT3 induced Akt phosphorylation and increased cyclin D1 protein levels in a Trk receptor-dependent fashion. Inhibition of Akt abrogated the effect of NT3 on cyclin D1 production. Furthermore, inhibition of either Akt or cyclin D1 suppressed NT3-induced cellular proliferation and MMP-9 and collagen production, as well as collagen deposition. Thus, NT3 upregulates cellular proliferation, ECM protein production, and collagen deposition in human AVICs. It exerts these effects through the Trk-Akt-cyclin D1 cascade. NT3 is a profibrogenic mediator in human aortic valve, and overproduction of NT3 by aortic valve tissue may contribute to the mechanism of valvular sclerosis. Copyright © 2017 the American Physiological Society.

  7. Synthesis of collagen by bovine chondrocytes cultured in alginate; posttranslational modifications and cell-matrix interaction

    NARCIS (Netherlands)

    Beekman, B.; Verzijl, N.; Bank, R.A.; Von Der Mark, K.; TeKoppele, J.M.

    1997-01-01

    The extracellular matrix synthesized by articular chondrocytes cultured in alginate beads was investigated. Collagen levels increased sigmoidally with time and remained constant after 2 weeks of culture. The presence of cartilage-specific type II collagen was confirmed immunohistochemically.

  8. Micrometer scale guidance of mesenchymal stem cells to form structurally oriented cartilage extracellular matrix.

    Science.gov (United States)

    Chou, Chih-Ling; Rivera, Alexander L; Sakai, Takao; Caplan, Arnold I; Goldberg, Victor M; Welter, Jean F; Baskaran, Harihara

    2013-05-01

    Tissue engineering is a possible method for long-term repair of cartilage lesions, but current tissue-engineered cartilage constructs have inferior mechanical properties compared to native cartilage. This problem may be due to the lack of an oriented structure in the constructs at the microscale that is present in the native tissue. In this study, we utilize contact guidance to develop constructs with microscale architecture for improved chondrogenesis and function. Stable channels of varying microscale dimensions were formed in collagen-based and polydimethylsiloxane membranes via a combination of microfabrication and soft-lithography. Human mesenchymal stem cells (MSCs) were selectively seeded in these channels. The chondrogenic potential of MSCs seeded in these channels was investigated by culturing them for 3 weeks under differentiating conditions, and then evaluating the subsequent synthesized tissue for mechanical function and by type II collagen immunohistochemistry. We demonstrate selective seeding of viable MSCs within the channels. MSC aligned and produced mature collagen fibrils along the length of the channel in smaller linear channels of widths 25-100 μm compared to larger linear channels of widths 500-1000 μm. Further, substrates with microchannels that led to cell alignment also led to superior mechanical properties compared to constructs with randomly seeded cells or selectively seeded cells in larger channels. The ultimate stress and modulus of elasticity of constructs with cells seeded in smaller channels increased by as much as fourfolds. We conclude that microscale guidance is useful to produce oriented cartilage structures with improved mechanical properties. These findings can be used to fabricate large clinically useful MSC-cartilage constructs with superior mechanical properties.

  9. Cartilage turnover and intra-articular corticosteroid injections in knee osteoarthritis.

    Science.gov (United States)

    Klocke, Rainer; Levasseur, Kirsty; Kitas, George D; Smith, Jacqueline P; Hirsch, George

    2018-02-02

    Intra-articular corticosteroid injections (IACI) are commonly used interventions for pain relief in patients with knee osteoarthritis (OA). Biomarkers may be helpful in further elucidating how IACI exert their effect. The aim of this study is to look at the response of biomarkers of cartilage and bone metabolism after IACI in knee OA. Eighty subjects with symptomatic knee OA [45% male, mean age (SD) 64 (11) years] underwent routine knee joint injection with 40 mg triamcinolone acetonide and 4 ml 1% lignocaine. Knee pain (as pain subscale of WOMAC VAS) and biomarkers [C-telopeptides of type-II collagen (uCTX-II), and N-telopeptides of type-I collagen in urine; cartilage oligomeric matrix protein (COMP), hyaluronic acid, N-terminal propeptide of type-IIA collagen, and human cartilage glycoprotein-39 (YKL-40) in serum] were measured at baseline and 3 weeks after IACI. Radiographic severity of disease was evaluated using knee radiographs. Median uCTX-II, a cartilage degradation marker, was lower at 3 weeks post IACI compared with baseline: 306.3 and 349.9 ng/mmol, respectively (p < 0.01), which remained significant after Bonferroni correction. Apart from a weak trend of lower sCOMP post IACI (p = 0.089), other biomarkers showed no change after IACI. Both baseline uCTX-II values and the change in uCTX-II from baseline to 3 weeks post injection correlated with radiographic severity of joint space narrowing, but not osteophyte grade. No association between uCTX-II and pain was observed. This observational study suggests that IACI in knee OA may reduce cartilage degradation in the short term.

  10. Mechanical properties of cross-linked collagen meshes after human adipose derived stromal cells seeding

    OpenAIRE

    Ochoa, I. (Ignacio); Peña, E. (Estefanía); Andreu, E.J. (Enrique José); Perez-Ilzarbe, M. (Maitane); Robles, J.E. (José Enrique); Alcaine, C. (C.); Lopez, T. (Tania); Prosper, F. (Felipe); Doblare, M. (M.)

    2010-01-01

    The main goal of this study was to evaluate the potential of collagen meshes derived from porcine dermis as scaffolds for repairing pelvic organ prolapses. Mechanical properties of collagen meshes with different cross-linking percentages before and after Adipose Derived Stromal Cells (ADSC) seeding were studied as well as the cell-scaffold interaction. Uniaxial tensile tests of the collagen meshes with three different cross-linking percentages (full-, partial-, and non...

  11. Localization of collagen type VIII in normal and pathological human cornea

    OpenAIRE

    Zenklová, Kateřina

    2007-01-01

    The aim of this work was to localize collagen type VIII in different layers of the cornea and to compare it's localization in normal corneas with pathological corneas obtained from patients with Fuchs endothelial dystrophy, posterior polymorphous dystrophy or keratoconus.The only comercially available antibody did not proove sufficient specifiky for collagen type VIII. With use of the antibody 9H3 anti alCVIII was collagen VIII evidenced in the cornea. This antibody can be used for detection ...

  12. Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase

    Directory of Open Access Journals (Sweden)

    Myllyharju Johanna

    2010-06-01

    Full Text Available Abstract Background We describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H, the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H α2β2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-α subunit antibody magnetic beads and an anti-β subunit antibody binds to the PDI/β subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation. Results We applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase® verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield. Conclusions Here we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due

  13. Effect of Tris-acetate buffer on endotoxin removal from human-like collagen used biomaterials

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Huizhi; Fan, Daidi, E-mail: fandaidi@nwu.edu.cn; Deng, Jianjun; Zhu, Chenghui; Hui, Junfeng; Ma, Xiaoxuan

    2014-09-01

    Protein preparation, which has active ingredients designated for the use of biomaterials and therapeutical protein, is obtained by genetic engineering, but products of genetic engineering are often contaminated by endotoxins. Because endotoxin is a ubiquitous and potent proinflammatory agent, endotoxin removal or depletion from protein is essential for researching any biomaterials. In this study, we have used Tris-acetate (TA) buffer of neutral pH value to evaluate endotoxins absorbed on the Pierce high-capacity endotoxin removal resin. The effects of TA buffer on pH, ionic strength, incubation time as well as human-like collagen (HLC) concentration on eliminating endotoxins are investigated. In the present experiments, we design an optimal method for TA buffer to remove endotoxin from recombinant collagen and use a chromogenic tachypleus amebocyte lysate (TAL) test kit to measure the endotoxin level of HLC. The present results show that, the endotoxins of HLC is dropped to 8.3 EU/ml at 25 mM TA buffer (pH 7.8) with 150 mM NaCl when setting incubation time at 6 h, and HLC recovery is about 96%. Under this experimental condition, it is proved to exhibit high efficiencies of both endotoxin removal and collagen recovery. The structure of treated HLC was explored by Transmission Electron Microscopy (TEM), demonstrating that the property and structure of HLC treated by TA buffer are maintained. Compared to the most widely used endotoxin removal method, Triton X-114 extraction, using TA buffer can obtain the non-toxic HLC without extra treatment for removing the toxic substances in Triton X-114. In addition, the present study aims at establishing a foundation for further work in laboratory animal science and providing a foundation for medical grade biomaterials. - Graphical abstract: The processes of endotoxins adsorbed from HLC. - Highlights: • TA buffer is a mild buffer system for endotoxins removal of HLC. • TA buffer may facilitate endotoxins adsorbed on the

  14. Development of a Reconstructed Cornea from Collagen-Chondroitin Sulfate Foams and Human Cell Cultures

    National Research Council Canada - National Science Library

    Vrana, N. Engin; Builles, Nicolas; Justin, Virginie; Bednarz, Jurgen; Pellegrini, Graziella; Ferrari, Barbara; Damour, Odile; Hulmes, David J. S; Hasirci, Vasif

    2008-01-01

    .... METHODS . Corneal extracellular matrix was simulated by a porous collagen/glycosaminoglycan-based scaffold seeded with stromal keratocytes and then, successively, epithelial and endothelial cells...

  15. Activated platelet-rich plasma improves adipose-derived stem cell transplantation efficiency in injured articular cartilage

    Science.gov (United States)

    2013-01-01

    Introduction Adipose-derived stem cells (ADSCs) have been isolated, expanded, and applied in the treatment of many diseases. ADSCs have also been used to treat injured articular cartilage. However, there is controversy regarding the treatment efficiency. We considered that ADSC transplantation with activated platelet-rich plasma (PRP) may improve injured articular cartilage compared with that of ADSC transplantation alone. In this study, we determined the role of PRP in ADSC transplantation to improve the treatment efficiency. Methods ADSCs were isolated and expanded from human adipose tissue. PRP was collected and activated from human peripheral blood. The effects of PRP were evaluated in vitro and in ADSC transplantation in vivo. In vitro, the effects of PRP on ADSC proliferation, differentiation into chondrogenic cells, and inhibition of angiogenic factors were investigated at three concentrations of PRP (10%, 15% and 20%). In vivo, ADSCs pretreated with or without PRP were transplanted into murine models of injured articular cartilage. Results PRP promoted ADSC proliferation and differentiation into chondrogenic cells that strongly expressed collagen II, Sox9 and aggrecan. Moreover, PRP inhibited expression of the angiogenic factor vascular endothelial growth factor. As a result, PRP-pretreated ADSCs improved healing of injured articular cartilage in murine models compared with that of untreated ADSCs. Conclusion Pretreatment of ADSCs with PRP is a simple method to efficiently apply ADSCs in cartilage regeneration. This study provides an important step toward the use of autologous ADSCs in the treatment of injured articular cartilage. PMID:23915433

  16. Contrast-Enhanced Computed Tomography Enables Quantitative Evaluation of Tissue Properties at Intrajoint Regions in Cadaveric Knee Cartilage.

    Science.gov (United States)

    Stewart, Rachel C; Honkanen, Juuso T J; Kokkonen, Harri T; Tiitu, Virpi; Saarakkala, Simo; Joukainen, Antti; Snyder, Brian D; Jurvelin, Jukka S; Grinstaff, Mark W; Töyräs, Juha

    2017-10-01

    Objective The aim of this study was to investigate whether the concentration of the anionic contrast agent ioxaglate, as quantitated by contrast-enhanced computed tomography (CECT) using a clinical cone-beam CT (CBCT) instrument, reflects biochemical, histological, and biomechanical characteristics of articular cartilage imaged in an ex vivo, intact human knee joint. Design An osteoarthritic human cadaveric knee joint (91 years old) was injected with ioxaglate (36 mg I/mL) and imaged using CBCT over 61 hours of ioxaglate diffusion into cartilage. Following imaging, the joint surfaces were excised, rinsed to remove contrast agent, and compressive stiffness (equilibrium and instantaneous compressive moduli) was measured via indentation testing ( n = 17 sites). Each site was sectioned for histology and assessed for glycosaminoglycan content using digital densitometry of Safranin-O stained sections, Fourier transform infrared spectroscopy for collagen content, and morphology using both the Mankin and OARSI semiquantitative scoring systems. Water content was determined using mass change after lyophilization. Results CECT attenuation at all imaging time points, including those cartilage water and glycosaminoglycan contents, Mankin score, and both equilibrium and instantaneous compressive moduli. Early time points (cartilage quality between intrajoint regions were distinguishable at diffusion equilibrium and after brief ioxaglate exposure. Conclusions CECT with ioxaglate affords biochemical and biomechanical measurements of cartilage health and performance even after short, clinically relevant exposure times, and may be useful in the clinic as a means for detecting early signs of cartilage pathology.

  17. Differences in collagen ultrastructure of human first trimester decidua basalis and parietalis: implications for trophoblastic invasion of the placental bed.

    Science.gov (United States)

    Sinai Talaulikar, Vikram; Kronenberger, Katrin; Bax, Bridget E; Moss, Raymond; Manyonda, Isaac

    2014-01-01

    The human embryo-maternal interface in the first trimester of pregnancy is an area of extensive tissue remodeling. Because collagen is the most abundant constituent of the extracellular matrix of the placental bed, successful invasion must involve its rapid turnover. We compared the nature and distribution of collagen fibrils in decidua basalis and parietalis. We used a direct-vision hysteroscopic technique to obtain biopsies of the decidua basalis and parietalis from 11 women undergoing pregnancy termination in the first trimester. The biopsies were subjected to light, transmission and scanning electron microscopy, and immunohistochemical studies using mouse monoclonal antibodies against cytokeratin 7 and collagen types I, III and V. Collagen fibrils in the stroma of decidua basalis were significantly thicker when compared to those in decidua parietalis (56.48 ± 1.37 nm vs 45.64 ± 0.85 nm; P collagen fibrils between basalis and parietalis, with thicker and disrupted fibrils within abundant amorphous tissue in basalis, and thinner uniform fibrils in parietalis. These differences may reflect an adaptive response by decidua or a direct consequence of the invading trophoblast cells. © 2013 The Authors. Journal of Obstetrics and Gynaecology Research © 2013 Japan Society of Obstetrics and Gynecology.

  18. Regeneration of human bones in hip osteonecrosis and human cartilage in knee osteoarthritis with autologous adipose-tissue-derived stem cells: a case series

    Directory of Open Access Journals (Sweden)

    Pak Jaewoo

    2011-07-01

    Full Text Available Abstract Introduction This is a series of clinical case reports demonstrating that a combination of percutaneously injected autologous adipose-tissue-derived stem cells, hyaluronic acid, platelet rich plasma and calcium chloride may be able to regenerate bones in human osteonecrosis, and with addition of a very low dose of dexamethasone, cartilage in human knee osteoarthritis. Case reports Stem cells were obtained from adipose tissue of abdominal origin by digesting lipoaspirate tissue with collagenase. These stem cells, along with hyaluronic acid, platelet rich plasma and calcium chloride, were injected into the right hip of a 29-year-old Korean woman and a 47-year-old Korean man. They both had a history of right hip osteonecrosis of the femoral head. For cartilage regeneration, a 70-year-old Korean woman and a 79-year-old Korean woman, both with a long history of knee pain due to osteoarthritis, were injected with stem cells along with hyaluronic acid, platelet rich plasma, calcium chloride and a nanogram dose of dexamethasone. Pre-treatment and post-treatment MRI scans, physical therapy, and pain score data were then analyzed. Conclusions The MRI data for all the patients in this series showed significant positive changes. Probable bone formation was clear in the patients with osteonecrosis, and cartilage regeneration in the patients with osteoarthritis. Along with MRI evidence, the measured physical therapy outcomes, subjective pain, and functional status all improved. Autologous mesenchymal stem cell injection, in conjunction with hyaluronic acid, platelet rich plasma and calcium chloride, is a promising minimally invasive therapy for osteonecrosis of femoral head and, with low-dose dexamethasone, for osteoarthritis of human knees.

  19. Pyropia yezoensis peptide promotes collagen synthesis by activating the TGF-?/Smad signaling pathway in the human dermal fibroblast cell line Hs27

    OpenAIRE

    Kim, Cho-Rong; Kim, Young-Min; Lee, Min-Kyeong; Kim, In-Hye; CHOI, YOUN-HEE; NAM, TAEK-JEONG

    2016-01-01

    Pyropia yezoensis (P. yezoensis) is a marine algae that exhibits antioxidant, anti-inflammatory, antitumor and anti-aging activities. In this study, we investigated the effects of the P. yezoensis peptide, PYP1-5, on collagen synthesis in the human dermal fibroblast cell line Hs27. Skin aging is related to reduced collagen production and the activities of multiple enzymes, including matrix metalloproteinases (MMPs), which degrade collagen structure in the dermis, and tissue inhibitor of tissu...

  20. Positive regulation of corneal type V collagen mRNA: analysis by chicken-human heterokaryon formation.

    Science.gov (United States)

    Linsenmayer, T F; Igoe, F; Gibney, E; Gordon, M K; Birk, D E

    1996-10-10

    Our previous studies have suggested that type V collagen is at least one factor responsible for the characteristically small, uniform diameter of striated collagen fibrils of the corneal stroma. These fibrils, which are heterotypic combinations of collagen types I and V, contain four- to fivefold more type V collagen than those of tendon and sclera. The latter are much larger and more heterodisperse. This high content of type V collagen in cornea is reflected by an equally elevated content of alpha1(V) chain mRNA in corneal fibroblasts. Thus, the increased production of the molecule in cornea appears to be regulated at the level of transcription and/or mRNA stability. One possible explanation for this is that corneal fibroblasts contain positive regulatory factors that specifically upregulate transcription of the type V collagen genes and/or increase their mRNA stability. To test this possibility, we have produced transient heterokaryons by fusing chicken corneal fibroblasts with two human noncorneal cell lines selected as containing little if any alpha1(V) mRNA. If the chicken corneal cells contain positive regulators that can act across species, these regulators should result in increased levels of the human alpha1(V) transcript. The results were evaluated by reverse transcript-polymerase chain reaction employing a primer pair selected for its ability specifically to amplify part of the human alpha1(V) mRNA. In fusions between chicken corneal fibroblasts and the human cell lines, after a lag of 10-14 h the heterokaryon-containing cultures showed de novo appearance or upregulation of human alpha1(V) chain mRNA, compared with that of the parental cell lines. Cultures of the mixed cell types that had not been fused showed no such upregulation, so the effect was not mediated by diffusible substances acting between the cells. Chicken tendon fibroblasts, a low producer of type V collagen, when tested in the same assay, evoked no detectible increase in the human

  1. Magnesium enhances adherence and cartilage formation of synovial mesenchymal stem cells through integrins.

    Science.gov (United States)

    Shimaya, M; Muneta, T; Ichinose, S; Tsuji, K; Sekiya, I

    2010-10-01

    We previously reported that more than 60% of synovial mesenchymal stem cells (MSCs) placed on osteochondral defects adhered to the defect within 10 min and promoted cartilage regeneration. The efficiency of adherence is considered to depend on the interaction between cells and extracellular matrix (ECM), in which integrins may play some important roles. Divalent cations such as calcium, magnesium, and manganese may affect functions of integrins, and the integrins may be involved in differentiation of MSCs. Among divalent cations, magnesium is used in clinical practice as a therapeutic agent and increases the affinity of integrin to ECM. In this study, we investigated whether magnesium enhanced adherence and chondrogenesis of synovial MSC through integrins. We performed assays for adherence of human synovial MSCs to collagen-coated slides, in vitro chondrogenesis, ex vivo assays for adherence of human synovial MSCs to osteochondral defect, and in vivo assays for adherence and cartilage formation of synovial MSCs in a rabbit osteochondral defect model. Magnesium increased adhesion of human synovial MSCs to collagen, and this effect was inhibited by neutralizing antibodies for integrin α3 and β1. Magnesium also promoted synthesis of cartilage matrix during in vitro chondrogenesis of synovial MSCs, which was diminished by neutralizing antibodies for integrin β1 but not for integrin α3. Ex vivo analyses demonstrated that magnesium enhanced adherence of human synovial MSCs to osteochondral defects. In vivo studies in rabbits showed that magnesium promoted adherence at 1 day and cartilage formation of synovial MSCs at 2 weeks. Magnesium enhanced adherence of synovial MSCs through integrins, which promoted synthesis of cartilage matrix at an early phase. Copyright © 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  2. Quantitative analysis of collagen lamellae in the normal and keratoconic human cornea by second harmonic generation imaging microscopy.

    Science.gov (United States)

    Morishige, Naoyuki; Shin-Gyou-Uchi, Ryutaro; Azumi, Haruya; Ohta, Hiroaki; Morita, Yukiko; Yamada, Naoyuki; Kimura, Kazuhiro; Takahara, Atsushi; Sonoda, Koh-Hei

    2014-11-25

    To characterize the structural properties of collagen lamellae in the normal and keratoconic human corneal stroma, we measured their width and angle relative to Bowman's layer (BL). Thirteen normal and four keratoconic corneas were examined. Collagen lamellae in tissue blocks from the central cornea were visualized by second harmonic generation imaging microscopy. Images obtained in 1-μm steps from BL to Descemet's membrane (DM) were subjected to three-dimensional reconstruction. The reconstructed data sets were divided into 10 layers of equal depth (L1-L10) for analysis. The width of lamellae adherent to BL (L0) was also determined. For the normal cornea, the width (mean ± SD) of collagen lamellae was 6.5 ± 1.7 μm at L0, decreased to 4.3 ± 1.3 μm at L1, and then increased gradually with progression toward DM to 122.2 ± 34.5 μm at L10, whereas the angle of lamellae was 20.9° ± 5.4° at L1 and decreased initially to 10.6° ± 3.2° at L2 before declining gradually to 2.7° ± 2.2° at L10. The width and angle of collagen lamellae in the keratoconic cornea were significantly larger and smaller, respectively, relative to those in the normal cornea. In the normal human cornea, collagen lamellae adjacent to BL are narrow and form a steep angle with BL, whereas they increase in width and their angle relative to BL flattens with progression toward DM. These properties of collagen lamellae are altered in keratoconus and are likely related to abnormalities of corneal shape. © ARVO.

  3. Myofibroblasts are responsible for collagen synthesis in the stroma of human hepatocellular carcinoma: an in vivo and in vitro study.

    Science.gov (United States)

    Faouzi, S; Le Bail, B; Neaud, V; Boussarie, L; Saric, J; Bioulac-Sage, P; Balabaud, C; Rosenbaum, J

    1999-02-01

    Marked changes in extracellular matrix occur in the stroma of hepatocellular carcinoma, as compared to normal or cirrhotic liver. The cell types responsible for extracellular matrix synthesis within hepatocellular carcinoma have not been clearly identified. In vivo collagen synthesis was studied by in situ hybridization and immunohistochemistry for types I, IV, V and VI collagen, together with immunolabeling of alpha-smooth muscle actin, a myofibroblast marker, and CD34, an endothelial cell marker. In vitro, extracellular matrix deposition by cultured myofibroblasts was studied by reticulin staining, immunocytochemistry and RNase protection. All collagens studied were expressed in the stroma of the tumor, with a higher level of type VI and IV collagens than of type I and V. The majority of the cells expressing collagen transcripts in human hepatocellular carcinoma stroma were alpha-actin positive and CD 34 negative. In vitro experiments demonstrated that the hepatocellular carcinoma cell lines HepG2, HuH7 and Hep3B markedly increased extracellular matrix deposition by human liver myofibroblasts. This increase was mediated by a soluble mediator present in tumor cell conditioned medium. It was not explained by an increase in mRNA levels of extracellular matrix components, nor by a decrease in the secretion of matrix-degrading proteinases by myofibroblasts. Myofibroblasts are the main source of collagens in the stroma of hepatocellular carcinoma. Our data also indicate that tumoral hepatocytes increase extracellular matrix deposition by cultured myofibroblasts, probably by post-transcriptional mechanisms. The generation of hepatocellular carcinoma stroma by myofibroblasts could thus be under control of tumoral cells.

  4. An in vitro comparative study of T2 and T2* mappings of human articular cartilage at 3-Tesla MRI using histology as the standard of reference

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Taehee; Park, Sunghoon [Ajou University School of Medicine, Department of Radiology, Suwon (Korea, Republic of); Ajou University Medical Center, Musculoskeletal Imaging Laboratory, Suwon (Korea, Republic of); Min, Byoung-Hyun [Ajou University School of Medicine, Department of Orthopaedic Surgery, Suwon (Korea, Republic of); Ajou University School of Medicine, Cartilage Regeneration Center, Suwon (Korea, Republic of); Yoon, Seung-Hyun [Ajou University School of Medicine, Cartilage Regeneration Center, Suwon (Korea, Republic of); Kim, Hakil [INHA University, School of Information and Communication Engineering, Incheon (Korea, Republic of); Lee, Hyun Young [Ajou University Medical Center, Regional Clinical Trial Center, Suwon (Korea, Republic of); Yonsei University College of Medicine, Department of Biostatistics, Seoul (Korea, Republic of); Kwack, Kyu-Sung [Ajou University School of Medicine, Department of Radiology, Suwon (Korea, Republic of); Ajou University Medical Center, Musculoskeletal Imaging Laboratory, Suwon (Korea, Republic of); Ajou University School of Medicine, Cartilage Regeneration Center, Suwon (Korea, Republic of)

    2014-07-15

    The aim of this study was to evaluate the correlations between T2 value, T2* value, and histological grades of degenerated human articular cartilage. T2 mapping and T2* mapping of nine tibial osteochondral specimens were obtained using a 3-T MRI after total knee arthroplasty. A total of 94 ROIs were analyzed. Histological grades were assessed using the David-Vaudey scale. Spearman's rho correlation analysis and Pearson's correlation analysis were performed. The mean relaxation values in T2 map with different histological grades (0, 1, 2) of the cartilage were 51.9 ± 9.2 ms, 55.8 ± 12.8 ms, and 59.6 ± 10.2 ms, respectively. The mean relaxation values in T2* map with different histological grades (0, 1, 2) of the cartilage were 20.3 ± 10.3 ms, 21.1 ± 12.4 ms, and 15.4 ± 8.5 ms, respectively. Spearman's rho correlation analysis confirmed a positive correlation between T2 value and histological grade (ρ = 0.313, p < 0.05). Pearson's correlation analysis revealed a significant negative correlation between T2 and T2* (r = -0.322, p < 0.05). Although T2* values showed a decreasing trend with an increase in cartilage degeneration, this correlation was not statistically significant in this study (ρ = -0.192, p = 0.129). T2 mapping was correlated with histological degeneration, and it may be a good biomarker for osteoarthritis in human articular cartilage. However, the strength of the correlation was weak (ρ = 0.313). Although T2* values showed a decreasing trend with an increase in cartilage degeneration, the correlation was not statistically significant. Therefore, T2 mapping may be more appropriate for the initial diagnosis of articular cartilage degeneration in the knee joint. Further studies on T2* mapping are needed to confirm its reliability and mechanism in cartilage degeneration. (orig.)

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

    Directory of Open Access Journals (Sweden)

    Xin-Yu Li

    2014-02-01

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

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

    Science.gov (United States)

    Li, Xin-Yu; Ji, Cai-Ni; Xu, Ling-Juan; Hu, Wei-Kun; Zhou, Bin; Li, Gui-Gang

    2014-01-01

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

  7. Human rheumatoid arthritis tissue production of IL-17A drives matrix and cartilage degradation: synergy with tumour necrosis factor-alpha, Oncostatin M and response to biologic therapies.

    LENUS (Irish Health Repository)

    Moran, Ellen M

    2009-01-01

    INTRODUCTION: The aim of this study was to examine IL-17A in patients, following anti-TNF-alpha therapy and the effect of IL-17A on matrix turnover and cartilage degradation. METHODS: IL-17A expression was examined by ELISA and immunohistology in the rheumatoid arthritis (RA) joints. RA whole synovial tissue explant (RA ST), primary synovial fibroblasts (RASFC), human cartilage and chondrocyte cultures were stimulated with IL-17A +\\/- TNF-alpha and Oncostatin M (OSM). Matrix metalloproteinase (MMP) and tissue inhibitor (TIMP-1) were assessed by ELISA and zymography. Cartilage proteoglycan release was assessed histologically by Safranin-O staining. Clinical parameters, IL-17A, MMP\\/TIMP were assessed in patients pre\\/post biologic therapy. RESULTS: IL-17A levels were higher in RA vs osteoarthritis (OA)\\/normal joints (P < 0.05). IL-17A up-regulated MMP-1, -2, -9, and -13 in RA ST, RASFC, cartilage and chondrocyte cultures (P < 0.05). In combination with TNF-alpha and OSM, IL-17A shifted the MMP:TIMP-1 ratio in favor of matrix degradation (all P < 0.05). Cartilage proteoglycan depletion in response to IL-17A was mild; however, in combination with TNF-alpha or OSM showed almost complete proteoglycan depletion. Serum IL-17A was detected in 28% of patients commencing biologic therapy. IL-17A negative patients demonstrated reductions post therapy in serum MMP1\\/TIMP4, MMP3\\/TIMP1 and MMP3\\/TIMP4 ratios and an increase in CS846 (all P < 0.05). No significant changes were observed in IL-17A positive patients. CONCLUSIONS: IL-17A is produced locally in the inflamed RA joint. IL-17A promotes matrix turnover and cartilage destruction, especially in the presence of other cytokines, mimicking the joint environment. IL-17A levels are modulated in vivo, following anti-TNF therapy, and may reflect changes in matrix turnover.

  8. Implementation of a gait cycle loading into healthy and meniscectomised knee joint models with fibril-reinforced articular cartilage.

    Science.gov (United States)

    Mononen, Mika E; Jurvelin, Jukka S; Korhonen, Rami K

    2015-01-01

    Computational models can be used to evaluate the functional properties of knee joints and possible risk locations within joints. Current models with fibril-reinforced cartilage layers do not provide information about realistic human movement during walking. This study aimed to evaluate stresses and strains within a knee joint by implementing load data from a gait cycle in healthy and meniscectomised knee joint models with fibril-reinforced cartilages. A 3D finite element model of a knee joint with cartilages and menisci was created from magnetic resonance images. The gait cycle data from varying joint rotations, translations and axial forces were taken from experimental studies and implemented into the model. Cartilage layers were modelled as a fibril-reinforced poroviscoelastic material with the menisci considered as a transversely isotropic elastic material. In the normal knee joint model, relatively high maximum principal stresses were specifically predicted to occur in the medial condyle of the knee joint during the loading response. Bilateral meniscectomy increased stresses, strains and fluid pressures in cartilage on the lateral side, especially during the first 50% of the stance phase of the gait cycle. During the entire stance phase, the superficial collagen fibrils modulated stresses of cartilage, especially in the medial tibial cartilage. The present computational model with a gait cycle and fibril-reinforced biphasic cartilage revealed time- and location-dependent differences in stresses, strains and fluid pressures occurring in cartilage during walking. The lateral meniscus was observed to have a more significant role in distributing loads across the knee joint than the medial meniscus, suggesting that meniscectomy might initiate a post-traumatic process leading to osteoarthritis at the lateral compartment of the knee joint.

  9. Trypsin-mediated enzymatic degradation of type II collagen in the human vitreous

    NARCIS (Netherlands)

    van Deemter, Marielle; Kuijer, Roel; Pas, Hendri Harm; van der Worp, Roelofje Jacoba; Hooymans, Johanna Martina Maria; Los, Leonoor Inge

    2013-01-01

    Purpose: Aging of the vitreous body can result in sight-threatening pathology. One aspect of vitreous aging is liquefaction, which results from the vanishing of collagen fibrils. We investigated the possibility that trypsins are involved in vitreous type II collagen degradation. Methods:

  10. Stable corneal regeneration four years after implantation of a cell-free recombinant human collagen scaffold.

    Science.gov (United States)

    Fagerholm, Per; Lagali, Neil S; Ong, Jeb A; Merrett, Kimberley; Jackson, W Bruce; Polarek, James W; Suuronen, Erik J; Liu, Yuwen; Brunette, Isabelle; Griffith, May

    2014-03-01

    We developed cell-free implants, comprising carbodiimide crosslinked recombinant human collagen (RHC), to enable corneal regeneration by endogenous cell recruitment, to address the worldwide shortage of donor corneas. Patients were grafted with RHC implants. Over four years, the regenerated neo-corneas were stably integrated without rejection, without the long immunosuppression regime needed by donor cornea patients. There was no recruitment of inflammatory dendritic cells into the implant area, whereas, even with immunosuppression, donor cornea recipients showed dendritic cell migration into the central cornea and a rejection episode was observed. Regeneration as evidenced by continued nerve and stromal cell repopulation occurred over the four years to approximate the micro-architecture of healthy corneas. Histopathology of a regenerated, clear cornea from a regrafted patient showed normal corneal architecture. Donor human cornea grafted eyes had abnormally tortuous nerves and stromal cell death was found. Implanted patients had a 4-year average corrected visual acuity of 20/54 and gained more than 5 Snellen lines of vision on an eye chart. The visual acuity can be improved with more robust materials for better shape retention. Nevertheless, these RHC implants can achieve stable regeneration and therefore, represent a potentially safe alternative to donor organ transplantation. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Chondroitin Sulfate- and Decorin-Based Self-Assembling Scaffolds for Cartilage Tissue Engineering.

    Directory of Open Access Journals (Sweden)

    Lourdes Recha-Sancho

    Full Text Available Cartilage injury and degenerative tissue progression remain poorly understood by the medical community. Therefore, various tissue engineering strategies aim to recover areas of damaged cartilage by using non-traditional approaches. To this end, the use of biomimetic scaffolds for recreating the complex in vivo cartilage microenvironment has become of increasing interest in the field. In the present study, we report the development of two novel biomaterials for cartilage tissue engineering (CTE with bioactive motifs, aiming to emulate the native cartilage extracellular matrix (ECM. We employed a simple mixture of the self-assembling peptide RAD16-I with either Chondroitin Sulfate (CS or Decorin molecules, taking advantage of the versatility of RAD16-I. After evaluating the structural stability of the bi-component scaffolds at a physiological pH, we characterized these materials using two different in vitro assessments: re-differentiation of human articular chondrocytes (AC and induction of human adipose derived stem cells (ADSC to a chondrogenic commitment. Interestingly, differences in cellular morphology and viability were observed between cell types and culture conditions (control and chondrogenic. In addition, both cell types underwent a chondrogenic commitment under inductive media conditions, and this did not occur under control conditions. Remarkably, the synthesis of important ECM constituents of mature cartilage, such as type II collagen and proteoglycans, was confirmed by gene and protein expression analyses and toluidine blue staining. Furthermore, the viscoelastic behavior of ADSC constructs after 4 weeks of culture was more similar to that of native articular cartilage than to that of AC constructs. Altogether, this comparative study between two cell types demonstrates the versatility of our novel biomaterials and suggests a potential 3D culture system suitable for promoting chondrogenic differentiation.

  12. Local changes in proteoglycan synthesis during culture are different for normal and osteoarthritic cartilage

    NARCIS (Netherlands)

    Lafeber, F. P.; van der Kraan, P. M.; van Roy, H. L.; Vitters, E. L.; Huber-Bruning, O.; van den Berg, W. B.; Bijlsma, J. W.

    1992-01-01

    Proteoglycan synthesis of mild-to-moderate osteoarthritic human knee cartilage was compared with that of normal cartilage of the same donor. Immediately after cartilage was obtained, the synthesis rate of proteoglycans was higher for osteoarthritic cartilage than for normal cartilage. Proteoglycan

  13. Effects of long-term immobilization and recovery on human triceps surae and collagen turnover in the Achilles tendon in patients with healing ankle fracture

    DEFF Research Database (Denmark)

    Christensen, Britt; Dyrberg, Eva; Aagaard, Per

    2008-01-01

    The aim of the present study was to analyze how human tendon connective tissue responds to an approximately 7-wk period of immobilization and a remobilization period of a similar length, in patients with unilateral ankle fracture, which is currently unknown. Calf muscle cross-sectional area (CSA...... or remobilization. Local collagen turnover was measured as the peritendinous concentrations of NH2-terminal propeptide of type I collagen (PINP) and COOH-terminal telopeptide region of type I collagen (ICTP), markers thought to be indexes of type I collagen synthesis and degradation, respectively. Both markers were...

  14. Effects of the Nd:YAG laser on DNA synthesis and collagen production in human skin fibroblast cultures

    Energy Technology Data Exchange (ETDEWEB)

    Castro, D.J.; Abergel, R.P.; Meeker, C.; Dwyer, R.M.; Lesavoy, M.A.; Uitto, J.

    1983-09-01

    Human skin fibroblasts were subjected to treatment with a Neodymium:YAG laser at 1060 nm with varying levels of energy determined by a reproducible method of dosimetry. DNA synthesis in the cells was measured by the incorporation of (3H)thymidine, and collagen production was monitored by the synthesis of nondialyzable (3H)hydroxyproline after incubation of cells with (3H)proline. Using energy levels equal to 1.7 X 10(3) J/cm2, a significant reduction in DNA synthesis was noted, while the cells remained viable as tested by the trypan blue exclusion test. With energy levels higher or equal to 2.3 X 10(3) J/cm2, the suppression of DNA synthesis was accompanied by cell nonviability. The collagen production, when measured immediately following the treatment with 1.7 X 10(3) J/cm2, was markedly reduced, and similar effects were observed with higher energy levels. However, when the cells were tested for collagen production at 20 hours following laser treatment, there was a significant decrease in collagen production at energy levels as low as 1.1 X 10(3) J/cm2, a dose that did not affect DNA synthesis or cell viability. Thus, the results indicate that the Nd:YAG laser can selectively suppress collagen production without affecting cell proliferation. These observations suggest that laser treatment could potentially be used to reduce collagen deposition in conditions such as keloids and hypertrophic scars.

  15. Chondroitin Sulfate Perlecan Enhances Collagen Fibril Formation

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  16. Photodynamic damage to cartilage and synovial tissue grafted on a chick's chorioallantoic membrane

    Science.gov (United States)

    Fisher, M.; Nahir, A. M.; Kimel, Sol

    1997-09-01

    Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovial joints causing pain deformities and disability. The highly vascular inflamed synovium has aggressive and destructive characteristics, it invades, erodes and gradually destroys cartilage and underlying bone. Photodynamic therapy (PDT) was performed using the chick chorioallantoic membrane (CAM) model to investigate the vitality of synovium and cartilage implanted on the CAM. Synovium, obtained from human patients, was grafted onto the CAM; gross microscopy and histology proved its vitality 7 days post grafting. Cartilage obtained from rabbit knee joint was also maintained on the CAM for 7 days. Its vitality was demonstrated by histology and by measuring metabolic and enzymatic activity of cartilage cells (chondrocytes) as well as the collagen and proteoglycans content. Selective PDT was performed using aluminum phthalocyanine tetrasulfonate (AlPcS4), a hydrophilic compound, soluble in biological solutions, as a photosensitizer. After irradiation with a diode laser (lambda equals 670 nm, 10 mW) damage was observed in vascularized synovium grafts, whereas avascular cartilage remained intact.

  17. Platelet lysate favours in vitro expansion of human bone marrow stromal cells for bone and cartilage engineering.

    Science.gov (United States)

    Zaky, S H; Ottonello, A; Strada, P; Cancedda, R; Mastrogiacomo, M

    2008-12-01

    The heterogeneous population of non-haematopoietic cells residing in the bone marrow (bone marrow stromal cells, BMSCs) and the different fractions and components obtained from platelet-rich plasma provide an invaluable source of autologous cells and growth factors for bone and other connective tissue reconstruction. In this study, we investigated the effect of an allogenic platelet lysate on human BMSCs proliferation and differentiation. Cell proliferation and number of performed cell doublings were enhanced in cultures supplemented with the platelet-derived growth factors (platelet lysate, PL), either with or without the concomitant addition of fetal bovine serum (FBS), compared to cultures performed in the presence of FBS and FGF2. Both in vitro and in vivo osteogenic differentiation were unaltered in cells maintained in medium supplemented with PL and not FBS (Only PL) and in cells maintained in medium containing FBS and FGF2. Interestingly, the in vitro cartilage formation was more effective in the pellet of BMSCs expanded in the Only PL medium. In particular, a chondrogenic differentiation was observed in pellets of some in vitro-expanded BMSCs in the Only PL medium, whereas pellets from parallel cell cultures in medium containing FBS did not respond to the chondrogenic induction. We conclude that the platelet lysate from human source is an effective and even more beneficial substitute for fetal bovine serum to support the in vitro expansion of human BMSCs for subsequent tissue-engineering applications. (c) 2008 John Wiley & Sons, Ltd.

  18. Collagenous Gastritis

    OpenAIRE

    Freeman, Hugh J.; Piercy, James R.A.; Raine, Robert J.

    1989-01-01

    A 54-year-old woman presented with nausea, vomiting and weight loss associated with impaired gastric emptying necessitating institution of parenteral nutrition. Subsequent studies revealed an unusual gastric mucosa! inflammatory process characterized by unique subepithelial collagenous deposits. Collagenous gastritis appears to be a distinct, possibly immune-mediated, chronic disorder, pathologically reminiscent of collagenous sprue and collagenous colitis.

  19. [Effects of alginate/collagen scaffold on cell proliferation and differentiation of human adipose-derived mesenchymal stem cells].

    Science.gov (United States)

    Cheng, W; Han, X P; Mou, S L; Yang, F; Liu, L P

    2017-04-09

    Objective: To build scaffold materials with different concentrations of alginate and collagen, and to observe the effects of alginate/collagen ratio on the proliferation of human adipose-derived mesenchymal stem cells (hAMSC) and osteogenic differentiation. The optimal concentration of alginate/collagen will be chosen for constructing hydrogel that will be used for bone tissue engineering. Methods: Soluble hydrogel scaffold materials containing alginate/collagen were prepared, and the following groups were established based on different alginate/collagen ratio: 4∶1 (group A), 2∶1 (group B), and 1∶1 (group C). Cell proliferation on the material surface was observed using the cell counting kit-8 (CCK-8) assay, while cell viability in each material group were observed using live/dead staining. Quantitative real-time PCR(qPCR) was used to measure the differential expression of osteogenesis-related genes on and in the materials. Immunofluorescence staining was used to measure the differential gene expression of osteogenesis-related proteins in each group. Results: The results from the CCK-8 assay showed increasing cell proliferation rate on the lyophilized hydrogel material surface as the collagen concentration increased, and the highest cell proliferation was observed in group C. Live/dead staining assay indicated that cells were able to proliferate in all three types of hydrogel materials, and the highest cell viability was found in material from group B ([87.50±2.65]%). qPCR showed that the expression of osteogenesis-related genes in group C was the highest, among the three groups, while the expression of osteocalcin in group B was significantly higher than those in the other two groups (Palginate/collagen scaffold materials did not show adverse effects on the cell proliferation of hAMSC and osteogenenic differentiation. Bone tissue engineering can use 10% hydrogel material, and when the sodium alginate and collagen have a ratio of 2∶1, the hydrogel can be

  20. [Urinary bladder substitution using combined membrane based on secretions of human mesenchymal stem cells and type I collagen].

    Science.gov (United States)

    Kirpatovckii, V I; Kamalov, D M; Efimenko, A Yu; Makarevich, P I; Sagaradze, G D; Makarevich, O A; Nimiritskii, P P; Osidak, E O; Domogatskii, S P; Karpov, V K; Akopyan, Z H A; Tkachuk, V A; Kamalov, A A

    2016-12-01

    Despite the widespread use of intestinal cystoplasty, urinary bladder substitution remains a challenging problem due to the complexity of operations and the potentially high risk of complications. A promising alternative may be bio-engineered collagen-based matrices containing stem cells or their secretions. To evaluate the effectiveness of this bladder substitution modality, an experiment was conducted on 14 male rabbits. The animals underwent resection of urinary bladder, and the formed defect was substituted with a membrane of type I collagen (series 1, 5 rabbits) or a membrane of the same composition containing a conditioned medium with secretion of mesenchymal stem/stromal cells derived from human adipose tissue (series 2, 5 rabbits). In the comparison group (4 rabbits) resection of the bladder and the closure of the defect was carried out without bladder substitution (series 3). At 1 month after surgery, there was a complete epithelization of the inner surface of the implant, and body tissues replaced the collagen matrix. In series 1, the collagen implant was replaced mainly by connective tissue ingrown with occasional solitary smooth muscle cells. In series 2, the newly formed bladder wall contained numerous smooth muscle cells, growing into the collagen matrix and forming the muscular coat. In series 3, the muscular layer regeneration at the scar site was also noted, but it was less intense, which was confirmed by morphometry. In series 2, more active vascularization of the collagen implant occurred due to neo-angiogenesis, which was more intense than that in series 3, and especially in series 1. Functional studies revealed a reduced bladder functional capacity in series 1 and 3, while in series 2 it was close to normal. During filling cystometry, changes in intra-vesical pressure profile in series 2 were close to normal, while in series 1 and 3 infusion of a small volume of saline resulted in a marked increase in intra-vesical pressure, showing a reduced

  1. Engineering superficial zone features in tissue engineered cartilage.

    Science.gov (United States)

    Chen, Tony; Hilton, Matthew J; Brown, Edward B; Zuscik, Michael J; Awad, Hani A

    2013-05-01

    A major challenge in cartilage tissue engineering is the need to recreate the native tissue's anisotropic extracellular matrix structure. This anisotropy has important mechanical and biological consequences and could be crucial for integrative repair. Here, we report that hydrodynamic conditions that mimic the motion-induced flow fields in between the articular surfaces in the synovial joint induce the formation of a distinct superficial layer in tissue engineered cartilage hydrogels, with enhanced production of cartilage matrix proteoglycan and Type II collagen. Moreover, the flow stimulation at the surface induces the production of the surface zone protein Proteoglycan 4 (aka PRG4 or lubricin). Analysis of second harmonic generation signature of collagen in this superficial layer reveals a highly aligned fibrillar matrix that resembles the alignment pattern in native tissue's surface zone, suggesting that mimicking synovial fluid flow at the cartilage surface in hydrodynamic bioreactors could be key to creating engineered cartilage with superficial zone features. Copyright © 2012 Wiley Periodicals, Inc.

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

    DEFF Research Database (Denmark)

    Langberg, Henning; Ellingsgaard, H; Madsen, T

    2007-01-01

    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, P0.05). 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...

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

    Science.gov (United States)

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

    2015-08-01

    Several diseases can lead to opacification of cornea requiring transplantation of donor tissue to restore vision. In this context, transparent collagen I fibrillated matrices have been synthesized at 15, 30, 60 and 90 mg/mL. The matrices were evaluated for fibril organizations, transparency, mechanical properties and ability to support corneal epithelial cell culture. The best results were obtained with 90 mg/mL scaffolds. At this concentration, the fibril organization presented some similarities to that found in corneal stroma. Matrices had a mean Young's modulus of 570 kPa and acellular scaffolds had a transparency of 87% in the 380-780 nm wavelength range. Human corneal epithelial cells successfully colonized the surface of the scaffolds and generated an epithelium with characteristics of corneal epithelial cells (i.e. expression of cytokeratin 3 and presence of desmosomes) and maintenance of stemness during culture (i.e. expression of ΔNp63α and formation of holoclones in colony formation assay). Presence of cultured epithelium on the matrices was associated with increased transparency (89%). Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  4. Transplantation of neurons derived from human iPS cells cultured on collagen matrix into guinea-pig cochleae.

    Science.gov (United States)

    Ishikawa, Masaaki; Ohnishi, Hiroe; Skerleva, Desislava; Sakamoto, Tatsunori; Yamamoto, Norio; Hotta, Akitsu; Ito, Juichi; Nakagawa, Takayuki

    2017-06-01

    The present study examined the efficacy of a neural induction method for human induced pluripotent stem (iPS) cells to eliminate undifferentiated cells and to determine the feasibility of transplanting neurally induced cells into guinea-pig cochleae for replacement of spiral ganglion neurons (SGNs). A stepwise method for differentiation of human iPS cells into neurons was used. First, a neural induction method was established on Matrigel-coated plates; characteristics of cell populations at each differentiation step were assessed. Second, neural stem cells were differentiated into neurons on a three-dimensional (3D) collagen matrix, using the same protocol of culture on Matrigel-coated plates; neuron subtypes in differentiated cells on a 3D collagen matrix were examined. Then, human iPS cell-derived neurons cultured on a 3D collagen matrix were transplanted into intact guinea-pig cochleae, followed by histological analysis. In vitro analyses revealed successful induction of neural stem cells from human iPS cells, with no retention of undifferentiated cells expressing OCT3/4. After the neural differentiation of neural stem cells, approximately 70% of cells expressed a neuronal marker, 90% of which were positive for vesicular glutamate transporter 1 (VGLUT1). The expression pattern of neuron subtypes in differentiated cells on a 3D collagen matrix was identical to that of the differentiated cells on Matrigel-coated plates. In addition, the survival of transplant-derived neurons was achieved when inflammatory responses were appropriately controlled. Our preparation method for human iPS cell-derived neurons efficiently eliminated undifferentiated cells and contributed to the settlement of transplant-derived neurons expressing VGLUT1 in guinea-pig cochleae. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  5. Intra-Articular Injections of Polyphenols Protect Articular Cartilage from Inflammation-Induced Degradation: Suggesting a Potential Role in Cartilage Therapeutics

    Science.gov (United States)

    Natarajan, Venkatachalam; Madhan, Balaraman; Tiku, Moti L.

    2015-01-01

    Arthritic diseases, such as osteoarthritis and rheumatoid arthritis, inflict an enormous health care burden on society. Osteoarthritis, a degenerative joint disease with high prevalence among older people, and rheumatoid arthritis, an autoimmune inflammatory disease, both lead to irreversible structural and functional damage to articular cartilage. The aim of this study was to investigate the effect of polyphenols such as catechin, quercetin, epigallocatechin gallate, and tannic acid, on crosslinking type II collagen and the roles of these agents in managing in vivo articular cartilage degradation. The thermal, enzymatic, and physical stability of bovine articular cartilage explants following polyphenolic treatment were assessed for efficiency. Epigallocatechin gallate and tannic acid-treated explants showed >12 °C increase over native cartilage in thermal stability, thereby confirming cartilage crosslinking. Polyphenol-treated cartilage also showed a significant reduction in the percentage of collagen degradation and the release of glycosaminoglycans against collagenase digestion, indicating the increase physical integrity and resistance of polyphenol crosslinked cartilage to enzymatic digestion. To examine the in vivo cartilage protective effects, polyphenols were injected intra-articularly before (prophylactic) and after (therapeutic) the induction of collagen-induced arthritis in rats. The hind paw volume and histomorphological scoring was done for cartilage damage. The intra-articular injection of epigallocatechin gallate and tannic acid did not significantly influence the time of onset or the intensity of joint inflammation. However, histomorphological scoring of the articular cartilage showed a significant reduction in cartilage degradation in prophylactic- and therapeutic-groups, indicating that intra-articular injections of polyphenols bind to articular cartilage and making it resistant to degradation despite ongoing inflammation. These studies establish

  6. Sericin Enhances the Bioperformance of Collagen-Based Matrices Preseeded with Human-Adipose Derived Stem Cells (hADSCs

    Directory of Open Access Journals (Sweden)

    Marieta Costache

    2013-01-01

    Full Text Available Current clinical strategies for adipose tissue engineering (ATE, including autologous fat implants or the use of synthetic surrogates, not only are failing in the long term, but also can’t face the latest requirements regarding the aesthetic restoration of the resulted imperfections. In this context, modern strategies in current ATE applications are based on the implantation of 3D cell-scaffold bioconstructs, designed for prospective achievement of in situ functional de novo tissue. Thus, in this paper, we reported for the first time the evaluation of a spongious 60% collagen and 40% sericin scaffold preseeded with human adipose-derived stem cells (hADSCs in terms of biocompatibility and adipogenic potential in vitro. We showed that the addition of the sticky protein sericin in the composition of a classical collagen sponge enhanced the adhesion and also the proliferation rate of the seeded cells, thus improving the biocompatibility of the novel scaffold. In addition, sericin stimulated PPARγ2 overexpression, triggering a subsequent upregulated expression profile of FAS, aP2 and perilipin adipogenic markers. These features, together with the already known sericin stimulatory potential on cellular collagen production, promote collagen-sericin biomatrix as a good candidate for soft tissue reconstruction and wound healing applications.

  7. Nanomechanics of the Cartilage Extracellular Matrix

    Science.gov (United States)

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

    2011-08-01

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

  8. Evidence of cartilage repair by joint distraction in a canine model of osteoarthritis.

    Science.gov (United States)

    Wiegant, Karen; Intema, Femke; van Roermund, Peter M; Barten-van Rijbroek, Angelique D; Doornebal, Arie; Hazewinkel, Herman A W; Lafeber, Floris P J G; Mastbergen, Simon C

    2015-02-01

    Knee osteoarthritis (OA) is a degenerative joint disorder characterized by cartilage, bone, and synovial tissue changes that lead to pain and functional impairment. Joint distraction is a treatment that provides long-term improvement in pain and function accompanied by cartilage repair, as evaluated indirectly by imaging studies and measurement of biochemical markers. The purpose of this study was to evaluate cartilage tissue repair directly by histologic and biochemical assessments after joint distraction treatment. In 27 dogs, OA was induced in the right knee joint (groove model; surgical damage to the femoral cartilage). After 10 weeks of OA development, the animals were randomized to 1 of 3 groups. Two groups were fitted with an external fixator, which they wore for a subsequent 10 weeks (one group with and one without joint distraction), and the third group had no external fixation (OA control group). Pain/function was studied by force plate analysis. Cartilage integrity and chondrocyte activity of the surgically untouched tibial plateaus were analyzed 25 weeks after removal of the fixator. Changes in force plate analysis values between the different treatment groups were not conclusive. Features of OA were present in the OA control group, in contrast to the generally less severe damage after joint distraction. Those treated with joint distraction had lower macroscopic and histologic damage scores, higher proteoglycan content, better retention of newly formed proteoglycans, and less collagen damage. In the fixator group without distraction, similarly diminished joint damage was found, although it was less pronounced. Joint distraction as a treatment of experimentally induced OA results in cartilage repair activity, which corroborates the structural observations of cartilage repair indicated by surrogate markers in humans. Copyright © 2015 by the American College of Rheumatology.

  9. Effect of collagen turnover on the accumulation of advanced glycation end products

    NARCIS (Netherlands)

    Verzijl, N.; Groot, J. de; Thorpe, S.R.; Bank, R.A.; Shaw, J.N.; Lyons, T.J.; Bijlsma, J.W.J.; Lafeber, F.P.J.G.; Baynes, J.W.; TeKoppele, J.M.

    2000-01-01

    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

  10. Regulation of migratory activity of human keratinocytes by topography of multiscale collagen-containing nanofibrous matrices.

    Science.gov (United States)

    Fu, Xiaoling; Xu, Meng; Liu, Jie; Qi, Yanmei; Li, Shaohua; Wang, Hongjun

    2014-02-01

    Nanofibrous matrices hold great promise in skin wound repair partially due to their capability of recapturing the essential attributes of native extracellular matrix (ECM). With regard to limited studies on the effect of nanofibrous matrices on keratinocytes, the present study was aimed to understand how the topographical feature of nanofibrous matrices regulates keratinocyte motility by culturing keratinocytes on polycaprolactone (PCL)/collagen nanofibrous matrices (rough surface with fiber diameters of 331 ± 112 nm) or the matrices coated with a thin layer of collagen gel to form a secondary ultrafine fibrous network (smooth surface with ultrafine fiber diameters of 55 ± 26 nm). It was found that the PCL/collagen nanofibrous matrices alone did not stimulate cell migration, while collagen gel coating could significantly increase cell motility. Further studies demonstrated that the ultrafine fibrous network of collagen gel coating significantly activated integrin β1, Rac1 and Cdc42, facilitated the deposition of laminin-332 (formerly called laminin-5), and promoted the expression of active matrix metalloproteinases (MMPs) (i.e., MMP-2 and 9). Neutralization of integrin β1 activity abrogated the gel coating-induced keratinocyte migration. These findings provide important evidence on the role of topographical features of nanofibrous matrices in regulating the phenotypic alteration of keratinocytes and suggest the possible utility of collagen-containing nanofibrous matrices for skin regeneration especially in re-epithelialization. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Mechanisms of self-organization for the collagen fibril lattice in the human cornea.

    Science.gov (United States)

    Cheng, Xi; Pinsky, Peter M

    2013-10-06

    The transparency of the human cornea depends on the regular lattice arrangement of the collagen fibrils and on the maintenance of an optimal hydration--the achievement of both depends on the presence of stromal proteoglycans (PGs) and their linear sidechains of negatively charged glycosaminoglycans (GAGs). Although the GAGs produce osmotic pressure by the Donnan effect, the means by which they exert positional control of the lattice is less clear. In this study, a theoretical model based on equilibrium thermodynamics is used to describe restoring force mechanisms that may control and maintain the fibril lattice and underlie corneal transparency. Electrostatic-based restoring forces that result from local charge density changes induced by fibril motion, and entropic elastic restoring forces that arise from duplexed GAG structures that bridge neighbouring fibrils, are described. The model allows for the possibility that fibrils have a GAG-dense coating that adds an additional fibril force mechanism preventing fibril aggregation. Swelling pressure predictions are used to validate the model with results showing excellent agreement with experimental data over a range of hydration from 30 to 200% of normal. The model suggests that the electrostatic restoring force is dominant, with the entropic forces from GAG duplexes being an order or more smaller. The effect of a random GAG organization, as observed in recent imaging, is considered in a dynamic model of the lattice that incorporates randomness in both the spatial distribution of GAG charge and the topology of the GAG duplexes. A striking result is that the electrostatic restoring forces alone are able to reproduce the image-based lattice distribution function for the human cornea, and thus dynamically maintain the short-range order of the lattice.

  12. Fibroblast activation protein peptide substrates identified from human collagen I derived gelatin cleavage sites.

    Science.gov (United States)

    Aggarwal, Saurabh; Brennen, W Nathaniel; Kole, Thomas P; Schneider, Elizabeth; Topaloglu, Ozlem; Yates, Melinda; Cotter, Robert J; Denmeade, Samuel R

    2008-01-22

    A highly consistent trait of tumor stromal fibroblasts is the induction of the membrane-bound serine protease fibroblast activation protein-alpha (FAP), which is overexpressed on the surface of reactive stromal fibroblasts present within the stroma of the majority of human epithelial tumors. In contrast, FAP is not expressed by tumor epithelial cells or by fibroblasts or other cell types in normal tissues. The proteolytic activity of FAP, therefore, represents a potential pan-tumor target that can be exploited for the release of potent cytotoxins from inactive prodrugs consisting of an FAP peptide substrate coupled to a cytotoxin. To identify FAP peptide substrates, we used liquid chromatography tandem mass spectroscopy based sequencing to generate a complete map of the FAP cleavage sites within human collagen I derived gelatin. Positional analysis of the frequency of each amino acid at each position within the cleavage sites revealed FAP consensus sequences PPGP and (D/E)-(R/K)-G-(E/D)-(T/S)-G-P. These studies further demonstrated that ranking cleavage sites based on the magnitude of the LC/MS/MS extracted ion current predicted FAP substrates that were cleaved with highest efficiency. Fluorescence-quenched peptides were synthesized on the basis of the cleavage sites with the highest ion current rankings, and kinetic parameters for FAP hydrolysis were determined. The substrate DRGETGP, which corresponded to the consensus sequence, had the lowest Km of 21 microM. Overall the Km values were relatively similar for both high and low ranked substrates, whereas the kcat values differed by up to 100-fold. On the basis of these results, the FAP consensus sequences are currently being evaluated as FAP-selective peptide carriers for incorporation into FAP-activated prodrugs.

  13. Contour interpolated radial basis functions with spline boundary correction for fast 3D reconstruction of the human articular cartilage from MR images

    Energy Technology Data Exchange (ETDEWEB)

    Javaid, Zarrar; Unsworth, Charles P., E-mail: c.unsworth@auckland.ac.nz [Department of Engineering Science, The University of Auckland, Auckland 1010 (New Zealand); Boocock, Mark G.; McNair, Peter J. [Health and Rehabilitation Research Center, Auckland University of Technology, Auckland 1142 (New Zealand)

    2016-03-15

    Purpose: The aim of this work is to demonstrate a new image processing technique that can provide a “near real-time” 3D reconstruction of the articular cartilage of the human knee from MR images which is user friendly. This would serve as a point-of-care 3D visualization tool which would benefit a consultant radiologist in the visualization of the human articular cartilage. Methods: The authors introduce a novel fusion of an adaptation of the contour method known as “contour interpolation (CI)” with radial basis functions (RBFs) which they describe as “CI-RBFs.” The authors also present a spline boundary correction which further enhances volume estimation of the method. A subject cohort consisting of 17 right nonpathological knees (ten female and seven male) is assessed to validate the quality of the proposed method. The authors demonstrate how the CI-RBF method dramatically reduces the number of data points required for fitting an implicit surface to the entire cartilage, thus, significantly improving the speed of reconstruction over the comparable RBF reconstruction method of Carr. The authors compare the CI-RBF method volume estimation to a typical commercial package (3D DOCTOR), Carr’s RBF method, and a benchmark manual method for the reconstruction of the femoral, tibial, and patellar cartilages. Results: The authors demonstrate how the CI-RBF method significantly reduces the number of data points (p-value < 0.0001) required for fitting an implicit surface to the cartilage, by 48%, 31%, and 44% for the patellar, tibial, and femoral cartilages, respectively. Thus, significantly improving the speed of reconstruction (p-value < 0.0001) by 39%, 40%, and 44% for the patellar, tibial, and femoral cartilages over the comparable RBF model of Carr providing a near real-time reconstruction of 6.49, 8.88, and 9.43 min for the patellar, tibial, and femoral cartilages, respectively. In addition, it is demonstrated how the CI-RBF method matches the volume

  14. International Cartilage Repair Society (ICRS) Recommended Guidelines for Histological Endpoints for Cartilage Repair Studies in Animal Models and Clinical Trials

    Science.gov (United States)

    Hoemann, Caroline; Kandel, Rita; Roberts, Sally; Saris, Daniel B.F.; Creemers, Laura; Mainil-Varlet, Pierre; Méthot, Stephane; Hollander, Anthony P.; Buschmann, Michael D.

    2011-01-01

    Cartilage repair strategies aim to resurface a lesion with osteochondral tissue resembling native cartilage, but a variety of repair tissues are usually observed. Histology is an important structural outcome that could serve as an interim measure of efficacy in randomized controlled clinical studies. The purpose of this article is to propose guidelines for standardized histoprocessing and unbiased evaluation of animal tissues and human biopsies. Methods were compiled from a literature review, and illustrative data were added. In animal models, treatments are usually administered to acute defects created in healthy tissues, and the entire joint can be analyzed at multiple postoperative time points. In human clinical therapy, treatments are applied to developed lesions, and biopsies are obtained, usually from a subset of patients, at a specific time point. In striving to standardize evaluation of structural endpoints in cartilage repair studies, 5 variables should be controlled: 1) location of biopsy/sample section, 2) timing of biopsy/sample recovery, 3) histoprocessing, 4) staining, and 5) blinded evaluation with a proper control group. Histological scores, quantitative histomorphometry of repair tissue thickness, percentage of tissue staining for collagens and glycosaminoglycan, polarized light microscopy for collagen fibril organization, and subchondral bone integration/structure are all relevant outcome measures that can be collected and used to assess the efficacy of novel therapeutics. Standardized histology methods could improve statistical analyses, help interpret and validate noninvasive imaging outcomes, and permit cross-comparison between studies. Currently, there are no suitable substitutes for histology in evaluating repair tissue quality and cartilaginous character. PMID:26069577

  15. Articular Cartilage of the Human Knee Joint: In Vivo Multicomponent T2 Analysis at 3.0 T

    Science.gov (United States)

    Choi, Kwang Won; Samsonov, Alexey; Spencer, Richard G.; Wilson, John J.; Block, Walter F.; Kijowski, Richard

    2015-01-01

    Purpose To compare multicomponent T2 parameters of the articular cartilage of the knee joint measured by using multicomponent driven equilibrium single-shot observation of T1 and T2 (mcDESPOT) in asymptomatic volunteers and patients with osteoarthritis. Materials and Methods This prospective study was performed with institutional review board approval and with written informed consent from all subjects. The mcDESPOT sequence was performed in the knee joint of 13 asymptomatic volunteers and 14 patients with osteoarthritis of the knee. Single-component T2 (T2Single), T2 of the fast-relaxing water component (T2F) and of the slow-relaxing water component (T2S), and the fraction of the fast-relaxing water component (FF) of cartilage were measured. Wilcoxon rank-sum tests and multivariate linear regression models were used to compare mcDESPOT parameters between volunteers and patients with osteoarthritis. Receiver operating characteristic analysis was used to assess diagnostic performance with mcDESPOT parameters for distinguishing morphologically normal cartilage from morphologically degenerative cartilage identified at magnetic resonance imaging in eight cartilage subsections of the knee joint. Results Higher cartilage T2Single (P < .001), lower cartilage FF (P < .001), and similar cartilage T2F (P = .079) and T2S (P = .124) values were seen in patients with osteoarthritis compared with those in asymptomatic volunteers. Differences in T2Single and FF remained significant (P < .05) after consideration of age differences between groups of subjects. Diagnostic performance was higher with FF than with T2Single for distinguishing between normal and degenerative cartilage (P < .05), with greater areas under the curve at receiver operating characteristic analysis. Conclusion Patients with osteoarthritis of the knee had significantly higher cartilage T2Single and significantly lower cartilage FF than did asymptomatic volunteers, and receiver operating characteristic analysis

  16. Fibronectin, laminin, and collagen IV as modulators of cell behavior during adrenal gland development in the human fetus.

    Science.gov (United States)

    Chamoux, Estelle; Narcy, Agnès; Lehoux, Jean-Guy; Gallo-Payet, Nicole

    2002-04-01

    The specific development of the human fetal adrenal gland requires cell proliferation, migration, apoptosis, and zone-specific steroidogenic activity. The present work was designed to determine the physiological significance of the previously identified spatial distribution of extracellular matrix components in the fetal gland. Primary cultures of human fetal adrenal cells grown on collagen IV, laminin, or fibronectin revealed that cell morphology was affected by environmental cues. Matrices also modulated the profile of steroid secretion by the fetal cells. Collagen IV favored cortisol secretion after ACTH or angiotensin II stimulation and increased dehydroepiandrosterone production when the AT(2) receptor of angiotensin II was specifically stimulated. These effects were correlated by changes in the mRNA levels of 3beta-hydroxysteroid dehydrogenase and cytochrome P450C17. In contrast, fibronectin and laminin decreased cell responsiveness to ACTH in terms of cortisol secretion, but enhanced ACTH-stimulated androgen secretion. Finally, extracellular matrices were able to orchestrate cell behavior. Collagen IV and laminin enhanced cell proliferation, and fibronectin increased cell death. This study is the first to demonstrate that the nature of extracellular matrix coordinates specific steroidogenic pathways and cell turnover in the developing human fetal adrenal gland.

  17. Serum Cartilage Biomarkers and Shoulder Instability.

    Science.gov (United States)

    Owens, Brett D; Cameron, Kenneth L; Bokshan, Steven L; Clifton, Kari B; Svoboda, Steven J; Wolf, Jennifer Moriatis

    2017-01-01

    Differences in cartilage biomarkers have been noted in patients with anterior cruciate ligament tears, but little is known about any similar relationship with shoulder instability. This study evaluated the relationship between serum cartilage biomarkers and shoulder instability. The authors present a prospective cohort study of young athletes followed from 2006 to 2010. A nested case-control analysis was conducted within this cohort to evaluate the association between preinjury collagen type II cleavage (a marker for type II collagen cleavage) and procollagen II carboxy propeptide (a marker of cartilage synthesis) and the subsequent likelihood of shoulder instability during the 4-year follow-up period. Preinjury collagen type II cleavage and procollagen II carboxy propeptide levels in 51 subjects who had shoulder instability were compared with levels in 210 subjects without documented anterior cruciate ligament or shoulder instability (control group) with commercially available enzyme-linked immunosorbent assay kits. Mean preinjury collagen type II cleavage levels in patients who subsequently had shoulder instability were significantly lower than those in the control group (73.91 vs 79.24 pg/mL, P=.03). No significant difference was found in preinjury procollagen II carboxy propeptide levels compared with the control group (359.94 vs 396.37, P=.24). This study is the first to examine the relationship between baseline collagen biomarkers and subsequent shoulder instability. The finding of lower baseline collagen type II cleavage levels in patients with subsequent shoulder instability may represent a genetic predisposition or a compensatory mechanism by which cartilage degradation is decreased in those who are more likely to have instability. [Orthopedics. 2017; 40(1):34-36.]. Copyright 2016, SLACK Incorporated.

  18. Second harmonic generation imaging in tissue engineering and cartilage pathologies

    Science.gov (United States)

    Lilledahl, Magnus; Olderøy, Magnus; Finnøy, Andreas; Olstad, Kristin; Brinchman, Jan E.

    2015-03-01

    The second harmonic generation from collagen is highly sensitive to what extent collagen molecules are ordered into fibrils as the SHG signal is approximately proportional to the square of the fibril thickness. This can be problematic when interpreting SHG images as thick fibers are much brighter than thinner fibers such that quantification of the amount of collagen present is difficult. On the other hand SHG is therefore also a very sensitive probe to determine whether collagen have assembled into fibrils or are still dissolved as individual collagen molecules. This information is not available from standard histology or immunohistochemical techniques. The degree for fibrillation is an essential component for proper tissue function. We will present the usefulness of SHG imaging in tissue engineering of cartilage as well as cartilage related pathologies. When engineering cartilage it is essential to have the appropriate culturing conditions which cause the collagen molecules to assemble into fibrils. By employing SHG imaging we have studied how cell seeding densities affect the fibrillation of collagen molecules. Furthermore we have used SHG to study pathologies in developing cartilage in a porcine model. In both cases SHG reveals information which is not visible in conventional histology or immunohistochemistry

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

    Science.gov (United States)

    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.

  20. Type III collagen is essential for growth acceleration of human osteoblastic cells by ascorbic acid 2-phosphate, a long-acting vitamin C derivative

    National Research Council Canada - National Science Library

    Maehata, Yojiro; Takamizawa, Shinji; Ozawa, Shigeyuki; Izukuri, Kazuhito; Kato, Yasumasa; Sato, Sadao; Lee, Masaichi-Chang-il; Kimura, Akinori; Hata, Ryu-Ichiro

    2007-01-01

    Collagen has been reported to be essential for the proliferation of various kinds of cells including human osteoblastic cells [Takamizawa, S., Maehata, Y., Imai, K., Senoo, H., Sato, S., Hata, R., 2004...

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

    Energy Technology Data Exchange (ETDEWEB)

    Bredfeldt, J; Liu, Y; Conklin, M; Keely, P; Eliceiri, K; Mackie, T [University of Wisconsin, Madison, WI (United States)

    2014-06-01

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

  2. Quantitative reconstruction of weaning ages in archaeological human populations using bone collagen nitrogen isotope ratios and approximate Bayesian computation.

    Directory of Open Access Journals (Sweden)

    Takumi Tsutaya

    Full Text Available BACKGROUND: Nitrogen isotope analysis of bone collagen has been used to reconstruct the breastfeeding practices of archaeological human populations. However, weaning ages have been estimated subjectively because of a lack of both information on subadult bone collagen turnover rates and appropriate analytical models. METHODOLOGY: Temporal changes in human subadult bone collagen turnover rates were estimated from data on tissue-level bone metabolism reported in previous studies. A model for reconstructing precise weaning ages was then developed using a framework of approximate Bayesian computation and incorporating the estimated turnover rates. The model is presented as a new open source R package, WARN (Weaning Age Reconstruction with Nitrogen isotope analysis, which computes the age at the start and end of weaning, (15N-enrichment through maternal to infant tissue, and [Formula: see text] value of collagen synthesized entirely from weaning foods with their posterior probabilities. The model was applied to 39 previously reported Holocene skeletal populations from around the world, and the results were compared with weaning ages observed in ethnographic studies. CONCLUSIONS: There were no significant differences in the age at the end of weaning between the archaeological (2.80±1.32 years and ethnographic populations. By comparing archaeological populations, it appears that weaning ages did not differ with the type of subsistence practiced (i.e., hunting-gathering or not. Most of [Formula: see text]-enrichment (2.44±0.90‰ was consistent with biologically valid values. The nitrogen isotope ratios of subadults after the weaning process were lower than those of adults in most of the archaeological populations (-0.48±0.61‰, and this depletion was greater in non-hunter-gatherer populations. Our results suggest that the breastfeeding period in humans had already been shortened by the early Holocene compared with those in extant great apes.

  3. Regulation of complement by cartilage oligomeric matrix protein allows for a novel molecular diagnostic principle in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Happonen, Kaisa E; Saxne, Tore; Aspberg, Anders

    2010-01-01

    Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage, where it catalyzes collagen fibrillogenesis. Elevated amounts of COMP are found in serum during increased turnover of cartilage associated with active joint disease, such as rheumatoid arthritis (RA) and osteoarthr...