FK506 protects against articular cartilage collagenous extra-cellular matrix degradation.

Science.gov (United States)

Siebelt, M; van der Windt, A E; Groen, H C; Sandker, M; Waarsing, J H; Müller, C; de Jong, M; Jahr, H; Weinans, H

2014-04-01

Osteoarthritis (OA) is a non-rheumatologic joint disease characterized by progressive degeneration of the cartilage extra-cellular matrix (ECM), enhanced subchondral bone remodeling, activation of synovial macrophages and osteophyte growth. Inhibition of calcineurin (Cn) activity through tacrolimus (FK506) in in vitro monolayer chondrocytes exerts positive effects on ECM marker expression. This study therefore investigated the effects of FK506 on anabolic and catabolic markers of osteoarthritic chondrocytes in 2D and 3D in vitro cultures, and its therapeutic effects in an in vivo rat model of OA. Effects of high and low doses of FK506 on anabolic (QPCR/histochemistry) and catabolic (QPCR) markers were evaluated in vitro on isolated (2D) and ECM-embedded chondrocytes (explants, 3D pellets). Severe cartilage damage was induced unilaterally in rat knees using papain injections in combination with a moderate running protocol. Twenty rats were treated with FK506 orally and compared to twenty untreated controls. Subchondral cortical and trabecular bone changes (longitudinal microCT) and macrophage activation (SPECT/CT) were measured. Articular cartilage was analyzed ex vivo using contrast enhanced microCT and histology. FK506 treatment of osteoarthritic chondrocytes in vitro induced anabolic (mainly collagens) and reduced catabolic ECM marker expression. In line with this, FK506 treatment clearly protected ECM integrity in vivo by markedly decreasing subchondral sclerosis, less development of subchondral pores, depletion of synovial macrophage activation and lower osteophyte growth. FK506 protected cartilage matrix integrity in vitro and in vivo. Additionally, FK506 treatment in vivo reduced OA-like responses in different articular joint tissues and thereby makes Cn an interesting target for therapeutic intervention of OA. Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

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

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

2017-11-01

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

Effect of collagen on magnetization transfer contrast assessed in cultured cartilage

International Nuclear Information System (INIS)

Aoki, Jun; Seo, Gwy-Suk; Karakida, Osamu; Ueda, Hitoshi; Sone, Shusuke; Hiraki, Yuji; Shukunami, Chisa; Moriya, Hiroto.

1996-01-01

We investigated the effect of collagen on magnetization transfer contrast (MTC) in cultured cartilage. In our culture system, only collagen synthesis was increased by the addition of vitamin C, while proteoglycan synthesis and the number of chondrocytes were unaffected. The MTC effect was assessed by using an off-resonance RF pulse (0.3 KHz off-resonance, sinc wave of 18 msec, maximum amplitude 4.61 x 10 -4 T) on a GRASS sequence. The cartilage cultured with vitamin C showed a higher MTC effect than that cultured without vitamin C. The major role of collagen on MTC was confirmed in living cartilage tissue. (author)

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

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

2014-04-01

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

The Secret Life of Collagen: Temporal Changes in Nanoscale Fibrillar Pre-Strain and Molecular Organization during Physiological Loading of Cartilage.

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Inamdar, Sheetal R; Knight, David P; Terrill, Nicholas J; Karunaratne, Angelo; Cacho-Nerin, Fernando; Knight, Martin M; Gupta, Himadri S

2017-10-24

Articular cartilage is a natural biomaterial whose structure at the micro- and nanoscale is critical for healthy joint function and where degeneration is associated with widespread disorders such as osteoarthritis. At the nanoscale, cartilage mechanical functionality is dependent on the collagen fibrils and hydrated proteoglycans that form the extracellular matrix. The dynamic response of these ultrastructural building blocks at the nanoscale, however, remains unclear. Here we measure time-resolved changes in collagen fibril strain, using small-angle X-ray diffraction during compression of bovine and human cartilage explants. We demonstrate the existence of a collagen fibril tensile pre-strain, estimated from the D-period at approximately 1-2%, due to osmotic swelling pressure from the proteoglycan. We reveal a rapid reduction and recovery of this pre-strain which occurs during stress relaxation, approximately 60 s after the onset of peak load. Furthermore, we show that this reduction in pre-strain is linked to disordering in the intrafibrillar molecular packing, alongside changes in the axial overlapping of tropocollagen molecules within the fibril. Tissue degradation in the form of selective proteoglycan removal disrupts both the collagen fibril pre-strain and the transient response during stress relaxation. This study bridges a fundamental gap in the knowledge describing time-dependent changes in collagen pre-strain and molecular organization that occur during physiological loading of articular cartilage. The ultrastructural details of this transient response are likely to transform our understanding of the role of collagen fibril nanomechanics in the biomechanics of cartilage and other hydrated soft tissues.

Postnatal development of collagen structure in ovine articular cartilage

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Kranenbarg Sander

2010-06-01

Full Text Available Abstract Background Articular cartilage (AC is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Across species, adult AC shows an arcade-like structure with collagen predominantly perpendicular to the subchondral bone near the bone, and collagen predominantly parallel to the articular surface near the articular surface. Recent studies into collagen fibre orientation in stillborn and juvenile animals showed that this structure is absent at birth. Since the collagen structure is an important factor for AC mechanics, the absence of the adult Benninghoff structure has implications for perinatal AC mechanobiology. The current objective is to quantify the dynamics of collagen network development in a model animal from birth to maturity. We further aim to show the presence or absence of zonal differentiation at birth, and to assess differences in collagen network development between different anatomical sites of a single joint surface. We use quantitative polarised light microscopy to investigate properties of the collagen network and we use the sheep (Ovis aries as our model animal. Results Predominant collagen orientation is parallel to the articular surface throughout the tissue depth for perinatal cartilage. This remodels to the Benninghoff structure before the sheep reach sexual maturity. Remodelling of predominant collagen orientation starts at a depth just below the future transitional zone. Tissue retardance shows a minimum near the articular surface at all ages, which indicates the presence of zonal differentiation at all ages. The absolute position of this minimum does change between birth and maturity. Between different anatomical sites, we find differences in the dynamics of collagen remodelling, but no differences in adult collagen structure. Conclusions The collagen network in articular cartilage remodels between birth and sexual maturity from a network with predominant orientation parallel to the

Endocytic collagen degradation

DEFF Research Database (Denmark)

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

2012-01-01

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

Postnatal development of collagen structure in ovine articular cartilage

NARCIS (Netherlands)

Turnhout, van M.C.; Schipper, H.; Engel, B.; Buist, W.; Kranenbarg, S.; Leeuwen, van J.L.

2010-01-01

Background Articular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Across species, adult AC shows an arcade-like structure with collagen predominantly perpendicular to the subchondral bone near the bone, and collagen predominantly

Postnatal development of collagen structure in ovine articular cartilage

NARCIS (Netherlands)

Turnhout, van M.C.; Schipper, H.; Engel, B.; Buist, W.; Kranenbarg, S.; Leeuwen, van J.L.

2010-01-01

BACKGROUND:Articular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Across species, adult AC shows an arcade-like structure with collagen predominantly perpendicular to the subchondral bone near the bone, and collagen predominantly

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

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

2017-12-01

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

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

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

2014-04-01

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

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

Calcified cartilage or bone? Collagens in the tessellated endoskeletons of cartilaginous fish (sharks and rays).

Science.gov (United States)

Seidel, Ronald; Blumer, Michael; Pechriggl, Elisabeth-Judith; Lyons, Kady; Hall, Brian K; Fratzl, Peter; Weaver, James C; Dean, Mason N

2017-10-01

The primary skeletal tissue in elasmobranchs -sharks, rays and relatives- is cartilage, forming both embryonic and adult endoskeletons. Only the skeletal surface calcifies, exhibiting mineralized tiles (tesserae) sandwiched between a cartilage core and overlying fibrous perichondrium. These two tissues are based on different collagens (Coll II and I, respectively), fueling a long-standing debate as to whether tesserae are more like calcified cartilage or bone (Coll 1-based) in their matrix composition. We demonstrate that stingray (Urobatis halleri) tesserae are bipartite, having an upper Coll I-based 'cap' that merges into a lower Coll II-based 'body' zone, although tesserae are surrounded by cartilage. We identify a 'supratesseral' unmineralized cartilage layer, between tesserae and perichondrium, distinguished from the cartilage core in containing Coll I and X (a common marker for mammalian mineralization), in addition to Coll II. Chondrocytes within tesserae appear intact and sit in lacunae filled with Coll II-based matrix, suggesting tesserae originate in cartilage, despite comprising a diversity of collagens. Intertesseral joints are also complex in their collagenous composition, being similar to supratesseral cartilage closer to the perichondrium, but containing unidentified fibrils nearer the cartilage core. Our results indicate a unique potential for tessellated cartilage in skeletal biology research, since it lacks features believed diagnostic for vertebrate cartilage mineralization (e.g. hypertrophic and apoptotic chondrocytes), while offering morphologies amenable for investigating the regulation of complex mineralized ultrastructure and tissues patterned on multiple collagens. Copyright © 2017 Elsevier Inc. All rights reserved.

Silencing of microRNA-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes by targeting FOXC1: miR-138 promotes cartilage degradation.

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Yuan, Y; Zhang, G Q; Chai, W; Ni, M; Xu, C; Chen, J Y

2016-10-01

Osteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage. Human articular cartilage was obtained from patients with and without OA, and chondrocytes were isolated and stimulated by IL-1β. The expression levels of miR-138-5p in cartilage and chondrocytes were both determined. After transfection with miR-138-5p mimics, allele-specific oligonucleotide (ASO)-miR-138-5p, or their negative controls, the messenger RNA (mRNA) levels of aggrecan (ACAN), collagen type II and alpha 1 (COL2A1), the protein levels of glycosaminoglycans (GAGs), and both the mRNA and protein levels of matrix metalloproteinase (MMP)-13 were evaluated. Luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot were performed to explore whether Forkhead Box C1 (FOCX1) was a target of miR-138-5p. Further, we co-transfected OA chondrocytes with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 and then stimulated with IL-1β to determine whether miR-138-5p-mediated IL-1β-induced cartilage matrix degradation resulted from targeting FOXC1. MiR-138-5p was significantly increased in OA cartilage and in chondrocytes in response to IL-1β-stimulation. Overexpression of miR-138-5p significantly increased the IL-1β-induced downregulation of COL2A1, ACAN, and GAGs, and increased the IL-1β-induced over expression of MMP-13.We found that FOXC1 is directly regulated by miR-138-5p. Additionally, co-transfection with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 resulted in higher levels of COL2A1, ACAN, and GAGs, but lower levels of MMP-13. miR-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes, possibly by targeting FOXC1.Cite this article: Y. Yuan, G. Q. Zhang, W. Chai,M. Ni, C. Xu, J

Chicken collagen type II reduces articular cartilage destruction in a model of osteoarthritis in rats.

Science.gov (United States)

Xu, D; Shen, W

2007-06-01

To evaluate the therapeutic effects of domestic chicken collagen type II (CCII) on rat osteoarthritis (OA) and analyze concomitant changes in the level of Matrix metalloproteinase (MMP)-13, MMP-9, Cathepsin K and their mRNA as well as the tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA in articular cartilage of osteoarthritic rats. Osteoarthritis models were surgically induced. Morphology of articular cartilage was done by haematoxylin and eosin staining and Mankin score was calculated, immunohistochemistry of MMP-13, MMP-9 and Cathepsin K was done by ABC method while the mRNA level for MMP-13, MMP-9, cathepsin K as well as TIMP-1 was evaluated by RT-PCR method. Oral administration of CCII reduced the morphological changes of osteoarthritic cartilage (shown by Mankin score), decreased levels of MMP-13, MMP-9, cathepsin K as well as their mRNA in articular cartilage from osteoarthritic rats while it exhibited no effect on TIMP-1 mRNA. Oral CCII reduced articular cartilage degradation of osteoarthritic rats and may probably be a potent drug candidate for OA treatment.

Mechanical stimulation to stimulate formation of a physiological collagen architecture in tissue-engineered cartilage; a numerical study

NARCIS (Netherlands)

Khoshgoftar, M.; Donkelaar, van C.C.; Ito, K.

2011-01-01

The load-bearing capacity of today's tissue-engineered (TE) cartilage is insufficient. The arcade-like collagen network in native cartilage plays an important role in its load-bearing properties. Inducing the formation of such collagen architecture in engineered cartilage can, therefore, enhance

Nondestructive evaluation of a new hydrolytically degradable and photo-clickable PEG hydrogel for cartilage tissue engineering.

Science.gov (United States)

Neumann, Alexander J; Quinn, Timothy; Bryant, Stephanie J

2016-07-15

Photopolymerizable and hydrolytically labile poly(ethylene glycol) (PEG) hydrogels formed from photo-clickable reactions were investigated as cell delivery platforms for cartilage tissue engineering (TE). PEG hydrogels were formed from thiol-norbornene PEG macromers whereby the crosslinks contained caprolactone segments with hydrolytically labile ester linkages. Juvenile bovine chondrocytes encapsulated in the hydrogels were cultured for up to four weeks and assessed biochemically and histologically, using standard destructive assays, and for mechanical and ultrasound properties, as nondestructive assays. Bulk degradation of acellular hydrogels was confirmed by a decrease in compressive modulus and an increase in mass swelling ratio over time. Chondrocytes deposited increasing amounts of sulfated glycosaminoglycans and collagens in the hydrogels with time. Spatially, collagen type II and aggrecan were present in the neotissue with formation of a territorial matrix beginning at day 21. Nondestructive measurements revealed an 8-fold increase in compressive modulus from days 7 to 28, which correlated with total collagen content. Ultrasound measurements revealed changes in the constructs over time, which differed from the mechanical properties, and appeared to correlate with ECM structure and organization shown by immunohistochemical analysis. Overall, non-destructive and destructive measurements show that this new hydrolytically degradable PEG hydrogel is promising for cartilage TE. Designing synthetic hydrogels whose degradation matches tissue growth is critical to maintaining mechanical integrity as the hydrogel degrades and new tissue forms, but is challenging due to the nature of the hydrogel crosslinks that inhibit diffusion of tissue matrix molecules. This study details a promising, new, photo-clickable and synthetic hydrogel whose degradation supports cartilaginous tissue matrix growth leading to the formation of a territorial matrix, concomitant with an

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

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

Postnatal development of depth-dependent collagen density in ovine articular cartilage

Directory of Open Access Journals (Sweden)

Kranenbarg Sander

2010-10-01

Full Text Available Abstract Background Articular cartilage (AC is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Adult AC is characterised by a depth-dependent composition and structure of the extracellular matrix that results in depth-dependent mechanical properties, important for the functions of adult AC. Collagen is the most abundant solid component and it affects the mechanical behaviour of AC. The current objective is to quantify the postnatal development of depth-dependent collagen density in sheep (Ovis aries AC between birth and maturity. We use Fourier transform infra-red micro-spectroscopy to investigate collagen density in 48 sheep divided over ten sample points between birth (stillborn and maturity (72 weeks. In each animal, we investigate six anatomical sites (caudal, distal and rostral locations at the medial and lateral side of the joint in the distal metacarpus of a fore leg and a hind leg. Results Collagen density increases from birth to maturity up to our last sample point (72 weeks. Collagen density increases at the articular surface from 0.23 g/ml ± 0.06 g/ml (mean ± s.d., n = 48 at 0 weeks to 0.51 g/ml ± 0.10 g/ml (n = 46 at 72 weeks. Maximum collagen density in the deeper cartilage increases from 0.39 g/ml ± 0.08 g/ml (n = 48 at 0 weeks to 0.91 g/ml ± 0.13 g/ml (n = 46 at 72 weeks. Most collagen density profiles at 0 weeks (85% show a valley, indicating a minimum, in collagen density near the articular surface. At 72 weeks, only 17% of the collagen density profiles show a valley in collagen density near the articular surface. The fraction of profiles with this valley stabilises at 36 weeks. Conclusions Collagen density in articular cartilage increases in postnatal life with depth-dependent variation, and does not stabilize up to 72 weeks, the last sample point in our study. We find strong evidence for a valley in collagen densities near the articular surface that is present in the youngest

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

ABSTRACT: INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a homopentameric protein in cartilage. The development of arthritis, like collagen-induced arthritis (CIA), involves cartilage as a target tissue. We have investigated the development of CIA in COMP-deficient mice. METHODS: COMP......-deficient mice in the 129/Sv background were backcrossed for 10 generations against B10.Q mice, which are susceptible to chronic CIA. COMP-deficient and wild-type mice were tested for onset, incidence, and severity of arthritis in both the collagen and collagen antibody-induced arthritis models. Serum anti......-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...

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.)

Effects of collagen matrix and bioreactor cultivation on cartilage regeneration of a full-thickness critical-size knee joint cartilage defects with subchondral bone damage in a rabbit model.

Directory of Open Access Journals (Sweden)

Kuo-Hwa Wang

Full Text Available Cartilage has limited self-repair ability. The purpose of this study was to investigate the effects of different species of collagen-engineered neocartilage for the treatment of critical-size defects in the articular joint in a rabbit model. Type II and I collagen obtained from rabbits and rats was mixed to form a scaffold. The type II/I collagen scaffold was then mixed with rabbit chondrocytes to biofabricate neocartilage constructs using a rotating cell culture system [three-dimensional (3D-bioreactor]. The rabbit chondrocytes were mixed with rabbit collagen scaffold and rat collagen scaffold to form neoRBT (neo-rabbit cartilage and neoRAT (neo-rat cartilage constructs, respectively. The neocartilage matrix constructs were implanted into surgically created defects in rabbit knee chondyles, and histological examinations were performed after 2 and 3 months. Cartilage-like lacunae formation surrounding the chondrocytes was noted in the cell cultures. After 3 months, both the neoRBT and neoRAT groups showed cartilage-like repair tissue covering the 5-mm circular, 4-mm-deep defects that were created in the rabbit condyle and filled with neocartilage plugs. Reparative chondrocytes were aligned as apparent clusters in both the neoRAT and neoRBT groups. Both neoRBT and neoRAT cartilage repair demonstrated integration with healthy adjacent tissue; however, more integration was obtained using the neoRAT cartilage. Our data indicate that different species of type II/I collagen matrix and 3D bioreactor cultivation can facilitate cartilage engineering in vitro for the repair of critical-size defect.

Calcitonin directly attenuates collagen type II degradation by inhibition of matrix metalloproteinase expression and activity in articular chondrocytes

DEFF Research Database (Denmark)

Sondergaard, B C; Wulf, H; Henriksen, K

2006-01-01

OBJECTIVE: Calcitonin was recently reported to counter progression of cartilage degradation in an experimental model of osteoarthritis, and the effects were primarily suggested to be mediated by inhibition of subchondral bone resorption. We investigated direct effects of calcitonin on chondrocytes...... by assessing expression of the receptor and pharmacological effects on collagen type II degradation under ex vivo and in vivo conditions. METHODS: Localization of the calcitonin receptor on articular chondrocytes was investigated by immunohistochemistry, and the expression by reverse transcriptase polymerase.......0001-1 microM]. In vivo, cartilage degradation was investigated in ovariectomized (OVX) rats administered with oral calcitonin [2 mg/kg calcitonin] for 9 weeks. RESULTS: The calcitonin receptor was identified in articular chondrocytes by immunohistochemistry and RT-PCR. Calcitonin concentration...

The architecture of cartilage: Elemental maps and scanning transmission ion microscopy/tomography

International Nuclear Information System (INIS)

Reinert, Tilo; Reibetanz, Uta; Schwertner, Michael; Vogt, Juergen; Butz, Tilman; Sakellariou, Arthur

2002-01-01

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

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

OpenAIRE

1986-01-01

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

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

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...

MAPKs are essential upstream signaling pathways in proteolytic cartilage degradation--divergence in pathways leading to aggrecanase and MMP-mediated articular cartilage degradation

DEFF Research Database (Denmark)

Sondergaard, B-C; Schultz, N; Madsen, S H

2010-01-01

Matrix metalloproteinases (MMPs) and aggrecanases are essential players in cartilage degradation. However, the signaling pathways that results in MMP and/or aggrecanase synthesis and activation are not well understood. We investigated the molecular events leading to MMP- and aggrecanase-mediated ......Matrix metalloproteinases (MMPs) and aggrecanases are essential players in cartilage degradation. However, the signaling pathways that results in MMP and/or aggrecanase synthesis and activation are not well understood. We investigated the molecular events leading to MMP- and aggrecanase......-mediated cartilage degradation....

Comparison of three types of chondrocytes in collagen scaffolds for cartilage tissue engineering

International Nuclear Information System (INIS)

Zhang Lu; Spector, Myron

2009-01-01

The objective of this study was to compare the chondrogenesis in type I and II collagen scaffolds seeded with chondrocytes from three types of cartilage, after four weeks of culture: auricular (AU), articular (AR) and meniscal (ME). Related aims were to investigate the expression of a contractile muscle actin isoform, α-smooth muscle actin (SMA), in the cells in the scaffold and to determine the presence of a lubricating glycoprotein, lubricin, in the constructs. Adult goat AU, AR and ME chondrocytes were seeded into two types of collagen scaffolds: type II collagen and type I/III collagen. After four weeks of culture, the constructs were prepared for histochemical and immunohistochemical analysis of the distribution of glycosaminoglycan (GAG), types I and II collagen, elastin, SM and lubricin. AU constructs contained substantially more tissue than the AR and ME samples. The AU constructs exhibited neocartilage, but no elastin. There were no notable differences between the type I and II collagen scaffolds. Novel findings were the expression of SMA by the AU cells in the scaffolds and the presence of lubricin in the AR and AU constructs. AU cells have the capability to produce cartilage in collagen scaffolds under conditions in which there is little histogenesis by AR and ME cells.

Comparison of three types of chondrocytes in collagen scaffolds for cartilage tissue engineering

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Zhang Lu [Department of Plastic and Reconstructive Surgery, Shanghai Tissue Engineering Center, Shanghai 9th People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Spector, Myron, E-mail: luzhangmd@gmail.co [Tissue Engineering, VA Boston Healthcare System, Boston, MA (United States)

2009-08-15

The objective of this study was to compare the chondrogenesis in type I and II collagen scaffolds seeded with chondrocytes from three types of cartilage, after four weeks of culture: auricular (AU), articular (AR) and meniscal (ME). Related aims were to investigate the expression of a contractile muscle actin isoform, alpha-smooth muscle actin (SMA), in the cells in the scaffold and to determine the presence of a lubricating glycoprotein, lubricin, in the constructs. Adult goat AU, AR and ME chondrocytes were seeded into two types of collagen scaffolds: type II collagen and type I/III collagen. After four weeks of culture, the constructs were prepared for histochemical and immunohistochemical analysis of the distribution of glycosaminoglycan (GAG), types I and II collagen, elastin, SM and lubricin. AU constructs contained substantially more tissue than the AR and ME samples. The AU constructs exhibited neocartilage, but no elastin. There were no notable differences between the type I and II collagen scaffolds. Novel findings were the expression of SMA by the AU cells in the scaffolds and the presence of lubricin in the AR and AU constructs. AU cells have the capability to produce cartilage in collagen scaffolds under conditions in which there is little histogenesis by AR and ME cells.

Stability of Collagen Scaffold Implants for Animals with Iatrogenic Articular Cartilage Defects

Directory of Open Access Journals (Sweden)

Josef Jančář

2009-01-01

Full Text Available Synthesis and characterization of biodegradable hydrogels based on collagen modified by addition of synthetic biodegradable copolymer intended for preparation of porous scaffolds for mesenchymal stem cells used for possible implantation to animals with articular surface defects was investigated. The synthetic biodegradable tri-block copolymer used was the block copolymer of polyethylene glycol (PEG, polylactic acid (PLA, polyglycolic acid (PGA (PEG-PLGA endcapped with itaconic acid (ITA. The water-soluble carbodiimide and N-hydroxysuccimide system (EDC-NHS was chosen as the cross-linking agent used to control the rate of hydrogel resorption. Dependence of the physical properties of the prepared hydrogels on the concentration of the EDC-NHS cross-linker, reaction time and concentration of PEG-PLGA-ITA copolymer was examined. Swelling behaviour, thermal stability, surface morphology and degradation rate were also characterized. Based on the obtained results, it can be concluded that increase in concentration of the cross-linking agent, as well as prolonged cross-linking time and increased amount of synthetic copolymer lead to enhanced thermal stability of the gels together with a reduced swelling ratio and degradation rate in saline. The resorption rate of these gels used in preparation of cartilage scaffolds can be controlled over a wide time interval by varying the collagen/(PEG-PLGA-ITA blend composition or the conditions of the cross-linking reaction.

The identification of CD163 expressing phagocytic chondrocytes in joint cartilage and its novel scavenger role in cartilage degradation.

Directory of Open Access Journals (Sweden)

Kai Jiao

Full Text Available BACKGROUND: Cartilage degradation is a typical characteristic of arthritis. This study examined whether there was a subset of phagocytic chondrocytes that expressed the specific macrophage marker, CD163, and investigated their role in cartilage degradation. METHODS: Cartilage from the knee and temporomandibular joints of Sprague-Dawley rats was harvested. Cartilage degradation was experimentally-induced in rat temporomandibular joints, using published biomechanical dental methods. The expression levels of CD163 and inflammatory factors within cartilage, and the ability of CD163(+ chondrocytes to conduct phagocytosis were investigated. Cartilage from the knees of patients with osteoarthritis and normal cartilage from knee amputations was also investigated. RESULTS: In the experimentally-induced degrading cartilage from temporomandibular joints, phagocytes were capable of engulfing neighboring apoptotic and necrotic cells, and the levels of CD163, TNF-α and MMPs were all increased (P0.05. CD163(+ chondrocytes were found in the cartilage mid-zone of temporomandibular joints and knee from healthy, three-week old rats. Furthermore, an increased number of CD163(+ chondrocytes with enhanced phagocytic activity were present in Col-II(+ chondrocytes isolated from the degraded cartilage of temporomandibular joints in the eight-week experimental group compared with their age-matched controls. Increased number with enhanced phagocytic activity of CD163(+ chondrocytes were also found in isolated Col-II(+ chondrocytes stimulated with TNF-α (P<0.05. Mid-zone distribution of CD163(+ cells accompanied with increased expression of CD163 and TNF-α were further confirmed in the isolated Col-II(+ chondrocytes from the knee cartilage of human patients with osteoarthritis, in contrast to the controls (both P<0.05. CONCLUSIONS: An increased number of CD163(+ chondrocytes with enhanced phagocytic activity were discovered within degraded joint cartilage, indicating a

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). Th...

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

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

2012-06-29

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.

Exosomes from embryonic mesenchymal stem cells alleviate osteoarthritis through balancing synthesis and degradation of cartilage extracellular matrix.

Science.gov (United States)

Wang, Yafei; Yu, Dongsheng; Liu, Zhiming; Zhou, Fang; Dai, Jun; Wu, Bingbing; Zhou, Jing; Heng, Boon Chin; Zou, Xiao Hui; Ouyang, Hongwei; Liu, Hua

2017-08-14

Mesenchymal stem cell therapy for osteoarthritis (OA) has been widely investigated, but the mechanisms are still unclear. Exosomes that serve as carriers of genetic information have been implicated in many diseases and are known to participate in many physiological processes. Here, we investigate the therapeutic potential of exosomes from human embryonic stem cell-induced mesenchymal stem cells (ESC-MSCs) in alleviating osteoarthritis (OA). Exosomes were harvested from conditioned culture media of ESC-MSCs by a sequential centrifugation process. Primary mouse chondrocytes treated with interleukin 1 beta (IL-1β) were used as an in vitro model to evaluate the effects of the conditioned medium with or without exosomes and titrated doses of isolated exosomes for 48 hours, prior to immunocytochemistry or western blot analysis. Destabilization of the medial meniscus (DMM) surgery was performed on the knee joints of C57BL/6 J mice as an OA model. This was followed by intra-articular injection of either ESC-MSCs or their exosomes. Cartilage destruction and matrix degradation were evaluated with histological staining and OARSI scores at the post-surgery 8 weeks. We found that intra-articular injection of ESC-MSCs alleviated cartilage destruction and matrix degradation in the DMM model. Further in vitro studies illustrated that this effect was exerted through ESC-MSC-derived exosomes. These exosomes maintained the chondrocyte phenotype by increasing collagen type II synthesis and decreasing ADAMTS5 expression in the presence of IL-1β. Immunocytochemistry revealed colocalization of the exosomes and collagen type II-positive chondrocytes. Subsequent intra-articular injection of exosomes derived from ESC-MSCs successfully impeded cartilage destruction in the DMM model. The exosomes from ESC-MSCs exert a beneficial therapeutic effect on OA by balancing the synthesis and degradation of chondrocyte extracellular matrix (ECM), which in turn provides a new target for OA drug

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

Science.gov (United States)

de Mulder, Eric L W; Hannink, Gerjon; van Kuppevelt, Toin H; Daamen, Willeke F; Buma, Pieter

2014-02-01

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 to the AC surface would result in qualitatively better tissue repair due to a guided cellular influx from the subchondral bone. By specific freezing protocols, type I collagen scaffolds with isotropic and anisotropic fiber architectures were produced. Rabbits were operated on bilaterally and two full thickness defects were created in each knee joint. The defects were filled with (1) an isotropic scaffold, (2) an anisotropic scaffold with pores parallel to the cartilage surface, and (3) an anisotropic scaffold with pores perpendicular to the cartilage surface. Empty defects served as controls. After 4 (n=13) and 12 (n=13) weeks, regeneration was scored qualitatively and quantitatively using histological analysis and a modified O'Driscoll score. After 4 weeks, all defects were completely filled with partially differentiated hyaline cartilage tissue. No differences in O'Driscoll scores were measured between empty defects and scaffold types. After 12 weeks, all treatments led to hyaline cartilage repair visualized by increased glycosaminoglycan staining. Total scores were significantly increased for parallel anisotropic and empty defects over time (phyaline-like cartilage repair. Fiber architecture had no effect on cartilage repair.

Autofluorescence lifetime metrology for label-free detection of cartilage matrix degradation

Science.gov (United States)

Nickdel, Mohammad B.; Lagarto, João. L.; Kelly, Douglas J.; Manning, Hugh B.; Yamamoto, Kazuhiro; Talbot, Clifford B.; Dunsby, Christopher; French, Paul; Itoh, Yoshifumi

2014-03-01

Degradation of articular cartilage extracellular matrix (ECM) by proteolytic enzyme is the hallmark of arthritis that leads to joint destruction. Detection of early biochemical changes in cartilage before irreversible structural damages become apparent is highly desirable. Here we report that the autofluorescence decay profile of cartilage is significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A multidimensional fluorometer utilizing ultraviolet excitation at 355 nm or 375 nm coupled to a fibreoptic probe was developed for single point time-resolved AFL measurements of porcine articular cartilage explants treated with different proteinases. Degradation of cartilage matrix components by treating with bacterial collagenase, matrix metalloproteinase 1, or trypsin resulted in significant reduction of AFL of the cartilage in both a dose and time dependent manner. Differences in cartilage AFL were also confirmed by fluorescence lifetime imaging microscopy (FLIM). Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may be utilized for diagnosis of arthritis as well as monitoring the efficacy of anti-arthritic therapeutic agents.

Induction of increased cAMP levels in articular chondrocytes blocks matrix metalloproteinase-mediated cartilage degradation, but not aggrecanase-mediated cartilage degradation

DEFF Research Database (Denmark)

Karsdal, Morten Asser; Sumer, Eren Ufuk; Wulf, Helle

2007-01-01

OBJECTIVE: Calcitonin has been suggested to have chondroprotective effects. One signaling pathway of calcitonin is via the second messenger cAMP. We undertook this study to investigate whether increased cAMP levels in chondrocytes would be chondroprotective. METHODS: Cartilage degradation......-dependently inhibited by forskolin and IBMX. The highest concentration of IBMX lowered cytokine-induced release of sGAG by 72%. CONCLUSION: Levels of cAMP in chondrocytes play a key role in controlling catabolic activity. Increased cAMP levels in chondrocytes inhibited MMP expression and activity and consequently...... strongly inhibited cartilage degradation. Specific cAMP modulators in chondrocytes may be potential treatments for cartilage degenerative diseases....

Analysis of human knee osteoarthritic cartilage using polarization sensitive second harmonic generation microscopy

Science.gov (United States)

Kumar, Rajesh; Grønhaug, Kirsten M.; Romijn, Elisabeth I.; Drogset, Jon O.; Lilledahl, Magnus B.

2014-05-01

Osteoarthritis is one of the most prevalent joint diseases in the world. Although the cause of osteoarthritis is not exactly clear, the disease results in a degradation of the quality of the articular cartilage including collagen and other extracellular matrix components. We have investigated alterations in the structure of collagen fibers in the cartilage tissue of the human knee using mulitphoton microscopy. Due to inherent high nonlinear susceptibility, ordered collagen fibers present in the cartilage tissue matrix produces strong second harmonic generation (SHG) signals. Significant morphological differences are found in different Osteoarthritic grades of cartilage by SHG microscopy. Based on the polarization analysis of the SHG signal, we find that a few locations of hyaline cartilage (mainly type II collagen) is being replaced by fibrocartilage (mainly type I cartilage), in agreement with earlier literature. To locate the different types and quantify the alteration in the structure of collagen fiber, we employ polarization-SHG microscopic analysis, also referred to as _-tensor imaging. The image analysis of p-SHG image obtained by excitation polarization measurements would represent different tissue constituents with different numerical values at pixel level resolution.

Selective laser sintered poly-ε-caprolactone scaffold hybridized with collagen hydrogel for cartilage tissue engineering

International Nuclear Information System (INIS)

Chen, Chih-Hao; Chen, Jyh-Ping; Shyu, Victor Bong-Hang; Lee, Ming-Yih

2014-01-01

Selective laser sintering (SLS), an additive manufacturing (AM) technology, can be used to produce tissue engineering scaffolds with pre-designed macro and micro features based on computer-aided design models. An in-house SLS machine was built and 3D poly-ε-caprolactone (PCL) scaffolds were manufactured using a layer-by-layer design of scaffold struts with varying orientations (0°/45°/0°/45°, 0°/90°/0°/90°, 0°/45°/90°/135°), producing scaffolds with pores of different shapes and distribution. To better enhance the scaffold properties, chondrocytes were seeded in collagen gel and loaded in scaffolds for cartilage tissue engineering. Gel uptake and dynamic mechanical analysis demonstrated the better suitability of the 0°/90°/0°/90° scaffolds for reconstructive cartilage tissue engineering purposes. Chondrocytes were then seeded onto the 0°/90°/0°/90° scaffolds in collagen I hydrogel (PCL/COL1) and compared to medium-suspended cells in terms of their cartilage-like tissue engineering parameters. PCL/COL1 allowed better cell proliferation when compared to PCL or two-dimensional tissue culture polystyrene. Scanning electron microscopy and confocal microscopy observations demonstrated a similar trend for extracellular matrix production and cell survival. Glycosaminoglycan and collagen II quantification also demonstrated the superior matrix secretion properties of PCL/COL1 hybrid scaffolds. Collagen-gel-suspended chondrocytes loaded in SLS-manufactured PCL scaffolds may provide a means of producing tissue-engineered cartilage with customized shapes and designs via AM technology. (paper)

Uncoupling of collagen II metabolism in newly diagnosed, untreated rheumatoid arthritis is linked to inflammation and antibodies against cyclic citrullinated peptides

DEFF Research Database (Denmark)

Christensen, Anne Friesgaard; Hørslev-Petersen, Kim; Christgau, Stephan

2010-01-01

. METHODS: One hundred sixty patients with newly diagnosed, untreated RA entered the Cyclosporine, Methotrexate, Steroid in RA (CIMESTRA) trial. Disease activity and radiograph status were measured at baseline and 4 years. The N-terminal propeptide of collagen IIA (PIIANP) and the cross-linked C...... associations of collagen II anabolism (PIIANP) and collagen II degradation (CTX-II) with anti-CCP, synovitis, and radiographic progression indicate that at this early stage of RA, cartilage collagen degradation is mainly driven by synovitis, while anti-CCP antibodies may interfere with cartilage regeneration...

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.

Changes in collagen synthesis and degradation during skeletal muscle growth

International Nuclear Information System (INIS)

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

1985-01-01

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

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.)

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

International Nuclear Information System (INIS)

Ohyabu, Yohimi; Adegawa, Takuro; Yoshioka, Tomohiko; Ikoma, Toshiyuki; Uemura, Toshimasa; Tanaka, Junzo

2010-01-01

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.

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.

A novel in vitro bovine cartilage punch model for assessing the regeneration of focal cartilage defects with biocompatible bacterial nanocellulose

Science.gov (United States)

2013-01-01

Introduction Current therapies for articular cartilage defects fail to achieve qualitatively sufficient tissue regeneration, possibly because of a mismatch between the speed of cartilage rebuilding and the resorption of degradable implant polymers. The present study focused on the self-healing capacity of resident cartilage cells in conjunction with cell-free and biocompatible (but non-resorbable) bacterial nanocellulose (BNC). This was tested in a novel in vitro bovine cartilage punch model. Methods Standardized bovine cartilage discs with a central defect filled with BNC were cultured for up to eight weeks with/without stimulation with transforming growth factor-β1 (TGF-β1. Cartilage formation and integrity were analyzed by histology, immunohistochemistry and electron microscopy. Content, release and neosynthesis of the matrix molecules proteoglycan/aggrecan, collagen II and collagen I were also quantified. Finally, gene expression of these molecules was profiled in resident chondrocytes and chondrocytes migrated onto the cartilage surface or the implant material. Results Non-stimulated and especially TGF-β1-stimulated cartilage discs displayed a preserved structural and functional integrity of the chondrocytes and surrounding matrix, remained vital in long-term culture (eight weeks) without signs of degeneration and showed substantial synthesis of cartilage-specific molecules at the protein and mRNA level. Whereas mobilization of chondrocytes from the matrix onto the surface of cartilage and implant was pivotal for successful seeding of cell-free BNC, chondrocytes did not immigrate into the central BNC area, possibly due to the relatively small diameter of its pores (2 to 5 μm). Chondrocytes on the BNC surface showed signs of successful redifferentiation over time, including increase of aggrecan/collagen type II mRNA, decrease of collagen type I mRNA and initial deposition of proteoglycan and collagen type II in long-term high-density pellet cultures

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

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

2016-07-20

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

Engineering Cartilage

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... 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 ...

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

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McKleroy, William; Lee, Ting-Hein

2013-01-01

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

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

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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.

Degradation of type IV collagen by neoplastic human skin fibroblasts

International Nuclear Information System (INIS)

Sheela, S.; Barrett, J.C.

1985-01-01

An assay for the degradation of type IV (basement membrane) collagen was developed as a biochemical marker for neoplastic cells from chemically transformed human skin fibroblasts. Type IV collagen was isolated from basement membrane of Syrian hamster lung and type I collagen was isolated from rat tails; the collagens were radioactively labelled by reductive alkylation. The abilities of normal (KD) and chemically transformed (Hut-11A) human skin fibroblasts to degrade the collagens were studied. A cell-associated assay was performed by growing either normal or transformed cells in the presence of radioactively labelled type IV collagen and measuring the released soluble peptides in the medium. This assay also demonstrated that KD cells failed to synthesize an activity capable of degrading type IV collagen whereas Hut-11A cells degraded type IV collagen in a linear manner for up to 4 h. Human serum at very low concentrations, EDTA and L-cysteine inhibited the enzyme activity, whereas protease inhibitors like phenylmethyl sulfonyl fluoride, N-ethyl maleimide or soybean trypsin inhibitor did not inhibit the enzyme from Hut-11A cells. These results suggest that the ability to degrade specifically type IV collagen may be an important marker for neoplastic human fibroblasts and supports a role for this collagenase in tumor cell invasion

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.

Differential association of the N-propeptide of collagen IIA (PIIANP) and collagen II C-telopeptide (CTX-II) with synovitis and erosions in early and longstanding rheumatoid arthritis

DEFF Research Database (Denmark)

Christensen, A F; Lottenburger, T; Lindegaard, H

2009-01-01

OBJECTIVES: To determine the N-terminal propeptide of collagen IIA (PIIANP) in early and established rheumatoid arthritis (RA) and to study the association with collagen II degradation assessed by its C-telopeptide (CTX-II), x-ray status and disease activity measures. METHODS: Two cohorts of RA......-ray progression (p=0.84). There was no correlation between PIIANP and CTX-II. CONCLUSION: Declining PIIANP with increasing RA duration and persistently increased CTX-II indicate that cartilage anabolic and degradative pathways are unbalanced from clinical RA onset. Furthermore, that collagen II depletion in RA...... is both mediated by anti-anabolic effects unassociated with synovitis (decreased PIIANP) and by excess collagen II degradation linked to synovitis (increased CTX-II)....

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

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

2017-02-01

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

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

Science.gov (United States)

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

2017-01-01

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

The metabolic dynamics of cartilage explants over a long-term culture period

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E.K Moo

2011-01-01

Full Text Available INTRODUCTION: Although previous studies have been performed on cartilage explant cultures, the generalized dynamics of cartilage metabolism after extraction from the host are still poorly understood due to differences in the experimental setups across studies, which in turn prevent building a complete picture. METHODS: In this study, we investigated the response of cartilage to the trauma sustained during extraction and determined the time needed for the cartilage to stabilize. Explants were extracted aseptically from bovine metacarpal-phalangeal joints and cultured for up to 17 days. RESULTS: The cell viability, cell number, proteoglycan content, and collagen content of the harvested explants were analyzed at 0, 2, 10, and 17 days after explantation. A high percentage of the cartilage explants were found to be viable. The cell density initially increased significantly but stabilized after two days. The proteoglycan content decreased gradually over time, but it did not decrease to a significant level due to leakage through the distorted peripheral collagen network and into the bathing medium. The collagen content remained stable for most of the culture period until it dropped abruptly on day 17. CONCLUSION: Overall, the tested cartilage explants were sustainable over long-term culture. They were most stable from day 2 to day 10. The degradation of the collagen on day 17 did not reach diseased levels, but it indicated the potential of the cultures to develop into degenerated cartilage. These findings have implications for the application of cartilage explants in pathophysiological fields.

Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs.

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Jabłońska-Trypuć, Agata; Matejczyk, Marzena; Rosochacki, Stanisław

2016-01-01

The main group of enzymes responsible for the collagen and other protein degradation in extracellular matrix (ECM) are matrix metalloproteinases (MMPs). Collagen is the main structural component of connective tissue and its degradation is a very important process in the development, morphogenesis, tissue remodeling, and repair. Typical structure of MMPs consists of several distinct domains. MMP family can be divided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other non-classified MMPs. MMPs and their inhibitors have multiple biological functions in all stages of cancer development: from initiation to outgrowth of clinically relevant metastases and likewise in apoptosis and angiogenesis. MMPs and their inhibitors are extensively examined as potential anticancer drugs. MMP inhibitors can be divided into two main groups: synthetic and natural inhibitors. Selected synthetic inhibitors are in clinical trials on humans, e.g. synthetic peptides, non-peptidic molecules, chemically modified tetracyclines, and bisphosphonates. Natural MMP inhibitors are mainly isoflavonoids and shark cartilage.

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

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

2016-08-01

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

Postnatal changes to the mechanical properties of articular cartilage are driven by the evolution of its collagen network

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AR Gannon

2015-01-01

Full Text Available While it is well established that the composition and organisation of articular cartilage dramatically change during skeletal maturation, relatively little is known about how this impacts the mechanical properties of the tissue. In this study, digital image correlation was first used to quantify spatial deformation within mechanically compressed skeletally immature (4 and 8 week old and mature (1 and 3 year old porcine articular cartilage. The compressive modulus of the immature tissue was relatively homogeneous, while the stiffness of mature articular cartilage dramatically increased with depth from the articular surface. Other, well documented, biomechanical characteristics of the tissue also emerged with skeletal maturity, such as strain-softening and a depth-dependent Poisson’s ratio. The most significant changes that occurred with age were in the deep zone of the tissue, where an order of magnitude increase in compressive modulus (from 0.97 MPa to 9.4 MPa for low applied strains was observed from 4 weeks postnatal to skeletal maturity. These temporal increases in compressive stiffness occurred despite a decrease in tissue sulphated glycosaminoglycan content, but were accompanied by increases in tissue collagen content. Furthermore, helium ion microscopy revealed dramatic changes in collagen fibril alignment through the depth of the tissue with skeletal maturity, as well as a fivefold increase in fibril diameter with age. Finally, computational modelling was used to demonstrate how both collagen network reorganisation and collagen stiffening play a key role in determining the final compressive mechanical properties of the tissue. Together these findings provide a unique insight into evolving structure-function relations in articular cartilage.

Tumor-Associated Macrophages Derived from Circulating Inflammatory Monocytes Degrade Collagen through Cellular Uptake

DEFF Research Database (Denmark)

Madsen, Daniel Hargbøl; Jürgensen, Henrik Jessen; Siersbæk, Majken Storm

2017-01-01

-associated macrophage (TAM)-like cells that degrade collagen in a mannose receptor-dependent manner. Accordingly, mannose-receptor-deficient mice display increased intratumoral collagen. Whole-transcriptome profiling uncovers a distinct extracellular matrix-catabolic signature of these collagen-degrading TAMs. Lineage......-ablation studies reveal that collagen-degrading TAMs originate from circulating CCR2+ monocytes. This study identifies a function of TAMs in altering the tumor microenvironment through endocytic collagen turnover and establishes macrophages as centrally engaged in tumor-associated collagen degradation. Madsen et...

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

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Kate Stuart

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

Hyaline cartilage cells outperform mandibular condylar cartilage cells in a TMJ fibrocartilage tissue engineering application.

Science.gov (United States)

Wang, L; Lazebnik, M; Detamore, M S

2009-03-01

To compare temporomandibular joint (TMJ) condylar cartilage cells in vitro to hyaline cartilage cells cultured in a three-dimensional (3D) environment for tissue engineering of mandibular condylar cartilage. Mandibular condylar cartilage and hyaline cartilage cells were harvested from pigs and cultured for 6 weeks in polyglycolic acid (PGA) scaffolds. Both types of cells were treated with glucosamine sulfate (0.4 mM), insulin-like growth factor-I (IGF-I) (100 ng/ml) and their combination. At weeks 0 and 6, cell number, glycosaminoglycan (GAG) and collagen content were determined, types I and II collagen were visualized by immunohistochemistry and GAGs were visualized by histology. Hyaline cartilage cells produced from half an order to a full order of magnitude more GAGs and collagen than mandibular condylar cartilage cells in 3D culture. IGF-I was a highly effective signal for biosynthesis with hyaline cartilage cells, while glucosamine sulfate decreased cell proliferation and biosynthesis with both types of cells. In vitro culture of TMJ condylar cartilage cells produced a fibrous tissue with predominantly type I collagen, while hyaline cartilage cells formed a fibrocartilage-like tissue with types I and II collagen. The combination of IGF and glucosamine had a synergistic effect on maintaining the phenotype of TMJ condylar cells to generate both types I and II collagen. Given the superior biosynthetic activity by hyaline cartilage cells and the practical surgical limitations of harvesting cells from the TMJ of a patient requiring TMJ reconstruction, cartilage cells from elsewhere in the body may be a potentially better alternative to cells harvested from the TMJ for TMJ tissue engineering. This finding may also apply to other fibrocartilages such as the intervertebral disc and knee meniscus in applications where a mature cartilage cell source is desired.

Complementary roles of intracellular and pericellular collagen degradation pathways in vivo

DEFF Research Database (Denmark)

Wagenaar-Miller, Rebecca A; Engelholm, Lars H; Gavard, Julie

2007-01-01

Collagen degradation is essential for cell migration, proliferation, and differentiation. Two key turnover pathways have been described for collagen: intracellular cathepsin-mediated degradation and pericellular collagenase-mediated degradation. However, the functional relationship between these ...

Micro-mechanical model for the tension-stabilized enzymatic degradation of collagen tissues

Science.gov (United States)

Nguyen, Thao; Ruberti, Jeffery

We present a study of how the collagen fiber structure influences the enzymatic degradation of collagen tissues. Experiments of collagen fibrils and tissues show that mechanical tension can slow and halt enzymatic degradation. Tissue-level experiments also show that degradation rate is minimum at a stretch level coincident with the onset of strain-stiffening in the stress response. To understand these phenomena, we developed a micro-mechanical model of a fibrous collagen tissue undergoing enzymatic degradation. Collagen fibers are described as sinusoidal elastica beams, and the tissue is described as a distribution of fibers. We assumed that the degradation reaction is inhibited by the axial strain energy of the crimped collagen fibers. The degradation rate law was calibrated to experiments on isolated single fibrils from bovine sclera. The fiber crimp and properties were fit to uniaxial tension tests of tissue strips. The fibril-level kinetic and tissue-level structural parameters were used to predict tissue-level degradation-induced creep rate under a constant applied force. We showed that we could accurately predict the degradation-induce creep rate of the pericardium and cornea once we accounted for differences in the fiber crimp structure and properties.

FT-IR Microspectroscopy of Rat Ear Cartilage.

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Benedicto de Campos Vidal

Full Text Available Rat ear cartilage was studied using Fourier transform-infrared (FT-IR microspectroscopy to expand the current knowledge which has been established for relatively more complex cartilage types. Comparison of the FT-IR spectra of the ear cartilage extracellular matrix (ECM with published data on articular cartilage, collagen II and 4-chondroitin-sulfate standards, as well as of collagen type I-containing dermal collagen bundles (CBs with collagen type II, was performed. Ear cartilage ECM glycosaminoglycans (GAGs were revealed histochemically and as a reduction in ECM FT-IR spectral band heights (1140-820 cm-1 after testicular hyaluronidase digestion. Although ear cartilage is less complex than articular cartilage, it contains ECM components with a macromolecular orientation as revealed using polarization microscopy. Collagen type II and GAGs, which play a structural role in the stereo-arrangement of the ear cartilage, contribute to its FT-IR spectrum. Similar to articular cartilage, ear cartilage showed that proteoglycans add a contribution to the collagen amide I spectral region, a finding that does not recommend this region for collagen type II quantification purposes. In contrast to articular cartilage, the symmetric stretching vibration of -SO3- groups at 1064 cm-1 appeared under-represented in the FT-IR spectral profile of ear cartilage. Because the band corresponding to the asymmetric stretching vibration of -SO3- groups (1236-1225 cm-1 overlapped with that of amide III bands, it is not recommended for evaluation of the -SO3- contribution to the FT-IR spectrum of the ear cartilage ECM. Instead, a peak (or shoulder at 1027-1016 cm-1 could be better considered for this intent. Amide I/amide II ratios as calculated here and data from the literature suggest that protein complexes of the ear cartilage ECM are arranged with a lower helical conformation compared to pure collagen II. The present results could motivate further studies on this tissue

FK506 protects against articular cartilage collagenous extra-cellular matrix degradation

NARCIS (Netherlands)

M. Siebelt (Michiel); A.E. van der Windt (Anna); H.C. Groen (Harald); M. Sandker (Marjan); J.H. Waarsing (Jan); C. Müller (Cristina); M. de Jong (Marcel); H. Jahr (Holger); H.H. Weinans (Harrie)

2014-01-01

textabstractObjective: Osteoarthritis (OA) is a non-rheumatologic joint disease characterized by progressive degeneration of the cartilage extra-cellular matrix (ECM), enhanced subchondral bone remodeling, activation of synovial macrophages and osteophyte growth. Inhibition of calcineurin (Cn)

Preparation, Cell Compatibility and Degradability of Collagen-Modified Poly(lactic acid

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Miaomiao Cui

2015-01-01

Full Text Available Poly(lactic acid (PLA was modified using collagen through a grafting method to improve its biocompatibility and degradability. The carboxylic group at the open end of PLA was transferred into the reactive acylchlorided group by a reaction with phosphorus pentachloride. Then, collagen-modified PLA (collagen-PLA was prepared by the reaction between the reactive acylchlorided group and amino/hydroxyl groups on collagen. Subsequently, the structure of collagen-PLA was confirmed by Fourier transform infrared spectroscopy, fluorescein isothiocyanate-labeled fluorescence spectroscopy, X-ray photoelectron spectroscopy, and DSC analyses. Finally, some properties of collagen-PLA, such as hydrophilicity, cell compatibility and degradability were characterized. Results showed that collagen had been grafted onto the PLA with 5% graft ratio. Water contact angle and water absorption behavior tests indicated that the hydrophilicity of collagen-PLA was significantly higher than that of PLA. The cell compatibility of collagen-PLA with mouse embryonic fibroblasts (3T3 was also significantly better than PLA in terms of cell morphology and cell proliferation, and the degradability of PLA was also improved after introducing collagen. Results suggested that collagen-PLA was a promising candidate for biomedical applications.

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

human recombinant cathepsins (Cats) and matrix-metalloproteases (MMPs). Next, we analyzed the spontaneous release of Helix-II and CTX-II from cartilage sections of patients with knee OA who were immediately deep frozen after joint replacement to preserve endogenous enzyme activity until assay. Cartilage....... Cat D was unable to digest intact cartilage. MMPs-1, -3, -7, -9, and -13 efficiently released CTX-II, but only small amount of Helix-II. Neither CTX-II nor Helix-II alone was able to reflect accurately the collagenolytic activity of Cats and MMPs as reflected by the release of hydroxyproline. In OA...

Scaffold-assisted cartilage tissue engineering using infant chondrocytes from human hip cartilage.

Science.gov (United States)

Kreuz, P C; Gentili, C; Samans, B; Martinelli, D; Krüger, J P; Mittelmeier, W; Endres, M; Cancedda, R; Kaps, C

2013-12-01

Studies about cartilage repair in the hip and infant chondrocytes are rare. The aim of our study was to evaluate the use of infant articular hip chondrocytes for tissue engineering of scaffold-assisted cartilage grafts. Hip cartilage was obtained from five human donors (age 1-10 years). Expanded chondrocytes were cultured in polyglycolic acid (PGA)-fibrin scaffolds. De- and re-differentiation of chondrocytes were assessed by histological staining and gene expression analysis of typical chondrocytic marker genes. In vivo, cartilage matrix formation was assessed by histology after subcutaneous transplantation of chondrocyte-seeded PGA-fibrin scaffolds in immunocompromised mice. The donor tissue was heterogenous showing differentiated articular cartilage and non-differentiated tissue and considerable expression of type I and II collagens. Gene expression analysis showed repression of typical chondrocyte and/or mesenchymal marker genes during cell expansion, while markers were re-induced when expanded cells were cultured in PGA-fibrin scaffolds. Cartilage formation after subcutaneous transplantation of chondrocyte loaded PGA-fibrin scaffolds in nude mice was variable, with grafts showing resorption and host cell infiltration or formation of hyaline cartilage rich in type II collagen. Addition of human platelet rich plasma (PRP) to cartilage grafts resulted robustly in formation of hyaline-like cartilage that showed type II collagen and regions with type X collagen. These results suggest that culture of expanded and/or de-differentiated infant hip cartilage cells in PGA-fibrin scaffolds initiates chondrocyte re-differentiation. The heterogenous donor tissue containing immature chondrocytes bears the risk of cartilage repair failure in vivo, which may be possibly overcome by the addition of PRP. Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Effect of antibiotics on in vitro and in vivo avian cartilage degradation.

Science.gov (United States)

Peters, T L; Fulton, R M; Roberson, K D; Orth, M W

2002-01-01

Antibiotics are used in the livestock industry not only to treat disease but also to promote growth and increase feed efficiency in less than ideal sanitary conditions. However, certain antibiotic families utilized in the poultry industry have recently been found to adversely affect bone formation and cartilage metabolism in dogs, rats, and humans. Therefore, the first objective of this study was to determine if certain antibiotics used in the poultry industry would inhibit in vitro cartilage degradation. The second objective was to determine if the antibiotics found to inhibit in vitro cartilage degradation also induced tibial dyschondroplasia in growing broilers. Ten antibiotics were studied by an avian explant culture system that is designed to completely degrade tibiae over 16 days. Lincomycin, tylosin tartrate, gentamicin, erythromycin, and neomycin sulfate did not inhibit degradation at any concentration tested. Doxycycline (200 microg/ml), oxytetracycline (200 microg/ml), enrofloxacin (200 and 400 microg/ml), ceftiofur (400 microg/ml), and salinomycin (10 microg/ml) prevented complete cartilage degradation for up to 30 days in culture. Thus, some of the antibiotics did inhibit cartilage degradation in developing bone. Day-old chicks were then administered the five antibiotics at 25%, 100%, or 400% above their recommended dose levels and raised until 21 days of age. Thiram, a fungicide known to induce experimental tibial dyschondroplasia (TD), was given at 20 ppm. Birds were then killed by cervical dislocation, and each proximal tibiotarsus was visually examined for TD lesions. The results showed that none of these antibiotics significantly induced TD in growing boilers at any concentration tested, whereas birds given 20 ppm thiram had a 92% incidence rate.

Evaluation of the effect of a chicken comb extract-containing supplement on cartilage and bone metabolism in athletes.

Science.gov (United States)

Yoshimura, Masafumi; Aoba, Yukihiro; Watari, Taiji; Momomura, Rei; Watanabe, Keita; Tomonaga, Akihito; Matsunaga, Michitaka; Suda, Yoshimasa; Lee, Woo Young; Asai, Katsuhito; Yoshimura, Kaori; Nakagawa, Takashi; Yamamoto, Tetsuro; Yamaguchi, Hideyo; Nagaoka, Isao

2012-10-01

In a previous study, we revealed that a commercially available product of dietary supplement containing a chicken comb extract (CCE), which is rich in hyaluronan, not only relieves joint pain and other symptoms, but also potentially improves the balance of type II collagen degradation/synthesis in patients with knee osteoarthritis. Since soccer is one of the sports most likely to cause knee osteoarthritis (OA), we evaluated the effect of a CCE-containing supplement on cartilage and bone metabolism in athletes. Fourteen and 15 subjects (all midfielders) were randomly assigned to receive the test product (test group) and the dummy placebo containing only vehicle (placebo group), respectively, for 12 weeks. The daily oral intake of the CCE-containing test product clearly decreased the urinary levels of both C-terminal crosslinked telopeptides of cartilage-specific type II collagen (CTX-II) as a type II collagen degradation marker and the N-terminal telopeptides of bone-specific type I collagen (NTx) as a marker of bone resorption at 12 weeks after the initiation of the intervention. By contrast, no significant reduction was detected in the placebo group at any timepoint during the intervention. These observations indicate that the test product is effective in inhibiting, not only cartilage degradation, but also bone remodeling. Thus, the CCE-containing supplement may be useful for the management of joint health in athletes.

Non-invasive monitoring of in vivo hydrogel degradation and cartilage regeneration by multiparametric MR imaging

Science.gov (United States)

Chen, Zelong; Yan, Chenggong; Yan, Shina; Liu, Qin; Hou, Meirong; Xu, Yikai; Guo, Rui

2018-01-01

Numerous biodegradable hydrogels for cartilage regeneration have been widely used in the field of tissue engineering. However, to non-invasively monitor hydrogel degradation and efficiently evaluate cartilage restoration in situ is still challenging. Methods: A ultrasmall superparamagnetic iron oxide (USPIO)-labeled cellulose nanocrystal (CNC)/silk fibroin (SF)-blended hydrogel system was developed to monitor hydrogel degradation during cartilage regeneration. The physicochemical characterization and biocompatibility of the hydrogel were evaluated in vitro. The in vivo hydrogel degradation and cartilage regeneration of different implants were assessed using multiparametric magnetic resonance imaging (MRI) and further confirmed by histological analysis in a rabbit cartilage defect model for 3 months. Results: USPIO-labeled hydrogels showed sufficient MR contrast enhancement and retained stability without loss of the relaxation rate. Neither the mechanical properties of the hydrogels nor the proliferation of bone-marrow mesenchymal stem cells (BMSCs) were affected by USPIO labeling in vitro. CNC/SF hydrogels with BMSCs degraded more quickly than the acellular hydrogels as reflected by the MR relaxation rate trends in vivo. The morphology of neocartilage was noninvasively visualized by the three-dimensional water-selective cartilage MRI scan sequence, and the cartilage repair was further demonstrated by macroscopic and histological observations. Conclusion: This USPIO-labeled CNC/SF hydrogel system provides a new perspective on image-guided tissue engineering for cartilage regeneration. PMID:29464005

On the role of hydrogel structure and degradation in controlling the transport of cell-secreted matrix molecules for engineered cartilage.

Science.gov (United States)

Dhote, Valentin; Skaalure, Stacey; Akalp, Umut; Roberts, Justine; Bryant, Stephanie J; Vernerey, Franck J

2013-03-01

Damage to cartilage caused by injury or disease can lead to pain and loss of mobility, diminishing one's quality of life. Because cartilage has a limited capacity for self-repair, tissue engineering strategies, such as cells encapsulated in synthetic hydrogels, are being investigated as a means to restore the damaged cartilage. However, strategies to date are suboptimal in part because designing degradable hydrogels is complicated by structural and temporal complexities of the gel and evolving tissue along multiple length scales. To address this problem, this study proposes a multi-scale mechanical model using a triphasic formulation (solid, fluid, unbound matrix molecules) based on a single chondrocyte releasing extracellular matrix molecules within a degrading hydrogel. This model describes the key players (cells, proteoglycans, collagen) of the biological system within the hydrogel encompassing different length scales. Two mechanisms are included: temporal changes of bulk properties due to hydrogel degradation, and matrix transport. Numerical results demonstrate that the temporal change of bulk properties is a decisive factor in the diffusion of unbound matrix molecules through the hydrogel. Transport of matrix molecules in the hydrogel contributes both to the development of the pericellular matrix and the extracellular matrix and is dependent on the relative size of matrix molecules and the hydrogel mesh. The numerical results also demonstrate that osmotic pressure, which leads to changes in mesh size, is a key parameter for achieving a larger diffusivity for matrix molecules in the hydrogel. The numerical model is confirmed with experimental results of matrix synthesis by chondrocytes in biodegradable poly(ethylene glycol)-based hydrogels. This model may ultimately be used to predict key hydrogel design parameters towards achieving optimal cartilage growth. Copyright © 2012 Elsevier Ltd. All rights reserved.

The collagen structure of equine articular cartilage, characterized using polarization-sensitive optical coherence tomography

International Nuclear Information System (INIS)

Ugryumova, Nadya; Attenburrow, Don P; Winlove, C Peter; Matcher, Stephen J

2005-01-01

Optical coherence tomography and polarization-sensitive optical coherence tomography images of equine articular cartilage are presented. Measurements were made on intact joint surfaces. Significant (e.g. x 2) variations in the intrinsic birefringence were found over spatial scales of a few millimetres, even on samples taken from young (18 month) animals that appeared visually homogeneous. A comparison of data obtained on a control tissue (equine flexor tendon) further suggests that significant variations in the orientation of the collagen fibres relative to the plane of the joint surface exist. Images of visually damaged cartilage tissue show characteristic features both in terms of the distribution of optical scatterers and of the birefringent components

The collagen structure of equine articular cartilage, characterized using polarization-sensitive optical coherence tomography

Energy Technology Data Exchange (ETDEWEB)

Ugryumova, Nadya; Attenburrow, Don P; Winlove, C Peter; Matcher, Stephen J [Biomedical Physics Group, School of Physics, University of Exeter, Stocker Road, Exeter EX4 4QL (United Kingdom)

2005-08-07

Optical coherence tomography and polarization-sensitive optical coherence tomography images of equine articular cartilage are presented. Measurements were made on intact joint surfaces. Significant (e.g. x 2) variations in the intrinsic birefringence were found over spatial scales of a few millimetres, even on samples taken from young (18 month) animals that appeared visually homogeneous. A comparison of data obtained on a control tissue (equine flexor tendon) further suggests that significant variations in the orientation of the collagen fibres relative to the plane of the joint surface exist. Images of visually damaged cartilage tissue show characteristic features both in terms of the distribution of optical scatterers and of the birefringent components.

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

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......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...

Cartilage proteoglycans inhibit fibronectin-mediated adhesion

Science.gov (United States)

Rich, A. M.; Pearlstein, E.; Weissmann, G.; Hoffstein, S. T.

1981-09-01

Normal tissues and organs show, on histological examination, a pattern of cellular and acellular zones that is characteristic and unique for each organ or tissue. This pattern is maintained in health but is sometimes destroyed by disease. For example, in mobile joints, the articular surfaces consist of relatively acellular hyaline cartilage, and the joint space is enclosed by a capsule of loose connective tissue with a lining of fibroblasts and macrophages. In the normal joint these cells are confined to the synovial lining and the articular surface remains acellular. In in vitro culture, macrophages and their precursor monocytes are very adhesive, and fibroblasts can migrate and overgrow surfaces such as collagen or plastic used for tissue culture. The fibroblasts adhere to collagen by means of fibronectin, which they synthesize and secrete1. Because the collagen of cartilage is capable of binding serum fibronectin2 and fibronectin is present in cartilage during its development3, these cells should, in theory, slowly migrate from the synovial lining to the articular surface. It is their absence from the articular cartilage in normal circumstances, and then presence in such pathological states as rheumatoid arthritis, that is striking. We therefore set out to determine whether a component of cartilage could prevent fibroblast adherence in a defined adhesion assay. As normal cartilage is composed of 50% proteoglycans and 50% collagen by dry weight4, we tested the possibility that the proteoglycans in cartilage inhibit fibroblast adhesion to collagen. We present here evidence that fibroblast spreading and adhesion to collagenous substrates is inhibited by cartilage proteoglycans.

Oral salmon calcitonin induced suppression of urinary collagen type II degradation in postmenopausal women: a new potential treatment of osteoarthritis.

Science.gov (United States)

Bagger, Yu Z; Tankó, László B; Alexandersen, Peter; Karsdal, Morten A; Olson, Melvin; Mindeholm, Linda; Azria, Moïse; Christiansen, Claus

2005-09-01

To assess the efficacy of 3 months of oral salmon calcitonin (sCT) on cartilage degradation as estimated by the changes in the urinary excretion of C-terminal telopeptide of collagen type II (CTX-II), and to investigate whether the response of oral sCT to urinary CTX-II depends on the baseline level of cartilage turnover. This was a randomized, double blind, placebo-controlled clinical setting including 152 Danish postmenopausal women aged 55-85. The subjects received treatment with the different doses of sCT (0.15, 0.4, 1.0, or 2.5 mg) combined with Eligen technology-based carrier molecule (200 mg), or placebo for 3 months. The efficacy parameter was the changes in the 24-h excretion of urinary CTX-I/II corrected for creatinine excretion at month 3. sCT induced a significant dose-dependent decrease in 24-h urinary CTX-II excretion. Similar dose-dependent responses were found in 24-h urinary CTX-I. When stratifying the study population into tertiles of baseline urinary CTX-II, the present osteoarthritic symptoms and definite cases of osteoarthritis (OA) were significantly more frequent in women in the highest tertile of CTX-II (mean 391 +/- 18 ng/mmol). Women who received 1.0 mg of sCT and had the highest cartilage turnover presented the greatest decrease in urinary CTX-II after 3 months of treatment. In addition to its pronounced effect on bone resorption, this novel oral sCT formulation may also reduce cartilage degradation and thereby provide therapeutic benefit in terms of chondroprotection. Women with high cartilage turnover are more likely to benefit from oral sCT treatment.

Relative contribution of matrix metalloprotease and cysteine protease activities to cytokine-stimulated articular cartilage degradation

DEFF Research Database (Denmark)

Sondergaard, B C; Henriksen, K; Wulf, H

2006-01-01

OBJECTIVE: Both matrix metalloprotease (MMP) activity and cathepsin K (CK) activity have been implicated in cartilage turnover. We investigated the relative contribution of MMP activity and CK activity in cartilage degradation using ex vivo and in vivo models. METHODS: Bovine articular cartilage...... explants were stimulated with oncostatin M (OSM) 10 ng/ml and tumor necrosis factor-alpha (TNF-alpha) 20 ng/ml in the presence or absence of the broad-spectrum MMP inhibitor GM6001 and the cysteine protease inhibitor, E64. Cartilage degradation was evaluated in the conditioned medium by glycosaminoglycans...... was measured from CK-deficient mice. RESULTS: OSM and TNF-alpha combined induced significant (Pcartilage degradation products measured by hydroxyproline and CTX-II compared to vehicle control. The cytokines potently induced MMP expression, assessed by zymography, and CK expression...

INVIVO DEGRADATION OF PROCESSED DERMAL SHEEP COLLAGEN EVALUATED WITH TRANSMISSION ELECTRON-MICROSCOPY

NARCIS (Netherlands)

VANWACHEM, PB; VANLUYN, MJA; NIEUWENHUIS, P; KOERTEN, HK; DAMINK, LO; TENHOOPEN, H; FEIJEN, J

The in vivo degradation of hexamethylenediisocyanate-tanned dermal sheep collagen was studied with transmission electron microscopy. Discs of hexamethylenediisocyanate-tanned dermal sheep collagen were subcutaneously implanted in rats. Both an intra- and an extracellular route of degradation could

Mechanical properties of the collagen network in human articular cartilage as measured by osmotic stress technique

NARCIS (Netherlands)

Basser, P.J.; Schneiderman, R.; Bank, R.A.; Wachtel, E.; Maroudas, A.

1998-01-01

We have used an isotropic osmotic stress technique to assess the swelling pressures of human articular cartilage over a wide range of hydrations in order to determine from these measurements, for the first time, the tensile stress in the collagen network, P(c), as a function of hydration. Osmotic

Importance of collagen orientation and depth-dependent fixed charge densities of cartilage on mechanical behavior of chondrocytes.

NARCIS (Netherlands)

Korhonen, R.K.; Julkunen, P.; Wilson, W.; Herzog, W.

2008-01-01

The collagen network and proteoglycan matrix of articular cartilage are thought to play an important role in controlling the stresses and strains in and around chondrocytes, in regulating the biosynthesis of the solid matrix, and consequently in maintaining the health of diarthrodial joints.

Type II collagen peptide is able to accelerate embryonic chondrocyte differentiation: an association with articular cartilage matrix resorption in osteoarthrosis

Directory of Open Access Journals (Sweden)

Elena Vasil'evna Chetina

2010-01-01

Conclusion. The effect of CP on gene expression and collagen decomposition activity depends on the morphotype of embryonic chondrocytes. Lack of effect of CP on collagen decomposition activity in both the embryonic hypertrophic chondrocytes and the cartilage explants from OA patients supports the hypothesis that the hypertrophic morphotype is a dominant morphotype of articular chondrocytes in OA. Moreover, collagen decomposition products can be involved in the resorption of matrix in OA and in the maintenance of chronic nature of the pathology.

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.

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

DEFF Research Database (Denmark)

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

2009-01-01

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

Collagen Structural Hierarchy and Susceptibility to Degradation by Ultraviolet Radiation.

Science.gov (United States)

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

2008-12-01

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

In vivo degradation of processed dermal sheep collagen evaluated with transmission electron microscopy

NARCIS (Netherlands)

van Wachem, P.B.; van Luyn, M.J.A.; Nieuwenhuis, P.; Koerten, H.K.; Olde damink, L.H.H.; Olde-Damink, L.; ten Hoopen, Hermina W.M.; Feijen, Jan

1991-01-01

The in vivo degradation of hexamethylenediisocyanate-tanned dermal sheep collagen was studied with transmission electron microscopy. Discs of hexamethylenediisocyanate-tanned dermal sheep collagen were subcutaneously implanted in rats. Both an intra- and an extracellular route of degradation could

uPARAP/endo180 directs lysosomal delivery and degradation of collagen IV

DEFF Research Database (Denmark)

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

2004-01-01

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

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.

Suramin Inhibits Osteoarthritic Cartilage Degradation by Increasing Extracellular Levels of Chondroprotective Tissue Inhibitor of Metalloproteinases 3.

Science.gov (United States)

Chanalaris, Anastasios; Doherty, Christine; Marsden, Brian D; Bambridge, Gabriel; Wren, Stephen P; Nagase, Hideaki; Troeberg, Linda

2017-10-01

Osteoarthritis is a common degenerative joint disease for which no disease-modifying drugs are currently available. Attempts to treat the disease with small molecule inhibitors of the metalloproteinases that degrade the cartilage matrix have been hampered by a lack of specificity. We aimed to inhibit cartilage degradation by augmenting levels of the endogenous metalloproteinase inhibitor, tissue inhibitor of metalloproteinases (TIMP)-3, through blocking its interaction with the endocytic scavenger receptor, low-density lipoprotein receptor-related protein 1 (LRP1). We discovered that suramin (C 51 H 40 N 6 O 23 S 6 ) bound to TIMP-3 with a K D value of 1.9 ± 0.2 nM and inhibited its endocytosis via LRP1, thus increasing extracellular levels of TIMP-3 and inhibiting cartilage degradation by the TIMP-3 target enzyme, adamalysin-like metalloproteinase with thrombospondin motifs 5. NF279 (8,8'-[carbonyl bis (imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)] bis -1,3,5-naphthalenetrisulfonic acid hexasodium salt), a structural analog of suramin, has an increased affinity for TIMP-3 and increased ability to inhibit TIMP-3 endocytosis and protect cartilage. Suramin is thus a promising scaffold for the development of novel therapeutics to increase TIMP-3 levels and inhibit cartilage degradation in osteoarthritis. Copyright © 2017 by The Author(s).

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.

Systems Based Study of the Therapeutic Potential of Small Charged Molecules for the Inhibition of IL-1 Mediated Cartilage Degradation

Science.gov (United States)

Kar, Saptarshi; Smith, David W.; Gardiner, Bruce S.; Grodzinsky, Alan J.

2016-01-01

Inflammatory cytokines are key drivers of cartilage degradation in post-traumatic osteoarthritis. Cartilage degradation mediated by these inflammatory cytokines has been extensively investigated using in vitro experimental systems. Based on one such study, we have developed a computational model to quantitatively assess the impact of charged small molecules intended to inhibit IL-1 mediated cartilage degradation. We primarily focus on the simplest possible computational model of small molecular interaction with the IL-1 system—direct binding of the small molecule to the active site on the IL-1 molecule itself. We first use the model to explore the uptake and release kinetics of the small molecule inhibitor by cartilage tissue. Our results show that negatively charged small molecules are excluded from the negatively charged cartilage tissue and have uptake kinetics in the order of hours. In contrast, the positively charged small molecules are drawn into the cartilage with uptake and release timescales ranging from hours to days. Using our calibrated computational model, we subsequently explore the effect of small molecule charge and binding constant on the rate of cartilage degradation. The results from this analysis indicate that the small molecules are most effective in inhibiting cartilage degradation if they are either positively charged and/or bind strongly to IL-1α, or both. Furthermore, our results showed that the cartilage structural homeostasis can be restored by the small molecule if administered within six days following initial tissue exposure to IL-1α. We finally extended the scope of the computational model by simulating the competitive inhibition of cartilage degradation by the small molecule. Results from this model show that small molecules are more efficient in inhibiting cartilage degradation by binding directly to IL-1α rather than binding to IL-1α receptors. The results from this study can be used as a template for the design and

Activation of Indian Hedgehog Promotes Chondrocyte Hypertrophy and Upregulation of MMP-13 in Human Osteoarthritic Cartilage

Science.gov (United States)

Wei, Fangyuan; Zhou, Jingming; Wei, Xiaochun; Zhang, Juntao; Fleming, Braden C.; Terek, Richard; Pei, Ming; Chen, Qian; Liu, Tao; Wei, Lei

2012-01-01

Objective The objectives of this study were to 1) determine the correlation between osteoarthritis (OA) and Ihh expression, and 2) establish the effects of Ihh on expression of markers of chondrocyte hypertrophy and MMP-13 in human OA cartilage. Design OA cartilage and synovial fluid samples were obtained during total knee arthroplasty. Normal cartilage samples were obtained from intra-articular tumor resections, and normal synovial fluid samples were obtained from healthy volunteers and the contralateral uninjured knee of patients undergoing anterior cruciate ligament reconstruction. OA was graded using the Mankin score. Expression of Ihh in synovial fluid was determined by western blot. Ihh, type X collagen and MMP-13 mRNA were determined by real time PCR. Protein expression of type X collagen and MMP-13 in cartilage samples were analyzed with immunohistochemistry. Chondrocyte size was measured using image analysis. Results Ihh expression was increased 2.6 fold in OA cartilage and 37% in OA synovial fluid when compared to normal control samples. Increased expression of Ihh was associated with the severity of OA and expression of markers of chondrocyte hypertrophy: type X collagen and MMP-13, and chondocyte size. Chondrocytes were more spherical with increasing severity of OA. There was a significant correlation between Mankin score and cell size (r2= 0.80) and Ihh intensity (r2 = 0.89). Exogenous Ihh induced a 6.8 fold increase of type X collagen and 2.8 fold increase of MMP-13 mRNA expression in cultured chondrocytes. Conversely, knockdown of Ihh by siRNA and Hh inhibitor Cyclopamine had the opposite effect. Conclusions Ihh expression correlates with OA progression and changes in chondrocyte morphology and gene expression consistent with chondrocyte hypertrophy and cartilage degradation seen in OA cartilage. Thus, Ihh may be a potential therapeutic target to prevent OA progression. PMID:22469853

Altering the swelling pressures within in vitro engineered cartilage is predicted to modulate the configuration of the collagen network and hence improve tissue mechanical properties.

Science.gov (United States)

Nagel, Thomas; Kelly, Daniel J

2013-06-01

Prestress in the collagen network has a significant impact on the material properties of cartilaginous tissues. It is closely related to the recruitment configuration of the collagen network which defines the transition from lax collagen fibres to uncrimped, load-bearing collagen fibres. This recruitment configuration can change in response to alterations in the external environmental conditions. In this study, the influence of changes in external salt concentration or sequential proteoglycan digestion on the configuration of the collagen network of tissue engineered cartilage is investigated using a previously developed computational model. Collagen synthesis and network assembly are assumed to occur in the tissue configuration present during in vitro culture. The model assumes that if this configuration is more compact due to changes in tissue swelling, the collagen network will adapt by lowering its recruitment stretch. When returned to normal physiological conditions, these tissues will then have a higher prestress in the collagen network. Based on these assumptions, the model demonstrates that proteoglycan digestion at discrete time points during culture as well as culture in a hypertonic medium can improve the functionality of tissue engineered cartilage, while culture in hypotonic solution is detrimental to the apparent mechanical properties of the graft. Copyright © 2013 Elsevier Ltd. All rights reserved.

Use of synovium-derived stromal cells and chitosan/collagen type I scaffolds for cartilage tissue engineering

Energy Technology Data Exchange (ETDEWEB)

Gong Zhongcheng; Lin Zhaoquan [Department of Oral and Maxillofacial Surgery, First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830054 (China); Xiong Hui; Long Xing; Wei Lili; Li Jian; Wu Yang, E-mail: xinglong1957@yahoo.com.c [State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine, Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei 430079 (China)

2010-10-01

The objective was to investigate synovium-derived stromal cells (SDSCs) coupled with chitosan/collagen type I (CS/COL-I) scaffolds for cartilage engineering. CS/COL-I scaffolds were fabricated through freeze-drying and cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. SDSCs were isolated from synovium and cultured onto CS/COL-I scaffolds, constructs of which were incubated in serum-free chondrogenic medium with sequential application of TGF-{beta}1 and bFGF for up to 21 days and then implanted into nude mice. The physical characteristics of the scaffolds were examined. The quality of the in vitro constructs was assessed in terms of DNA content by PicoGreen assay and cartilaginous matrix by histological examination. The implants of the constructs were evaluated by histological and immunohistochemical examinations and reverse transcription PCR. Results indicated that the CS/COL-I scaffold showed porous structures, and the DNA content of SDSCs in CS/COL-I scaffolds increased at 1 week culture time. Both of the constructs in vitro and the implants were examined with positive stained GAGs histologically and the implants with positive collagen type II immunohistochemically. RT-PCR of the implants indicated that aggrecan and collagen type II expressed. It suggested that SDSCs coupled with CS/COL-I scaffolds treated sequentially with TGF-{beta}1 and bFGF in vitro were highly competent for engineered cartilage formation in vitro and in vivo.

Synthesis and characterization of a lubricin mimic (mLub) to reduce friction and adhesion on the articular cartilage surface.

Science.gov (United States)

Lawrence, Alexandra; Xu, Xin; Bible, Melissa D; Calve, Sarah; Neu, Corey P; Panitch, Alyssa

2015-12-01

The lubricating proteoglycan, lubricin, facilitates the remarkable low friction and wear properties of articular cartilage in the synovial joints of the body. Lubricin lines the joint surfaces and plays a protective role as a boundary lubricant in sliding contact; decreased expression of lubricin is associated with cartilage degradation and the pathogenesis of osteoarthritis. An unmet need for early osteoarthritis treatment is the development of therapeutic molecules that mimic lubricin function and yet are also resistant to enzymatic degradation common in the damaged joint. Here, we engineered a lubricin mimic (mLub) that is less susceptible to enzymatic degradation and binds to the articular surface to reduce friction. mLub was synthesized using a chondroitin sulfate backbone with type II collagen and hyaluronic acid (HA) binding peptides to promote interaction with the articular surface and synovial fluid constituents. In vitro and in vivo characterization confirmed the binding ability of mLub to isolated type II collagen and HA, and to the cartilage surface. Following trypsin treatment to the cartilage surface, application of mLub, in combination with purified or commercially available hyaluronan, reduced the coefficient of friction, and adhesion, to control levels as assessed over macro-to micro-scales by rheometry and atomic force microscopy. In vivo studies demonstrate an mLub residency time of less than 1 week. Enhanced lubrication by mLub reduces surface friction and adhesion, which may suppress the progression of degradation and cartilage loss in the joint. mLub therefore shows potential for treatment in early osteoarthritis following injury. Copyright © 2015 Elsevier Ltd. All rights reserved.

Matrix development in self-assembly of articular cartilage.

Directory of Open Access Journals (Sweden)

Gidon Ofek

2008-07-01

Full Text Available Articular cartilage is a highly functional tissue which covers the ends of long bones and serves to ensure proper joint movement. A tissue engineering approach that recapitulates the developmental characteristics of articular cartilage can be used to examine the maturation and degeneration of cartilage and produce fully functional neotissue replacements for diseased tissue.This study examined the development of articular cartilage neotissue within a self-assembling process in two phases. In the first phase, articular cartilage constructs were examined at 1, 4, 7, 10, 14, 28, 42, and 56 days immunohistochemically, histologically, and through biochemical analysis for total collagen and glycosaminoglycan (GAG content. Based on statistical changes in GAG and collagen levels, four time points from the first phase (7, 14, 28, and 56 days were chosen to carry into the second phase, where the constructs were studied in terms of their mechanical characteristics, relative amounts of collagen types II and VI, and specific GAG types (chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and hyaluronan. Collagen type VI was present in initial abundance and then localized to a pericellular distribution at 4 wks. N-cadherin activity also spiked at early stages of neotissue development, suggesting that self-assembly is mediated through a minimization of free energy. The percentage of collagen type II to total collagen significantly increased over time, while the proportion of collagen type VI to total collagen decreased between 1 and 2 wks. The chondroitin 6- to 4- sulfate ratio decreased steadily during construct maturation. In addition, the compressive properties reached a plateau and tensile characteristics peaked at 4 wks.The indices of cartilage formation examined in this study suggest that tissue maturation in self-assembled articular cartilage mirrors known developmental processes for native tissue. In terms of tissue engineering, it is

Subchondral Bone Plate Thickening Precedes Chondrocyte Apoptosis and Cartilage Degradation in Spontaneous Animal Models of Osteoarthritis

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Zaitunnatakhin Zamli

2014-01-01

Full Text Available Osteoarthritis (OA is the most common joint disorder characterised by bone remodelling and cartilage degradation and associated with chondrocyte apoptosis. These processes were investigated at 10, 16, 24, and 30 weeks in Dunkin Hartley (DH and Bristol Strain 2 (BS2 guinea pigs that develop OA spontaneously. Both strains had a more pronounced chondrocyte apoptosis, cartilage degradation, and subchondral bone changes in the medial than the lateral side of the tibia, and between strains, the changes were always greater and faster in DH than BS2. In the medial side, a significant increase of chondrocyte apoptosis and cartilage degradation was observed in DH between 24 and 30 weeks of age preceded by a progressive thickening and stiffening of subchondral bone plate (Sbp. The Sbp thickness consistently increased over the 30-week study period but the bone mineral density (BMD of the Sbp gradually decreased after 16 weeks. The absence of these changes in the medial side of BS2 may indicate that the Sbp of DH was undergoing remodelling. Chondrocyte apoptosis was largely confined to the deep zone of articular cartilage and correlated with thickness of the subchondral bone plate suggesting that cartilage degradation and chondrocyte apoptosis may be a consequence of continuous bone remodelling during the development of OA in these animal models of OA.

Changes in Structural-Mechanical Properties and Degradability of Collagen during Aging-associated Modifications*

Science.gov (United States)

Panwar, Preety; Lamour, Guillaume; Mackenzie, Neil C. W.; Yang, Heejae; Ko, Frank; Li, Hongbin; Brömme, Dieter

2015-01-01

During aging, changes occur in the collagen network that contribute to various pathological phenotypes in the skeletal, vascular, and pulmonary systems. The aim of this study was to investigate the consequences of age-related modifications on the mechanical stability and in vitro proteolytic degradation of type I collagen. Analyzing mouse tail and bovine bone collagen, we found that collagen at both fibril and fiber levels varies in rigidity and Young's modulus due to different physiological changes, which correlate with changes in cathepsin K (CatK)-mediated degradation. A decreased susceptibility to CatK-mediated hydrolysis of fibrillar collagen was observed following mineralization and advanced glycation end product-associated modification. However, aging of bone increased CatK-mediated osteoclastic resorption by ∼27%, and negligible resorption was observed when osteoclasts were cultured on mineral-deficient bone. We observed significant differences in the excavations generated by osteoclasts and C-terminal telopeptide release during bone resorption under distinct conditions. Our data indicate that modification of collagen compromises its biomechanical integrity and affects CatK-mediated degradation both in bone and tissue, thus contributing to our understanding of extracellular matrix aging. PMID:26224630

Type II collagen in cartilage evokes peptide-specific tolerance and skews the immune response.

Science.gov (United States)

Malmström, V; Kjellén, P; Holmdahl, R

1998-06-01

T cell recognition of type II collagen (CII) is a crucial event in the induction of collagen-induced arthritis in the mouse. Several CII peptides have been shown to be of importance, dependent on which MHC haplotype the mouse carries. By sequencing the rat CII and comparing the sequence with mouse, human, bovine and chicken CII, we have found that the immunodominant peptides all differ at critical positions compared with the autologous mouse sequence. Transgenic expression of the immunodominant Aq-restricted heterologous CII 256-270 epitope inserted into type I collagen (TSC mice) or type II collagen (MMC-1 mice) led to epitope-specific tolerance. Immunization of TSC mice with chick CII led to arthritis and immune responses, dependent on the subdominant, Aq-restricted and chick-specific CII 190-200 epitope. Immunization of F1 mice, expressing both H-2q and H-2r as well as transgenic expression of the Aq-restricted CII 256-270 epitope in cartilage, with bovine CII, led to arthritis, dependent on the Ar-restricted, bovine-specific epitope CII 607-621. These data show that the immunodominance of CII recognition is directed towards heterologous determinants, and that T cells directed towards the corresponding autologous epitopes are tolerated without evidence of active suppression.

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

Science.gov (United States)

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

2017-09-01

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

Collagen degradation in the abdominal aneurysm: A conspiracy of matrix metalloproteinase and cysteine collagenases

NARCIS (Netherlands)

Abdul-Hussien, H.; Soekhoe, R.G.V.; Weber, E.; Thüsen, J.H. von der; Kleemann, R.; Mulder, A.; Hajo Van Bockel, J.; Hanemaaijer, R.; Lindeman, J.H.N.

2007-01-01

Growth and rupture of abdominal aortic aneurysms (AAAs) result from increased collagen turnover. Collagen turnover critically depends on specific collagenases that cleave the triple helical region of fibrillar collagen. As yet, the collagenases responsible for collagen degradation in AAAs have not

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

Directory of Open Access Journals (Sweden)

Erica P Homan

2014-01-01

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

Engineering of hyaline cartilage with a calcified zone using bone marrow stromal cells.

Science.gov (United States)

Lee, W D; Hurtig, M B; Pilliar, R M; Stanford, W L; Kandel, R A

2015-08-01

In healthy joints, a zone of calcified cartilage (ZCC) provides the mechanical integration between articular cartilage and subchondral bone. Recapitulation of this architectural feature should serve to resist the constant shear force from the movement of the joint and prevent the delamination of tissue-engineered cartilage. Previous approaches to create the ZCC at the cartilage-substrate interface have relied on strategic use of exogenous scaffolds and adhesives, which are susceptible to failure by degradation and wear. In contrast, we report a successful scaffold-free engineering of ZCC to integrate tissue-engineered cartilage and a porous biodegradable bone substitute, using sheep bone marrow stromal cells (BMSCs) as the cell source for both cartilaginous zones. BMSCs were predifferentiated to chondrocytes, harvested and then grown on a porous calcium polyphosphate substrate in the presence of triiodothyronine (T3). T3 was withdrawn, and additional predifferentiated chondrocytes were placed on top of the construct and grown for 21 days. This protocol yielded two distinct zones: hyaline cartilage that accumulated proteoglycans and collagen type II, and calcified cartilage adjacent to the substrate that additionally accumulated mineral and collagen type X. Constructs with the calcified interface had comparable compressive strength to native sheep osteochondral tissue and higher interfacial shear strength compared to control without a calcified zone. This protocol improves on the existing scaffold-free approaches to cartilage tissue engineering by incorporating a calcified zone. Since this protocol employs no xenogeneic material, it will be appropriate for use in preclinical large-animal studies. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Radiation cross-linked collagen/dextran dermal scaffolds: effects of dextran on cross-linking and degradation.

Science.gov (United States)

Zhang, Yaqing; Zhang, Xiangmei; Xu, Ling; Wei, Shicheng; Zhai, Maolin

2015-01-01

Ionizing radiation effectively cross-links collagen into network with enhanced anti-degradability and biocompatibility, while radiation-cross-linked collagen scaffold lacks flexibility, satisfactory surface appearance, and performs poor in cell penetration and ingrowth. To make the radiation-cross-linked collagen scaffold to serve as an ideal artificial dermis, dextran was incorporated into collagen. Scaffolds with the collagen/dextran (Col/Dex) ratios of 10/0, 7/3, and 5/5 were fabricated via (60)Co γ-irradiation cross-linking, followed by lyophilization. The morphology, microstructure, physicochemical, and biological properties were investigated. Compared with pure collagen, scaffolds with dextran demonstrated more porous appearance, enhanced hydrophilicity while the cross-linking density was lower with the consequence of larger pore size, higher water uptake, as well as reduced stiffness. Accelerated degradation was observed when dextran was incorporated in both the in vitro and in vivo assays, which led to earlier integration with cell and host tissue. The effect of dextran on degradation was ascribed to the decreased cross-linking density, looser microstructure, more porous and hydrophilic surface. Considering the better appearance, softness, moderate degradation rate due to controllable cross-linking degree and good biocompatibility as well, radiation-cross-linked collagen/dextran scaffolds are expected to serve as promising artificial dermal substitutes.

Surface modification of polycaprolactone scaffolds fabricated via selective laser sintering for cartilage tissue engineering

International Nuclear Information System (INIS)

Chen, Chih-Hao; Lee, Ming-Yih; Shyu, Victor Bong-Hang; Chen, Yi-Chieh; Chen, Chien-Tzung; Chen, Jyh-Ping

2014-01-01

Surface modified porous polycaprolactone scaffolds fabricated via rapid prototyping techniques were evaluated for cartilage tissue engineering purposes. Polycaprolactone scaffolds manufactured by selective laser sintering (SLS) were surface modified through immersion coating with either gelatin or collagen. Three groups of scaffolds were created and compared for both mechanical and biological properties. Surface modification with collagen or gelatin improved the hydrophilicity, water uptake and mechanical strength of the pristine scaffold. From microscopic observations and biochemical analysis, collagen-modified scaffold was the best for cartilage tissue engineering in terms of cell proliferation and extracellular matrix production. Chondrocytes/collagen-modified scaffold constructs were implanted subdermally in the dorsal spaces of female nude mice. Histological and immunohistochemical staining of the retrieved implants after 8 weeks revealed enhanced cartilage tissue formation. We conclude that collagen surface modification through immersion coating on SLS-manufactured scaffolds is a feasible scaffold for cartilage tissue engineering in craniofacial reconstruction. - Highlights: • Selective laser sintered polycaprolactone scaffolds are prepared. • Scaffolds are surface modified through immersion coating with gelatin or collagen. • Collagen-scaffold is the best for cartilage tissue engineering in vitro. • Chondrocytes/collagen-scaffold reveals enhanced cartilage tissue formation in vivo

Surface modification of polycaprolactone scaffolds fabricated via selective laser sintering for cartilage tissue engineering

Energy Technology Data Exchange (ETDEWEB)

Chen, Chih-Hao [Department of Chemical and Materials Engineering, Chang Gung University, Kweishan, Taoyuan 333, Taiwan, ROC (China); Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Craniofacial Research Center, Chang Gung University, Kweishann, Taoyuan 333, Taiwan, ROC (China); Lee, Ming-Yih [Graduate Institute of Medical Mechatronics, Chang Gung University, Kweishan, Taoyuan 333, Taiwan, ROC (China); Shyu, Victor Bong-Hang; Chen, Yi-Chieh; Chen, Chien-Tzung [Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Craniofacial Research Center, Chang Gung University, Kweishann, Taoyuan 333, Taiwan, ROC (China); Chen, Jyh-Ping, E-mail: jpchen@mail.cgu.edu.tw [Department of Chemical and Materials Engineering, Chang Gung University, Kweishan, Taoyuan 333, Taiwan, ROC (China); Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kweishan, Taoyuan 333, Taiwan, ROC (China)

2014-07-01

Surface modified porous polycaprolactone scaffolds fabricated via rapid prototyping techniques were evaluated for cartilage tissue engineering purposes. Polycaprolactone scaffolds manufactured by selective laser sintering (SLS) were surface modified through immersion coating with either gelatin or collagen. Three groups of scaffolds were created and compared for both mechanical and biological properties. Surface modification with collagen or gelatin improved the hydrophilicity, water uptake and mechanical strength of the pristine scaffold. From microscopic observations and biochemical analysis, collagen-modified scaffold was the best for cartilage tissue engineering in terms of cell proliferation and extracellular matrix production. Chondrocytes/collagen-modified scaffold constructs were implanted subdermally in the dorsal spaces of female nude mice. Histological and immunohistochemical staining of the retrieved implants after 8 weeks revealed enhanced cartilage tissue formation. We conclude that collagen surface modification through immersion coating on SLS-manufactured scaffolds is a feasible scaffold for cartilage tissue engineering in craniofacial reconstruction. - Highlights: • Selective laser sintered polycaprolactone scaffolds are prepared. • Scaffolds are surface modified through immersion coating with gelatin or collagen. • Collagen-scaffold is the best for cartilage tissue engineering in vitro. • Chondrocytes/collagen-scaffold reveals enhanced cartilage tissue formation in vivo.

A novel surface modification on calcium polyphosphate scaffold for articular cartilage tissue engineering

International Nuclear Information System (INIS)

Lien, S.-M.; Liu, C.-K.; Huang, T.-J.

2007-01-01

The surface of porous three-dimensional (3D) calcium polyphosphate (CPP) scaffold was modified by treatment of quenching-after-sintering in the fabrication process. Scanning electron microscopic examination and degradation tests confirmed a new type of surface modification. A rotary-shaking culture was compared to that of a stationary culture and the results showed that rotary shaking led to enhanced extracellular matrices (ECM) secretion of both proteoglycans and collagen. Rotary-shaking cultured results showed that the quenching-treated CPP scaffold produced a better cartilage tissue, with both proteoglycans and collagen secretions enhanced, than the air-cooled-after-sintering scaffolds. Moreover, β-CPP scaffolds were better for the ECM secretion of both proteoglycans and collagen than the β-CPP + γ-CPP multiphase scaffold. However, the multiphase scaffold led to higher growth rate than that of β-CPP scaffold; the quenching-after-sintering treatment reversed this. In addition, the ECM secretions of both proteoglycans and collagen in the quenching-treated β-CPP scaffold were higher than those in the air-cooled one. Thus, the novel treatment of quenching-after-sintering has shown merits to the porous 3D CPP scaffolds for articular cartilage tissue engineering

Subchondral Bone Plate Thickening Precedes Chondrocyte Apoptosis and Cartilage Degradation in Spontaneous Animal Models of Osteoarthritis

OpenAIRE

Zamli, Zaitunnatakhin; Robson Brown, Kate; Tarlton, John F.; Adams, Mike A.; Torlot, Georgina E.; Cartwright, Charlie; Cook, William A.; Vassilevskaja, Kristiina; Sharif, Mohammed

2014-01-01

Osteoarthritis (OA) is the most common joint disorder characterised by bone remodelling and cartilage degradation and associated with chondrocyte apoptosis. These processes were investigated at 10, 16, 24, and 30 weeks in Dunkin Hartley (DH) and Bristol Strain 2 (BS2) guinea pigs that develop OA spontaneously. Both strains had a more pronounced chondrocyte apoptosis, cartilage degradation, and subchondral bone changes in the medial than the lateral side of the tibia, and between strains, the ...

Articular cartilage repair with recombinant human type II collagen/polylactide scaffold in a preliminary porcine study.

Science.gov (United States)

Muhonen, Virpi; Salonius, Eve; Haaparanta, Anne-Marie; Järvinen, Elina; Paatela, Teemu; Meller, Anna; Hannula, Markus; Björkman, Mimmi; Pyhältö, Tuomo; Ellä, Ville; Vasara, Anna; Töyräs, Juha; Kellomäki, Minna; Kiviranta, Ilkka

2016-05-01

The purpose of this study was to investigate the potential of a novel recombinant human type II collagen/polylactide scaffold (rhCo-PLA) in the repair of full-thickness cartilage lesions with autologous chondrocyte implantation technique (ACI). The forming repair tissue was compared to spontaneous healing (spontaneous) and repair with a commercial porcine type I/III collagen membrane (pCo). Domestic pigs (4-month-old, n = 20) were randomized into three study groups and a circular full-thickness chondral lesion with a diameter of 8 mm was created in the right medial femoral condyle. After 3 weeks, the chondral lesions were repaired with either rhCo-PLA or pCo together with autologous chondrocytes, or the lesion was only debrided and left untreated for spontaneous repair. The repair tissue was evaluated 4 months after the second operation. Hyaline cartilage formed most frequently in the rhCo-PLA treatment group. Biomechanically, there was a trend that both treatment groups resulted in better repair tissue than spontaneous healing. Adverse subchondral bone reactions developed less frequently in the spontaneous group (40%) and the rhCo-PLA treated group (50%) than in the pCo control group (100%). However, no statistically significant differences were found between the groups. The novel rhCo-PLA biomaterial showed promising results in this proof-of-concept study, but further studies will be needed in order to determine its effectiveness in articular cartilage repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:745-753, 2016. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

A Novel Method to Simulate the Progression of Collagen Degeneration of Cartilage in the Knee: Data from the Osteoarthritis Initiative

Science.gov (United States)

Mononen, Mika E.; Tanska, Petri; Isaksson, Hanna; Korhonen, Rami K.

2016-02-01

We present a novel algorithm combined with computational modeling to simulate the development of knee osteoarthritis. The degeneration algorithm was based on excessive and cumulatively accumulated stresses within knee joint cartilage during physiological gait loading. In the algorithm, the collagen network stiffness of cartilage was reduced iteratively if excessive maximum principal stresses were observed. The developed algorithm was tested and validated against experimental baseline and 4-year follow-up Kellgren-Lawrence grades, indicating different levels of cartilage degeneration at the tibiofemoral contact region. Test groups consisted of normal weight and obese subjects with the same gender and similar age and height without osteoarthritic changes. The algorithm accurately simulated cartilage degeneration as compared to the Kellgren-Lawrence findings in the subject group with excess weight, while the healthy subject group’s joint remained intact. Furthermore, the developed algorithm followed the experimentally found trend of cartilage degeneration in the obese group (R2 = 0.95, p osteoarthritis (0-2 years, p  0.05). The proposed algorithm revealed a great potential to objectively simulate the progression of knee osteoarthritis.

Ectopic mineralization of cartilage and collagen-rich tendons and ligaments in Enpp1asj-2J mice.

Science.gov (United States)

Zhang, Jieyu; Dyment, Nathaniel A; Rowe, David W; Siu, Sarah Y; Sundberg, John P; Uitto, Jouni; Li, Qiaoli

2016-03-15

Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder caused by mutations in the ENPP1 gene, manifests with extensive mineralization of the cardiovascular system. A spontaneous asj-2J mutant mouse has been characterized as a model for GACI. Previous studies focused on phenotypic characterization of skin and vascular tissues. This study further examined the ectopic mineralization phenotype of cartilage, collagen-rich tendons and ligaments in this mouse model. The mice were placed on either control diet or the "acceleration diet" for up to 12 weeks of age. Soft connective tissues, such as ear (elastic cartilage) and trachea (hyaline cartilage), were processed for standard histology. Assessment of ectopic mineralization in articular cartilage and fibrocartilage as well as tendons and ligaments which are attached to long bones were performed using a novel cryo-histological method without decalcification. These analyses demonstrated ectopic mineralization in cartilages as well as tendons and ligaments in the homozygous asj-2J mice at 12 weeks of age, with the presence of immature osteophytes displaying alkaline phosphatase and tartrate-resistant acid phosphatase activities as early as at 6 weeks of age. Alkaline phosphatase activity was significantly increased in asj-2J mouse serum as compared to wild type mice, indicating increased bone formation rate in these mice. Together, these data highlight the key role of ENPP1 in regulating calcification of both soft and skeletal tissues.

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.

The collagen receptor uPARAP/Endo180 in tissue degradation and cancer (Review)

DEFF Research Database (Denmark)

Carlsen Melander, Eva Maria; Jürgensen, Henrik J; Madsen, Daniel H

2015-01-01

The collagen receptor uPARAP/Endo180, the product of the MRC2 gene, is a central component in the collagen turnover process governed by various mesenchymal cells. Through the endocytosis of collagen or large collagen fragments, this recycling receptor serves to direct basement membrane collagen...... as well as interstitial collagen to lysosomal degradation. This capacity, shared only with the mannose receptor from the same protein family, endows uPARAP/Endo180 with a critical role in development and homeostasis, as well as in pathological disruptions of the extracellular matrix structure. Important...

Hyaline Articular Matrix Formed by Dynamic Self-Regenerating Cartilage and Hydrogels.

Science.gov (United States)

Meppelink, Amanda M; Zhao, Xing; Griffin, Darvin J; Erali, Richard; Gill, Thomas J; Bonassar, Lawrence J; Redmond, Robert W; Randolph, Mark A

2016-07-01

Injuries to the articular cartilage surface are challenging to repair because cartilage possesses a limited capacity for self-repair. The outcomes of current clinical procedures aimed to address these injuries are inconsistent and unsatisfactory. We have developed a novel method for generating hyaline articular cartilage to improve the outcome of joint surface repair. A suspension of 10(7) swine chondrocytes was cultured under reciprocating motion for 14 days. The resulting dynamic self-regenerating cartilage (dSRC) was placed in a cartilage ring and capped with fibrin and collagen gel. A control group consisted of chondrocytes encapsulated in fibrin gel. Constructs were implanted subcutaneously in nude mice and harvested after 6 weeks. Gross, histological, immunohistochemical, biochemical, and biomechanical analyses were performed. In swine patellar groove, dSRC was implanted into osteochondral defects capped with collagen gel and compared to defects filled with osteochondral plugs, collagen gel, or left empty after 6 weeks. In mice, the fibrin- and collagen-capped dSRC constructs showed enhanced contiguous cartilage matrix formation over the control of cells encapsulated in fibrin gel. Biochemically, the fibrin and collagen gel dSRC groups were statistically improved in glycosaminoglycan and hydroxyproline content compared to the control. There was no statistical difference in the biomechanical data between the dSRC groups and the control. The swine model also showed contiguous cartilage matrix in the dSRC group but not in the collagen gel and empty defects. These data demonstrate the survivability and successful matrix formation of dSRC under the mechanical forces experienced by normal hyaline cartilage in the knee joint. The results from this study demonstrate that dSRC capped with hydrogels successfully engineers contiguous articular cartilage matrix in both nonload-bearing and load-bearing environments.

Tranexamic acid, an inhibitor of plasminogen activation, reduces urinary collagen cross-link excretion in both experimental and rheumatoid arthritis

NARCIS (Netherlands)

Ronday, H.K.; TeKoppele, J.M.; Greenwald, R.A.; Moak, S.A.; Roos, J.A.D.M. de; Dijkmans, B.A.C.; Breedveld, F.C.; Verheijen, J.H.

1998-01-01

The plasminogen activation system is one of the enzyme systems held responsible for bone and cartilage degradation in rheumatoid arthritis (RA). In this study, we evaluated the effect of tranexamic acid (TEA), an inhibitor of plasminogen activation, on urinary collagen cross-link excretion and

Experimental resin cements containing bioactive fillers reduce matrix metalloproteinase-mediated dentin collagen degradation.

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Osorio, Raquel; Yamauti, Monica; Sauro, Salvatore; Watson, Thimoty F; Toledano, Manuel

2012-09-01

Collagen dentin matrix may represent a suitable scaffold to be remineralized in the presence of bioactive materials. The purpose of this study was to determine if experimental resin cements containing bioactive fillers may modulate matrix metalloproteinase-mediated collagen degradation of etched dentin. Human dentin beams demineralized using 10% phosphoric acid or 0.5 mol/L EDTA were infiltrated with the following experimental resins: (1) unfilled resin, (2) resin with Bioglass 45S5 particles (Sylc; OSspray Ltd, London, UK), and (3) resin with β-tricalcium phosphate-modified calcium silicate cement (HCAT-β) particles. The filler/resin ratio was 40/60 wt%. The specimens were stored in artificial saliva, and the determination of C-terminal telopeptide (ICTP) was performed by radioimmunoassay after 24 hours, 1 week, and 4 weeks. Scanning electron microscopic analysis of dentin surfaces after 4 weeks of storage was also executed. Collagen degradation was prominent both in phosphoric acid and EDTA-treated dentin. Resin infiltration strongly reduced the MMP activity in demineralized dentin. Resin-containing Bioglass 45S5 particles exerted higher and more stable protection of collagen at all tested dentin states and time points. HCAT-β induced collagen protection from MMPs only in EDTA-treated specimens. Dentin remineralization was achieved when dentin was infiltrated with the resin cements containing bioactive fillers. MMP degradation of dentin collagen is strongly reduced in resin-infiltrated dentin. The inclusion of Bioglass 45S5 particles exerted an additional protection of collagen during dentin remineralization. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Mechano growth factor (MGF) and transforming growth factor (TGF)-β3 functionalized silk scaffolds enhance articular hyaline cartilage regeneration in rabbit model.

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Luo, Ziwei; Jiang, Li; Xu, Yan; Li, Haibin; Xu, Wei; Wu, Shuangchi; Wang, Yuanliang; Tang, Zhenyu; Lv, Yonggang; Yang, Li

2015-06-01

Damaged cartilage has poor self-healing ability and usually progresses to scar or fibrocartilaginous tissue, and finally degenerates to osteoarthritis (OA). Here we demonstrated that one of alternative isoforms of IGF-1, mechano growth factor (MGF) acted synergistically with transforming growth factor β3 (TGF-β3) embedded in silk fibroin scaffolds to induce chemotactic homing and chondrogenic differentiation of mesenchymal stem cells (MSCs). Combination of MGF and TGF-β3 significantly increased cell recruitment up to 1.8 times and 2 times higher than TGF-β3 did in vitro and in vivo. Moreover, MGF increased Collagen II and aggrecan secretion of TGF-β3 induced hMSCs chondrogenesis, but decreased Collagen I in vitro. Silk fibroin (SF) scaffolds have been widely used for tissue engineering, and we showed that methanol treated pured SF scaffolds were porous, similar to compressive module of native cartilage, slow degradation rate and excellent drug released curves. At 7 days after subcutaneous implantation, TGF-β3 and MGF functionalized silk fibroin scaffolds (STM) recruited more CD29+/CD44+cells (Pcartilage-like extracellular matrix and less fibrillar collagen were detected in STM scaffolds than that in TGF-β3 modified scaffolds (ST) at 2 months after subcutaneous implantation. When implanted into articular joints in a rabbit osteochondral defect model, STM scaffolds showed the best integration into host tissues, similar architecture and collagen organization to native hyaline cartilage, as evidenced by immunostaining of aggrecan, collagen II and collagen I, as well as Safranin O and Masson's trichrome staining, and histological evalution based on the modified O'Driscoll histological scoring system (Pcartilage regeneration. This study demonstrated that TGF-β3 and MGF functionalized silk fibroin scaffolds enhanced endogenous stem cell recruitment and facilitated in situ articular cartilage regeneration, thus providing a novel strategy for cartilage repair

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

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

2012-06-01

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

A method of experimental rheumatoid arthritis induction using collagen type II isolated from chicken sternal cartilage.

Science.gov (United States)

Su, Zhaoliang; Shotorbani, Siamak Sandoghchian; Jiang, Xugan; Ma, Rui; Shen, Huiling; Kong, Fanzhi; Xu, Huaxi

2013-07-01

At present, collagen‑induced arthritis (CIA) is the best known and most extensively used model for the immunological and pathological characteristics of human rheumatoid arthritis (RA). This model is useful not only in aiding our understanding of the pathogenesis of this disease, but also in the development of new therapies. Bovine, porcine and human collagen has been used to induce CIA; however, response has been identified to vary between strains and injection conditions, and false positive results and reduced potency are common as a result of minor contaminants or deglycosylated protein. Therefore, in the present study, type II collagen (CII) was isolated and purified from chicken sternal cartilage and was found to successfully induce the RA model. Furthermore, T helper 17 (Th17) cells were observed to infiltrate the joint on day 45 following induction by CII. In vitro, expression of toll‑like receptor 2 (TLR2) increased in peritoneal macrophages stimulated by CII. In addition, blockage of TLR2 was identified to markedly decrease levels of TGF‑β and IL‑6 in the cell culture supernatant. The results indicate that CII isolated from chicken sternal cartilage may be recognized by TLR2 on macrophages, leading to TGF‑β and IL‑6 production and subsequent activation of Th17 cells which mediates CIA development.

Engineering endostatin-producing cartilaginous constructs for cartilage repair using nonviral transfection of chondrocyte-seeded and mesenchymal-stem-cell-seeded collagen scaffolds.

Science.gov (United States)

Jeng, Lily; Olsen, Bjorn R; Spector, Myron

2010-10-01

Although there is widespread recognition of the importance of angiogenesis in tissue repair, there is little work on the inhibition of angiogenesis in the context of tissue engineering of naturally avascular tissues, like articular cartilage. The objective was to engineer a collagen-scaffold-based cartilaginous construct overexpressing a potent antiangiogenic factor, endostatin, using nonviral transfection. Endostatin-plasmid-supplemented collagen scaffolds were seeded with mesenchymal stem cells and chondrocytes and cultured for 20–22 days. The effects of the following variables on endostatin expression and chondrogenesis were examined: collagen scaffold material, method of nonviral vector incorporation, plasmid load, culture medium, and oxygen tension. An increase and peak of endostatin protein was observed during the first week of culture, followed by a decrease to low levels, suggesting that overexpression of endostatin could be sustained for several days using the nonviral vector. The amount of endostatin produced was tunable with the external factors. Chondrogenesis was observed in the engineered constructs cultured in chondrogenic medium at the 3-week time point, demonstrating that endostatin did not inhibit the chondrogenic potential of mesenchymal stem cells or the general viability of the cells. The ability to engineer endostatin-expressing cartilaginous constructs will be of value for future work exercising regulatory control of angiogenesis in cartilage repair.

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...

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

International Nuclear Information System (INIS)

Julkunen, P; Korhonen, R K; Nissi, M J; Jurvelin, J S

2008-01-01

Magnetic resonance imaging (MRI) provides a method for non-invasive characterization of cartilage composition and structure. We aimed to see whether T 1 and T 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 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 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 1 . In bovine cartilage, T 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 2 . Based on the simulations, cartilage with a complex collagen architecture (5 or 7 laminae), leading to high bulk T 2 due to magic angle effects, provided higher compressive stiffness than tissue with a simple collagen architecture (3 laminae). Our results suggest that T 1 reflects PG-specific mechanical properties of cartilage. High T 2 is characteristic to soft cartilage with a classical collagen architecture. Contradictorily, high bulk T 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

Trend of Cadherin-11 expression and its impact on cartilage degradation in the temporomandibular joints of guinea pigs with spontaneous osteoarthritis.

Science.gov (United States)

Wu, Mengjie; Lu, Haiping; Yu, Fengyang; Zhou, Yiqun

2016-08-01

This study aims to investigate spatial and temporal changes in cadherin-11 (CAD-11) expression and their effects on cartilage degeneration in the temporomandibular joint (TMJ) of guinea pigs with spontaneous osteoarthritis (OA). Dunkin-Hartley (DH) and Bristol strain 2 (BS2) guinea pigs at ages of 1, 3, 6, 9, and 12 months were categorized into two groups and analyzed. The bilateral TMJ condyles of DH and BS2 guinea pigs were harvested and fixed. The distribution and expression profiles of CAD-11, collagen type II, and matrix metalloproteinase 3 (MMP-3) were detected by immunohistological assays. Histological micrographs of the condyle cartilage were obtained and analyzed. Osteoarthritis can be spontaneously induced by mechanical stress in DH guinea pigs. The main histopathological changes in the TMJ structure and increased expression of MMP-3 occurred within 6-9 months of ages in DH guinea pigs with spontaneous OA. By contrast, minimal to mild cartilage degradations were observed in the TMJ of BS2 guinea pigs even at the age of 12 months. From as early as 3 months of age, the expression levels of CAD-11 were upregulated in the TMJ of DH guinea pigs compared with those in BS2 animals. CAD-11 expression differed between the two groups at 12 months of age. Increased CAD-11 expression within cartilage is associated with the development and progression of OA between the two strains of guinea pigs. Therefore, CAD-11 expression in TMJ could be an important predisposing factor for the development of spontaneous OA. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Shortwave-infrared Raman spectroscopic classification of water fractions in articular cartilage ex vivo

Science.gov (United States)

Unal, Mustafa; Akkus, Ozan

2018-01-01

Water loss is an early onset indicator of osteoarthritis. Although Raman spectroscopy (RS) holds the potential for measurement of cartilage hydration, the knowledge of Raman OH-stretch bands of biological tissue is very limited. We assesed here the sensitivity of RS to identify and classify water types in the cartilage. Raman spectrum measurements over the high wavenumber range were employed to identify different water fractions in articular cartilage. Raman spectra were collected from wet and sequentially dehydrated cartilage along with pure collagen type II and chondroitin sulfate standards. OH-stretch band of cartilage is dominated by mobile water, up to 95% of total intensities. We identified six peaks in cartilage spectrum using second-derivative analysis: peaks at 3200 and 3650 cm-1 are associated with organic matrix (both collagen and proteglycan) and matrix-bound water molecules. Peaks at 3250, 3453, and 3630 cm-1 are associated with collagen and collagen-related water molecules, whereas the peak at 3520 cm-1 is associated with proteoglycan (PG) and PG-related water molecules. The current work is the first thorough analysis of the Raman OH-stretch band of the cartilage and with the knowledge generated by this study, it may now be possible to study on cartilage hydration by RS.

Cartilaginous extracellular matrix-modified chitosan hydrogels for cartilage tissue engineering.

Science.gov (United States)

Choi, Bogyu; Kim, Soyon; Lin, Brian; Wu, Benjamin M; Lee, Min

2014-11-26

Cartilaginous extracellular matrix (ECM) components such as type-II collagen (Col II) and chondroitin sulfate (CS) play a crucial role in chondrogenesis. However, direct clinical use of natural Col II or CS as scaffolds for cartilage tissue engineering is limited by their instability and rapid enzymatic degradation. Here, we investigate the incorporation of Col II and CS into injectable chitosan hydrogels designed to gel upon initiation by exposure to visible blue light (VBL) in the presence of riboflavin. Unmodified chitosan hydrogel supported proliferation and deposition of cartilaginous ECM by encapsulated chondrocytes and mesenchymal stem cells. The incorporation of native Col II or CS into chitosan hydrogels further increased chondrogenesis. The incorporation of Col II, in particular, was found to be responsible for the enhanced cellular condensation and chondrogenesis observed in modified hydrogels. This was mediated by integrin α10 binding to Col II, increasing cell-matrix adhesion. These findings demonstrate the potential of cartilage ECM-modified chitosan hydrogels as biomaterials to promote cartilage regeneration.

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

Science.gov (United States)

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

2013-01-01

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

The relationship between shear force, compression, collagen characteristics, desmin degradation and sarcomere length in lamb biceps femoris.

Science.gov (United States)

Starkey, Colin P; Geesink, Geert H; van de Ven, Remy; Hopkins, David L

2017-04-01

This study aimed to identity the relationships between known variants of tenderness (collagen content (total and soluble), desmin degradation and sarcomere length) and shear force and compression in the biceps femoris aged for 14days from 112 mixed sex lambs. Desmin degradation was related to compression (Pcompression decreased. Sarcomere length (SL) was related to shear force (Pcompression (Pcompression, sarcomere length and soluble collagen. The findings from this experiment indicate that the known variants (soluble collagen, sarcomere length and desmin degradation) are related to shear force and compression in ovine biceps femoris. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

PIXE and cSAXS studies at the bone-cartilage interface

International Nuclear Information System (INIS)

Kaabar, W.; Gundogdu, O.; Bradley, D.A.; Bunk, O.; Pfeiffer, F.; Farquharson, M.J.; Webb, M.; Jeynes, C.

2008-01-01

Full text: Divalent cations such as Zn and Ca play a central role both in the normal processes of growth and remodelling as well as in the degenerative and inflammatory processes of articular cartilage during arthritis. These cations act as co-factors of a class of enzymes known as metalloproteinases, believed to be active during the initiation, progress and remodelling processes associated with osteoarthritis. Other important enzymes such as alkaline phosphatase, involved in cartilage mineralization, are also associated with the presence of these metallic co-factors. A number of authors have used X-ray fluorescence, employing synchrotron radiation sources to map metal ion distributions in bone and cartilage. In the present work, investigations were carried out on the distribution of metallic ions (Zn, Ca, P and S) in articular cartilage samples at the University of Surrey hosted EPSRC national ion beam facility based on a 2 MV Tandetron accelerator. An in-air beam line was used, with proton energy of 2.5 MeV. Micro Proton-Induced X-ray Emission (μ-PIXE) analysis has been made of the bone-cartilage interface for samples taken from the human femoral head. The bone-cartilage interface region between uncalcified and mineralized cartilage regions has attracted particular interest, being identified to be an active site of remodelling. Here coherent small angle X-ray scattering (cSAXS) has also been employed to investigate the structure and organization of the collagen network in decalcified diseased human femoral heads and the equine metacarpus joint, study being carried out at the Paul Scherrer Institute (PSI) synchrotron beamline cSAXS. (Fig. 1: cSAXS over a 1 mm x 1.5 mm area of a cartilage/bone sample; the left- and right hand panels corresponds to the length scales 658-568 A and 962-833 A respectively. The bar scale indicates relative orientation, from 0 deg (blue) to 90 deg (red)). The results of Fig. 1 are plotted in terms of orientation of cartilage and bone

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.

Assessment of hyaline cartilage matrix composition using near infrared spectroscopy.

Science.gov (United States)

Palukuru, Uday P; McGoverin, Cushla M; Pleshko, Nancy

2014-09-01

Changes in the composition of the extracellular matrix (ECM) are characteristic of injury or disease in cartilage tissue. Various imaging modalities and biochemical techniques have been used to assess the changes in cartilage tissue but lack adequate sensitivity, or in the case of biochemical techniques, result in destruction of the sample. Fourier transform near infrared (FT-NIR) spectroscopy has shown promise for the study of cartilage composition. In the current study NIR spectroscopy was used to identify the contributions of individual components of cartilage in the NIR spectra by assessment of the major cartilage components, collagen and chondroitin sulfate, in pure component mixtures. The NIR spectra were obtained using homogenous pellets made by dilution with potassium bromide. A partial least squares (PLS) model was calculated to predict composition in bovine cartilage samples. Characteristic absorbance peaks between 4000 and 5000 cm(-1) could be attributed to components of cartilage, i.e. collagen and chondroitin sulfate. Prediction of the amount of collagen and chondroitin sulfate in tissues was possible within 8% (w/dw) of values obtained by gold standard biochemical assessment. These results support the use of NIR spectroscopy for in vitro and in vivo applications to assess matrix composition of cartilage tissues, especially when tissue destruction should be avoided. Copyright © 2014. Published by Elsevier B.V.

Effects of type I collagen degradation on the durability of three adhesive systems in the early phase of dentin bonding.

Directory of Open Access Journals (Sweden)

Lin Hu

Full Text Available This study was designed to evaluate the effects of type I collagen degradation on the durability of three adhesive systems in the early phase of dentin bonding.Bonded dentin specimens were prepared using three different types of adhesive systems. Micro-tensile bond strength and degradation of collagen were tested before, and after 1 month or 4 months of aging in artificial saliva. The relationship between micro-tensile bond strength and collagen degradation was analyzed by calculating their Pearson's correlation coefficient.Aging induced time-dependent reduction in micro-tensile bond strengths for all the tested adhesive systems, although such reduction for the single-step self-etching adhesive G-Bond (GB was not statistically significant. The bond strength of the two-step self-etching primer adhesive system Clearfil SE Bond (SEB was similar to that of the two-step etch-and-rinse self-priming adhesive system Single Bond 2 (SB, and they were both significantly reduced after one or four months of aging. A negative correlation was found between the degree of collagen degradation and magnitude of micro-tensile bond strength (r = -0.65, p = 0.003. The Pearson's correlation coefficient was 0.426, indicating that 42.6% of the aging-induced reduction in bond strength can be explained by the degradation of collagen.In the early phase of dentin bonding, there was a negative correlation between the degree of collagen degradation and the magnitude of micro-tensile bond strength. The reduction of bond strength was accompanied by the degradation of collagen. These results provide evidence for the causative relationship between the degradation of collagen and the deterioration of dentin-adhesive interface.

X-ray phase contrast imaging of the bone-cartilage interface

International Nuclear Information System (INIS)

Ismail, E.C.; Kaabar, W.; Garrity, D.; Gundogdu, O.; Bradley, D.A.; Bunk, O.; Pfeiffer, F.; Farquharson, M.J.

2008-01-01

Full text: Synovial joints articulate in a lubricating environment, the system providing for smooth articulation. The articular cartilage overlying the bone consists of a network of collagen fibres. This network is essential to cartilage integrity, suffering damage in degenerative joint disease such as osteoarthritis. At Surrey and also in work conducted by this group at the Paul Scherrer Institute (PSI) synchrotron site we have been applying a number of techniques in studying the bone-cartilage interface and of changes occurring in this with disease. One technique attracting particular interest is X-ray phase contrast imaging, yielding information on anatomical features that manifest from the large scale organisation of collagen and the mineralised phase contained within the collagen fibres in the deep cartilage zone. This work will briefly review some of the basic supporting physics and then shows some of the images and other results that we have obtained to-date

Enhanced hyaline cartilage matrix synthesis in collagen sponge scaffolds by using siRNA to stabilize chondrocytes phenotype cultured with bone morphogenetic protein-2 under hypoxia.

Science.gov (United States)

Legendre, Florence; Ollitrault, David; Hervieu, Magalie; Baugé, Catherine; Maneix, Laure; Goux, Didier; Chajra, Hanane; Mallein-Gerin, Frédéric; Boumediene, Karim; Galera, Philippe; Demoor, Magali

2013-07-01

Cartilage healing by tissue engineering is an alternative strategy to reconstitute functional tissue after trauma or age-related degeneration. However, chondrocytes, the major player in cartilage homeostasis, do not self-regenerate efficiently and lose their phenotype during osteoarthritis. This process is called dedifferentiation and also occurs during the first expansion step of autologous chondrocyte implantation (ACI). To ensure successful ACI therapy, chondrocytes must be differentiated and capable of synthesizing hyaline cartilage matrix molecules. We therefore developed a safe procedure for redifferentiating human chondrocytes by combining appropriate physicochemical factors: hypoxic conditions, collagen scaffolds, chondrogenic factors (bone morphogenetic protein-2 [BMP-2], and insulin-like growth factor I [IGF-I]) and RNA interference targeting the COL1A1 gene. Redifferentiation of dedifferentiated chondrocytes was evaluated using gene/protein analyses to identify the chondrocyte phenotypic profile. In our conditions, under BMP-2 treatment, redifferentiated and metabolically active chondrocytes synthesized a hyaline-like cartilage matrix characterized by type IIB collagen and aggrecan molecules without any sign of hypertrophy or osteogenesis. In contrast, IGF-I increased both specific and noncharacteristic markers (collagens I and X) of chondrocytes. The specific increase in COL2A1 gene expression observed in the BMP-2 treatment was shown to involve the specific enhancer region of COL2A1 that binds the trans-activators Sox9/L-Sox5/Sox6 and Sp1, which are associated with a decrease in the trans-inhibitors of COL2A1, c-Krox, and p65 subunit of NF-kappaB. Our procedure in which BMP-2 treatment under hypoxia is associated with a COL1A1 siRNA, significantly increased the differentiation index of chondrocytes, and should offer the opportunity to develop new ACI-based therapies in humans.

Quantitative Raman characterization of cross-linked collagen thin films as a model system for diagnosing early osteoarthritis

Science.gov (United States)

Wang, Chao; Durney, Krista M.; Fomovsky, Gregory; Ateshian, Gerard A.; Vukelic, Sinisa

2016-03-01

The onset of osteoarthritis (OA)in articular cartilage is characterized by degradation of extracellular matrix (ECM). Specifically, breakage of cross-links between collagen fibrils in the articular cartilage leads to loss of structural integrity of the bulk tissue. Since there are no broadly accepted, non-invasive, label-free tools for diagnosing OA at its early stage, Raman spectroscopyis therefore proposed in this work as a novel, non-destructive diagnostic tool. In this study, collagen thin films were employed to act as a simplified model system of the cartilage collagen extracellular matrix. Cross-link formation was controlled via exposure to glutaraldehyde (GA), by varying exposure time and concentration levels, and Raman spectral information was collected to quantitatively characterize the cross-link assignments imparted to the collagen thin films during treatment. A novel, quantitative method was developed to analyze the Raman signal obtained from collagen thin films. Segments of Raman signal were decomposed and modeled as the sum of individual bands, providing an optimization function for subsequent curve fitting against experimental findings. Relative changes in the concentration of the GA-induced pyridinium cross-links were extracted from the model, as a function of the exposure to GA. Spatially resolved characterization enabled construction of spectral maps of the collagen thin films, which provided detailed information about the variation of cross-link formation at various locations on the specimen. Results showed that Raman spectral data correlate with glutaraldehyde treatment and therefore may be used as a proxy by which to measure loss of collagen cross-links in vivo. This study proposes a promising system of identifying onset of OA and may enable early intervention treatments that may serve to slow or prevent osteoarthritis progression.

Effect of collagen turnover on the accumulation of advanced glycation end products

NARCIS (Netherlands)

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

2000-01-01

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

Which elements are involved in reversible and irreversible cartilage degradation in osteoarthritis?

DEFF Research Database (Denmark)

Bay-Jensen, Anne-Christine; Hoegh-Madsen, Suzi; Dam, Erik

2010-01-01

-physiology of the joint and whether the joint damage is reversible or irreversible. In this review, we compile emerging data on cellular and pathological aspects of OA, and ask whether these data could give clue to when cartilage degradation is reversible and whether a point-of-no-return exists. We highlight different...

Fourier transform infrared imaging and infrared fiber optic probe spectroscopy identify collagen type in connective tissues.

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Arash Hanifi

Full Text Available Hyaline cartilage and mechanically inferior fibrocartilage consisting of mixed collagen types are frequently found together in repairing articular cartilage. The present study seeks to develop methodology to identify collagen type and other tissue components using Fourier transform infrared (FTIR spectral evaluation of matrix composition in combination with multivariate analyses. FTIR spectra of the primary molecular components of repair cartilage, types I and II collagen, and aggrecan, were used to develop multivariate spectral models for discrimination of the matrix components of the tissues of interest. Infrared imaging data were collected from bovine bone, tendon, normal cartilage, meniscus and human repair cartilage tissues, and composition predicted using partial least squares analyses. Histology and immunohistochemistry results were used as standards for validation. Infrared fiber optic probe spectral data were also obtained from meniscus (a tissue with mixed collagen types to evaluate the potential of this method for identification of collagen type in a minimally-invasive clinical application. Concentration profiles of the tissue components obtained from multivariate analysis were in excellent agreement with histology and immunohistochemistry results. Bone and tendon showed a uniform distribution of predominantly type I collagen through the tissue. Normal cartilage showed a distribution of type II collagen and proteoglycan similar to the known composition, while in repair cartilage, the spectral distribution of both types I and II collagen were similar to that observed via immunohistochemistry. Using the probe, the outer and inner regions of the meniscus were shown to be primarily composed of type I and II collagen, respectively, in accordance with immunohistochemistry data. In summary, multivariate analysis of infrared spectra can indeed be used to differentiate collagen type I and type II, even in the presence of proteoglycan, in

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage.

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Arbabi, Vahid; Pouran, Behdad; Zadpoor, Amir A; Weinans, Harrie

2017-04-23

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

Studies of mineralization in tissue culture: optimal conditions for cartilage calcification

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Boskey, A. L.; Stiner, D.; Doty, S. B.; Binderman, I.; Leboy, P.

1992-01-01

The optimal conditions for obtaining a calcified cartilage matrix approximating that which exists in situ were established in a differentiating chick limb bud mesenchymal cell culture system. Using cells from stage 21-24 embryos in a micro-mass culture, at an optimal density of 0.5 million cells/20 microliters spot, the deposition of small crystals of hydroxyapatite on a collagenous matrix and matrix vesicles was detected by day 21 using X-ray diffraction, FT-IR microscopy, and electron microscopy. Optimal media, containing 1.1 mM Ca, 4 mM P, 25 micrograms/ml vitamin C, 0.3 mg/ml glutamine, no Hepes buffer, and 10% fetal bovine serum, produced matrix resembling the calcifying cartilage matrix of fetal chick long bones. Interestingly, higher concentrations of fetal bovine serum had an inhibitory effect on calcification. The cartilage phenotype was confirmed based on the cellular expression of cartilage collagen and proteoglycan mRNAs, the presence of type II and type X collagen, and cartilage type proteoglycan at the light microscopic level, and the presence of chondrocytes and matrix vesicles at the EM level. The system is proposed as a model for evaluating the events in cell mediated cartilage calcification.

Effects of growth factors and glucosamine on porcine mandibular condylar cartilage cells and hyaline cartilage cells for tissue engineering applications.

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Wang, Limin; Detamore, Michael S

2009-01-01

Temporomandibular joint (TMJ) condylar cartilage is a distinct cartilage that has both fibrocartilaginous and hyaline-like character, with a thin proliferative zone that separates the fibrocartilaginous fibrous zone at the surface from the hyaline-like mature and hypertrophic zones below. In this study, we compared the effects of insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF), transforming growth factor beta1 (TGF-beta1), and glucosamine sulphate on porcine TMJ condylar cartilage and ankle cartilage cells in monolayer culture. In general, TMJ condylar cartilage cells proliferated faster than ankle cartilage cells, while ankle cells produced significantly greater amounts of glycosaminoglycans (GAGs) and collagen than TMJ condylar cartilage cells. IGF-I and bFGF were potent stimulators of TMJ cell proliferation, while no signals statistically outperformed controls for ankle cell proliferation. IGF-I was the most effective signal for GAG production with ankle cells, and the most potent upregulator of collagen synthesis for both cell types. Glucosamine sulphate promoted cell proliferation and biosynthesis at specific concentrations and outperformed growth factors in certain instances. In conclusion, hyaline cartilage cells had lower cell numbers and superior biosynthesis compared to TMJ condylar cartilage cells, and we have found IGF-I at 100 ng/mL and glucosamine sulphate at 100 microg/mL to be the most effective signals for these cells under the prescribed conditions.

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

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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.

Occlusal loading and cross-linking effects on dentin collagen degradation in physiological conditions.

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Turco, Gianluca; Frassetto, Andrea; Fontanive, Luca; Mazzoni, Annalisa; Cadenaro, Milena; Di Lenarda, Roberto; Tay, Franklin R; Pashley, David H; Breschi, Lorenzo

2016-02-01

This study evaluated the ability of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) to improve the stability of demineralized dentin collagen matrices when subjected to mechanical cycling by means of Chewing Simulation (CS). Demineralized dentin disks were randomly assigned to four groups (N=4): (1) immersion in artificial saliva at 37°C for 30 days; (2) pre-treatment with 0.5 M EDC for 60 s, then stored as in Group 1; (3) CS challenge (50 N occlusal load, 30 s occlusal time plus 30 s with no load, for 30 days); (4) pre-treatment with 0.5 M EDC as in Group 2 and CS challenge as in Group 3. Collagen degradation was evaluated by sampling storage media for ICTP and CTX telopeptides. EDC treated specimens showed no significant telopeptides release, irrespective of the aging method. Cyclic stressing of EDC-untreated specimens caused significantly higher ICTP release at day 1, compared to static storage, while by days 3 and 4, the ICTP release in the cyclic group fell significantly below the static group, and then remained undetectable from 5 to 30 days. CTX release in the cyclic groups, on EDC-untreated control specimens was always lower than in the static group in days 1-4, and then fell to undetectable for 30 days. This study showed that chewing stresses applied to control untreated demineralized dentin increased degradation of collagen in terms of CTX release, while collagen crosslinking agents may prevent dentin collagen degradation, irrespective of simulated occlusal function. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Anti-Inflammatory Inhibitors Targeting Jak and Ikk Have An Anabolic Effect on Type II Collagen Turnover ex Vivo

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Kjelgaard-Petersen, Cecilie Freja; Bay-Jensen, Anne-Christine; Karsdal, M.A.

2016-01-01

be beneficial for the selection of novel anti-inflammatory treatments for RA and iOA. Objectives The aim of this study was to investigate the direct effect of the anti-inflammatory inhibitors R406 (the active metabolite of Fostamatinib), Tofacitinib, TPCA-1 and SB203580 on the cartilage ECM turnover. Methods...... Full depth bovine cartilage ex vivo cultures were cultured for 3 weeks with OSM [10 ng/mL] and TNFα [2 ng/mL] (O+T) or together with R406, Tofacitinib or TPCA-1 at 10 μM and a two-fold dilution to 0.16 μM. SB203580 was tested at 3 μM, 1 μM and 0.3 μM. As negative control, untreated explants were...... R406, the Jak inhibitor Tofacitinib, and the IKK inhibitor TPCA-1 inhibited the release of ARGS or AGNx1, while the p38 inhibitor, SB203580, had no effect. The turnover of type II collagen was measured by the formation of type II collagen (ProC2) and MMP-mediated degradation of type II collagen (C2M...

Effect of supramolecular organization of a cartilaginous tissue on thermal stability of collagen II

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Ignat'eva, N. Yu.; Averkiev, S. V.; Lunin, V. V.; Grokhovskaya, T. E.; Obrezkova, M. V.

2006-08-01

The thermal stability of collagen II in various cartilaginous tissues was studied. It was found that heating a tissue of nucleus pulposus results in collagen II melting within a temperature range of 60-70°C; an intact tissue of hyaline cartilage (of nasal septum and cartilage endplates) is a thermally stable system, where collagen II is not denatured completely up to 100°C. It was found that partial destruction of glycosaminoglycans in hyaline cartilage leads to an increase in the degree of denaturation of collagen II upon heating, although a significant fraction remains unchanged. It was shown that electrostatic interactions of proteoglycans and collagen only slightly affect the thermal stability of collagen II in the tissues. Evidently, proteoglycan aggregates play a key role: they create topological hindrances for moving polypeptide chains, thereby reducing the configurational entropy of collagen macromolecules in the state of a random coil.

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

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

2008-10-01

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

The effect of fixed charge density and cartilage swelling on mechanics of knee joint cartilage during simulated gait.

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Räsänen, Lasse P; Tanska, Petri; Zbýň, Štefan; van Donkelaar, Corrinus C; Trattnig, Siegfried; Nieminen, Miika T; Korhonen, Rami K

2017-08-16

The effect of swelling of articular cartilage, caused by the fixed charge density (FCD) of proteoglycans, has not been demonstrated on knee joint mechanics during simulated walking before. In this study, the influence of the depth-wise variation of FCD was investigated on the internal collagen fibril strains and the mechanical response of the knee joint cartilage during gait using finite element (FE) analysis. The FCD distribution of tibial cartilage was implemented from sodium ( 23 Na) MRI into a 3-D FE-model of the knee joint ("Healthy model"). For comparison, models with decreased FCD values were created according to the decrease in FCD associated with the progression of osteoarthritis (OA) ("Early OA" and "Advanced OA" models). In addition, a model without FCD was created ("No FCD" model). The effect of FCD was studied with five different collagen fibril network moduli of cartilage. Using the reference fibril network moduli, the decrease in FCD from "Healthy model" to "Early OA" and "Advanced OA" models resulted in increased axial strains (by +2 and +6%) and decreased fibril strains (by -3 and -13%) throughout the stance, respectively, calculated as mean values through cartilage depth in the tibiofemoral contact regions. Correspondingly, compared to the "Healthy model", the removal of the FCD altogether in "NoFCD model" resulted in increased mean axial strains by +16% and decreased mean fibril strains by -24%. This effect was amplified as the fibril network moduli were decreased by 80% from the reference. Then mean axial strains increased by +6, +19 and +49% and mean fibril strains decreased by -9, -20 and -32%, respectively. Our results suggest that the FCD in articular cartilage has influence on cartilage responses in the knee during walking. Furthermore, the FCD is suggested to have larger impact on cartilage function as the collagen network degenerates e.g. in OA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Preparation and characterization of a decellularized cartilage scaffold for ear cartilage reconstruction

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Utomo, Lizette; Pleumeekers, Mieke M; Van Osch, Gerjo J V M; Nimeskern, Luc; Stok, Kathryn S; Nürnberger, Sylvia; Hildner, Florian

2015-01-01

Scaffolds are widely used to reconstruct cartilage. Yet, the fabrication of a scaffold with a highly organized microenvironment that closely resembles native cartilage remains a major challenge. Scaffolds derived from acellular extracellular matrices are able to provide such a microenvironment. Currently, no report specifically on decellularization of full thickness ear cartilage has been published. In this study, decellularized ear cartilage scaffolds were prepared and extensively characterized. Cartilage decellularization was optimized to remove cells and cell remnants from elastic cartilage. Following removal of nuclear material, the obtained scaffolds retained their native collagen and elastin contents as well as their architecture and shape. High magnification scanning electron microscopy showed no obvious difference in matrix density after decellularization. However, glycosaminoglycan content was significantly reduced, resulting in a loss of viscoelastic properties. Additionally, in contact with the scaffolds, human bone-marrow-derived mesenchymal stem cells remained viable and are able to differentiate toward the chondrogenic lineage when cultured in vitro. These results, including the ability to decellularize whole human ears, highlight the clinical potential of decellularization as an improved cartilage reconstruction strategy. (paper)

Study on de novo collagen biosynthesis and degradation markers of bone

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Hanna, L.S.; Matta, T.F.; Ibrahim, I.; Meky, N.H.

2003-01-01

This investigation was carried out to study the performance of de novo biochemical markers of serum pro collagen type-1 amino terminal extension (PINP), as a marker of collagen biosynthesis, and urinary collagen crosslink free deoxypyridinoline (DPD) as a marker of collagen degradation. Moreover, urinary calcium C Ca) and inorganic phosphorus (P), as markers of bone demineralization, in addition to urinary creatinine (Cr), to reflect status of renal function, were also studied in order to assess the activity of bone turnover in osteoporotic (OST), postmenopausal (POST), peri menopausal(PERI), premenopausal (PRE) women and also in young adult (YON) ones. The obtained results showed that urinary creatinine levels were within the normal ranges in all women even in the elderly osteoporotic and postmenopausal women. Serum PINP did not reflect osteoblastic activity. Urinary DPD proved to be a good marker in monitoring the postmenopausal bone resorption and urinary Ca was a reliable marker for bone loss in osteoporosis and bone turnover in the postmenopausal status

NONLINEAR SPECTRAL IMAGING OF ELASTIC CARTILAGE IN RABBIT EARS

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JING CHEN

2013-07-01

Full Text Available Elastic cartilage in the rabbit external ear is an important animal model with attractive potential value for researching the physiological and pathological states of cartilages especially during wound healing. In this work, nonlinear optical microscopy based on two-photon excited fluorescence and second harmonic generation were employed for imaging and quantifying the intact elastic cartilage. The morphology and distribution of main components in elastic cartilage including cartilage cells, collagen and elastic fibers were clearly observed from the high-resolution two-dimensional nonlinear optical images. The areas of cell nuclei, a parameter related to the pathological changes of normal or abnormal elastic cartilage, can be easily quantified. Moreover, the three-dimensional structure of chondrocytes and matrix were displayed by constructing three-dimensional image of cartilage tissue. At last, the emission spectra from cartilage were obtained and analyzed. We found that the different ratio of collagen over elastic fibers can be used to locate the observed position in the elastic cartilage. The redox ratio based on the ratio of nicotinamide adenine dinucleotide (NADH over flavin adenine dinucleotide (FAD fluorescence can also be calculated to analyze the metabolic state of chondrocytes in different regions. Our results demonstrated that this technique has the potential to provide more accurate and comprehensive information for the physiological states of elastic cartilage.

Chondrogenic differentiation of mesenchymal stem cells in a leakproof collagen sponge

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Chen Guoping; Akahane, Daisuke; Kawazoe, Naoki; Yamamoto, Katsuyuki; Tateishi, Tetsuya

2008-01-01

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-β3 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-β3 and BMP6

Mastication markedly affects mandibular condylar cartilage growth, gene expression, and morphology.

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Enomoto, Akiko; Watahiki, Junichi; Nampo, Tomoki; Irie, Tarou; Ichikawa, Yuuta; Tachikawa, Tetsuhiko; Maki, Koutaro

2014-09-01

Mandibular growth is believed to be strongly related to mastication. Furthermore, mandibular condylar cartilage is known to be derived from neural crest cells. We examined whether the degree of chewing affects condylar cartilage growth of the mandible. Mice were fed diets with varying hardness. Genes specific to neural crest-derived cells were measured by real-time polymerase chain reaction to compare the expression changes between the mandibular and tibia cartilages. The mandibular condylar cartilage was then evaluated histologically, and proliferation was evaluated using proliferating cell nuclear antigen. Immunostaining was conducted for osteopontin, type X collagen, and Musashi1, and real-time polymerase chain reaction was used to assess the expression levels of osteopontin and type X collagen. Markers including P75, Wnt-1, Musashi1, and Nestin were upregulated in the mandibular condylar cartilage as compared with the tibial cartilage. Histologic assessment of the mandibular cartilage showed that the hypertrophic chondrocyte zone was statistically significantly thicker in mice fed a hard diet. Chondrocyte proliferation and Musashi1 expression were lower in mice fed a hard diet. After 4 weeks, numerous osteopontin and type X collagen-positive cells were observed in mice fed a mixed diet. Mastication affects the balance between differentiation and proliferation in the mandibular condylar cartilage. This phenomenon might be attributed to the presence of neural crest-derived cells. Copyright © 2014 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.

Extracellular collagenases and the endocytic receptor, urokinase plasminogen activator receptor-associated protein/Endo180, cooperate in fibroblast-mediated collagen degradation

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Madsen, Daniel H; Engelholm, Lars H; Ingvarsen, Signe

2007-01-01

in these events. A recently discovered turnover route with importance for tumor growth involves intracellular collagen degradation and is governed by the collagen receptor, urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180). The interplay between this mechanism and extracellular...... collagenolysis is not known. In this report, we demonstrate the existence of a new, composite collagen breakdown pathway. Thus, fibroblast-mediated collagen degradation proceeds preferentially as a sequential mechanism in which extracellular collagenolysis is followed by uPARAP/Endo180-mediated endocytosis......The collagens of the extracellular matrix are the most abundant structural proteins in the mammalian body. In tissue remodeling and in the invasive growth of malignant tumors, collagens constitute an important barrier, and consequently, the turnover of collagen is a rate-limiting process...

Melanocortin 1 receptor-signaling deficiency results in an articular cartilage phenotype and accelerates pathogenesis of surgically induced murine osteoarthritis.

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Lorenz, Julia; Seebach, Elisabeth; Hackmayer, Gerit; Greth, Carina; Bauer, Richard J; Kleinschmidt, Kerstin; Bettenworth, Dominik; Böhm, Markus; Grifka, Joachim; Grässel, Susanne

2014-01-01

Proopiomelanocortin-derived peptides exert pleiotropic effects via binding to melanocortin receptors (MCR). MCR-subtypes have been detected in cartilage and bone and mediate an increasing number of effects in diathrodial joints. This study aims to determine the role of MC1-receptors (MC1) in joint physiology and pathogenesis of osteoarthritis (OA) using MC1-signaling deficient mice (Mc1re/e). OA was surgically induced in Mc1re/e and wild-type (WT) mice by transection of the medial meniscotibial ligament. Histomorphometry of Safranin O stained articular cartilage was performed with non-operated controls (11 weeks and 6 months) and 4/8 weeks past surgery. µCT-analysis for assessing epiphyseal bone architecture was performed as a longitudinal study at 4/8 weeks after OA-induction. Collagen II, ICAM-1 and MC1 expression was analysed by immunohistochemistry. Mc1re/e mice display less Safranin O and collagen II stained articular cartilage area compared to WT prior to OA-induction without signs of spontaneous cartilage surface erosion. This MC1-signaling deficiency related cartilage phenotype persisted in 6 month animals. At 4/8 weeks after OA-induction cartilage erosions were increased in Mc1re/e knees paralleled by weaker collagen II staining. Prior to OA-induction, Mc1re/e mice do not differ from WT with respect to bone parameters. During OA, Mc1re/e mice developed more osteophytes and had higher epiphyseal bone density and mass. Trabecular thickness was increased while concomitantly trabecular separation was decreased in Mc1re/e mice. Numbers of ICAM-positive chondrocytes were equal in non-operated 11 weeks Mc1re/e and WT whereas number of positive chondrocytes decreased during OA-progression. Unchallenged Mc1re/e mice display smaller articular cartilage covered area without OA-related surface erosions indicating that MC1-signaling is critical for proper cartilage matrix integrity and formation. When challenged with OA, Mc1re/e mice develop a more severe OA

Species-Independent Modeling of High-Frequency Ultrasound Backscatter in Hyaline Cartilage.

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Männicke, Nils; Schöne, Martin; Liukkonen, Jukka; Fachet, Dominik; Inkinen, Satu; Malo, Markus K; Oelze, Michael L; Töyräs, Juha; Jurvelin, Jukka S; Raum, Kay

2016-06-01

Apparent integrated backscatter (AIB) is a common ultrasound parameter used to assess cartilage matrix degeneration. However, the specific contributions of chondrocytes, proteoglycan and collagen to AIB remain unknown. To reveal these relationships, this work examined biopsies and cross sections of human, ovine and bovine cartilage with 40-MHz ultrasound biomicroscopy. Site-matched estimates of collagen concentration, proteoglycan concentration, collagen orientation and cell number density were employed in quasi-least-squares linear regression analyses to model AIB. A positive correlation (R(2) = 0.51, p 70°) to the sound beam direction. These findings indicate causal relationships between AIB and cartilage structural parameters and could aid in more sophisticated future interpretations of ultrasound backscatter. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

New tools for non-invasive exploration of collagen network in cartilaginous tissue-engineered substitute.

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Henrionnet, Christel; Dumas, Dominique; Hupont, Sébastien; Stoltz, Jean François; Mainard, Didier; Gillet, Pierre; Pinzano, Astrid

2017-01-01

In tissue engineering approaches, the quality of substitutes is a key element to determine its ability to treat cartilage defects. However, in clinical practice, the evaluation of tissue-engineered cartilage substitute quality is not possible due to the invasiveness of the standard procedure, which is to date histology. The aim of this work was to validate a new innovative system performed from two-photon excitation laser adapted to an optical macroscope to evaluate at macroscopic scale the collagen network in cartilage tissue-engineered substitutes in confrontation with gold standard histologic techniques or immunohistochemistry to visualize type II collagen. This system permitted to differentiate the quality of collagen network between ITS and TGF-β1 treatments. Multiscale large field imaging combined to multimodality approaches (SHG-TCSPC) at macroscopical scale represent an innovative and non-invasive technique to monitor the quality of collagen network in cartilage tissue-engineered substitutes before in vivo implantation.

Quantification of the optical surface reflection and surface roughness of articular cartilage using optical coherence tomography

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Saarakkala, Simo; Wang Shuzhe; Huang Yanping; Zheng Yongping [Department of Health Technology and Informatics, Hong Kong Polytechnic University, Hong Kong (China)], E-mail: simo.saarakkala@uku.fi, E-mail: ypzheng@ieee.org

2009-11-21

Optical coherence tomography (OCT) is a promising new technique for characterizing the structural changes of articular cartilage in osteoarthritis (OA). The calculation of quantitative parameters from the OCT signal is an important step to develop OCT as an effective diagnostic technique. In this study, two novel parameters for the quantification of optical surface reflection and surface roughness from OCT measurements are introduced: optical surface reflection coefficient (ORC), describing the amount of a ratio of the optical reflection from cartilage surface with respect to that from a reference material, and OCT roughness index (ORI) indicating the smoothness of the cartilage surface. The sensitivity of ORC and ORI to detect changes in bovine articular cartilage samples after enzymatic degradations of collagen and proteoglycans using collagenase and trypsin enzymes, respectively, was tested in vitro. A significant decrease (p < 0.001) in ORC as well as a significant increase (p < 0.001) in ORI was observed after collagenase digestion. After trypsin digestion, no significant changes in ORC or ORI were observed. To conclude, the new parameters introduced were demonstrated to be feasible and sensitive to detect typical OA-like degenerative changes in the collagen network. From the clinical point of view, the quantification of OCT measurements is of great interest since OCT probes have been already miniaturized and applied in patient studies during arthroscopy or open knee surgery in vivo. Further studies are still necessary to demonstrate the clinical capability of the introduced parameters for naturally occurring early OA changes in the cartilage.

Techniques and Applications of in vivo Diffusion Imaging of Articular Cartilage

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Raya, José G.

2014-01-01

Early in the process of osteoarthritis (OA) the composition (water, proteoglycan [PG], and collagen) and structure of articular cartilage is altered leading to changes in its mechanical properties. A technique that can assess the composition and structure of the cartilage in vivo can provide insight in the mechanical integrity of articular cartilage and become a powerful tool for the early diagnosis of OA. Diffusion tensor imaging (DTI) has been proposed as a biomarker for cartilage composition and structure. DTI is sensitive to the PG content through the mean diffusivity (MD) and to the collagen architecture through the fractional anisotropy (FA). However, the acquisition of DTI of articular cartilage in vivo is challenging due to the short T2 of articular cartilage (~40 ms at 3 T) and the high resolution needed (0.5–0.7 mm in plane) to depict the cartilage anatomy. We describe the pulse sequences used for in vivo DTI of articular cartilage and discus general strategies for protocol optimization. We provide a comprehensive review of measurements of DTI of articular cartilage from ex vivo validation experiments to its recent clinical applications. PMID:25865215

Microstructural and compositional features of the fibrous and hyaline cartilage on the medial tibial plateau imply a unique role for the hopping locomotion of kangaroo.

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Bo He

Full Text Available Hopping provides efficient and energy saving locomotion for kangaroos, but it results in great forces in the knee joints. A previous study has suggested that a unique fibrous cartilage in the central region of the tibial cartilage could serve to decrease the peak stresses generated within kangaroo tibiofemoral joints. However, the influences of the microstructure, composition and mechanical properties of the central fibrous and peripheral hyaline cartilage on the function of the knee joints are still to be defined. The present study showed that the fibrous cartilage was thicker and had a lower chondrocyte density than the hyaline cartilage. Despite having a higher PG content in the middle and deep zones, the fibrous cartilage had an inferior compressive strength compared to the peripheral hyaline cartilage. The fibrous cartilage had a complex three dimensional collagen meshwork with collagen bundles parallel to the surface in the superficial zone, and with collagen bundles both parallel and perpendicular to the surface in the middle and deep zones. The collagen in the hyaline cartilage displayed a typical Benninghoff structure, with collagen fibres parallel to the surface in the superficial zone and collagen fibres perpendicular to the surface in the deep zone. Elastin fibres were found throughout the entire tissue depth of the fibrous cartilage and displayed a similar alignment to the adjacent collagen bundles. In comparison, the elastin fibres in the hyaline cartilage were confined within the superficial zone. This study examined for the first time the fibrillary structure, PG content and compressive properties of the central fibrous cartilage pad and peripheral hyaline cartilage within the kangaroo medial tibial plateau. It provided insights into the microstructure and composition of the fibrous and peripheral hyaline cartilage in relation to the unique mechanical properties of the tissues to provide for the normal activities of kangaroos.

Microstructural and compositional features of the fibrous and hyaline cartilage on the medial tibial plateau imply a unique role for the hopping locomotion of kangaroo.

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He, Bo; Wu, Jian Ping; Xu, Jiake; Day, Robert E; Kirk, Thomas Brett

2013-01-01

Hopping provides efficient and energy saving locomotion for kangaroos, but it results in great forces in the knee joints. A previous study has suggested that a unique fibrous cartilage in the central region of the tibial cartilage could serve to decrease the peak stresses generated within kangaroo tibiofemoral joints. However, the influences of the microstructure, composition and mechanical properties of the central fibrous and peripheral hyaline cartilage on the function of the knee joints are still to be defined. The present study showed that the fibrous cartilage was thicker and had a lower chondrocyte density than the hyaline cartilage. Despite having a higher PG content in the middle and deep zones, the fibrous cartilage had an inferior compressive strength compared to the peripheral hyaline cartilage. The fibrous cartilage had a complex three dimensional collagen meshwork with collagen bundles parallel to the surface in the superficial zone, and with collagen bundles both parallel and perpendicular to the surface in the middle and deep zones. The collagen in the hyaline cartilage displayed a typical Benninghoff structure, with collagen fibres parallel to the surface in the superficial zone and collagen fibres perpendicular to the surface in the deep zone. Elastin fibres were found throughout the entire tissue depth of the fibrous cartilage and displayed a similar alignment to the adjacent collagen bundles. In comparison, the elastin fibres in the hyaline cartilage were confined within the superficial zone. This study examined for the first time the fibrillary structure, PG content and compressive properties of the central fibrous cartilage pad and peripheral hyaline cartilage within the kangaroo medial tibial plateau. It provided insights into the microstructure and composition of the fibrous and peripheral hyaline cartilage in relation to the unique mechanical properties of the tissues to provide for the normal activities of kangaroos.

Intracellular collagen degradation mediated by uPARAP/Endo180 is a major pathway of extracellular matrix turnover during malignancy

DEFF Research Database (Denmark)

Curino, Alejandro C; Engelholm, Lars H; Yamada, Susan S

2005-01-01

We recently reported that uPARAP/Endo180 can mediate the cellular uptake and lysosomal degradation of collagen by cultured fibroblasts. Here, we show that uPARAP/Endo180 has a key role in the degradation of collagen during mammary carcinoma progression. In the normal murine mammary gland, uPARAP/...

Decellularized cartilage may be a chondroinductive material for osteochondral tissue engineering.

Directory of Open Access Journals (Sweden)

Amanda J Sutherland

Full Text Available Extracellular matrix (ECM-based materials are attractive for regenerative medicine in their ability to potentially aid in stem cell recruitment, infiltration, and differentiation without added biological factors. In musculoskeletal tissue engineering, demineralized bone matrix is widely used, but recently cartilage matrix has been attracting attention as a potentially chondroinductive material. The aim of this study was thus to establish a chemical decellularization method for use with articular cartilage to quantify removal of cells and analyze the cartilage biochemical content at various stages during the decellularization process, which included a physically devitalization step. To study the cellular response to the cartilage matrix, rat bone marrow-derived mesenchymal stem cells (rBMSCs were cultured in cell pellets containing cells only (control, chondrogenic differentiation medium (TGF-β, chemically decellularized cartilage particles (DCC, or physically devitalized cartilage particles (DVC. The chemical decellularization process removed the vast majority of DNA and about half of the glycosaminoglycans (GAG within the matrix, but had no significant effect on the amount of hydroxyproline. Most notably, the DCC group significantly outperformed TGF-β in chondroinduction of rBMSCs, with collagen II gene expression an order of magnitude or more higher. While DVC did not exhibit a chondrogenic response to the extent that DCC did, DVC had a greater down regulation of collagen I, collagen X and Runx2. A new protocol has been introduced for cartilage devitalization and decellularization in the current study, with evidence of chondroinductivity. Such bioactivity along with providing the 'raw material' building blocks of regenerating cartilage may suggest a promising role for DCC in biomaterials that rely on recruiting endogenous cell recruitment and differentiation for cartilage regeneration.

Enzyme-linked immunosorbent assay (ELISAs) for metalloproteinase derived type II collagen neoepitope, CIIM--increased serum CIIM in subjects with severe radiographic osteoarthritis

DEFF Research Database (Denmark)

Bay-Jensen, Anne-Christine; Liu, Qi; Byrjalsen, Inger

2011-01-01

OBJECTIVES: In joint degenerative diseases, the collagens are degraded by matrix metalloproteinases and protein fragments are released to serum as potential biomarkers. METHODS: A collagen type II specific neoepitope, CIIM, was identified (…RDGAAG(1053)) by mass spectrometry. Two ELISAs against...... the neoepitope were developed. CIIM was measured in cartilage explants in the presence or absence of protease inhibitors. CIIM was measured in OA synovial fluid (n=51) and serum (n=156). Knee OA was graded by standard Kellgren-Lawrence (KL) score. RESULTS: The ELISAs showed good technical performance; CV%,

Evaluation of degenerative changes in articular cartilage of osteoarthritis by Raman spectroscopy

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Oshima, Yusuke; Ishimaru, Yasumitsu; Kiyomatsu, Hiroshi; Hino, Kazunori; Miura, Hiromasa

2018-02-01

Osteoarthritis (OA) is a 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 proposal, we aim to develop Raman spectroscopic system for the quality assessment of articular cartilage during arthroscopic surgery. Toward this goal, we are focusing on the proteoglycan content and collagen fiber alignment in cartilage matrix which may be associated with degenerative changes in OA, and we designed an original Raman device for remote sensing during arthroscopic surgery. In this project, we define the grading system for cartilage defect based on Raman spectroscopy, and we complete the evaluation of the Raman probing system which makes it possible to detect early stage of degenerative cartilage as a novel tool for OA diagnosis using human subject.

Evaluation of early changes of cartilage biomarkers following ...

African Journals Online (AJOL)

Hamdy Khamis Koryem

2014-08-15

Aug 15, 2014 ... resulting in structural, biochemical and mechanical changes that can progress from pre-clinical, to pre-radiographic .... blasts/chondrocytes) that produce an extracellular matrix of ... to represent an adequate index of the rate of type II collagen ... collagenous proteins (e.g. cartilage oligomeric matrix protein,.

X-ray phase contrast imaging of the bone-cartilage interface

International Nuclear Information System (INIS)

Ismail, Elna Che; Kaabar, W.; Garrity, D.; Gundogdu, O.; Bunk, O.; Pfeiffer, F.; Farquharson, M.J.; Bradley, D.A.

2010-01-01

Synovial joints articulate in a lubricating environment, the system providing for smooth articulation. The articular cartilage overlying the bone consists of a network of collagen fibres. This network is essential to cartilage integrity, suffering damage in degenerative joint disease such as osteoarthritis. At Surrey and also in work conducted by this group at the Paul Scherrer Institute (PSI) synchrotron site we have been applying a number of techniques to study the bone-cartilage interface and of changes occurring in this with disease. One of the techniques attracting particular interest is X-ray phase contrast imaging, yielding information on anatomical features that manifest from the large scale organisation of collagen and the mineralised phase contained within the collagen fibres in the deep cartilage zone. This work briefly reviews some of the basic supporting physics of X-ray phase contrast imaging and then shows example images of the articular surface and subchondral bone and other supporting results obtained to-date. Present results have been obtained on sections of bone not displaying evidence of an osteoarthritic lesion and can be used as a baseline against which diseased bone can be compared.

X-ray phase contrast imaging of the bone-cartilage interface

Energy Technology Data Exchange (ETDEWEB)

Ismail, Elna Che; Kaabar, W.; Garrity, D.; Gundogdu, O. [Department of Physics, University of Surrey, Guildford GU2 7XH (United Kingdom); Bunk, O. [Paul Scherrer Institut, CH-5232 Villigen (Switzerland); Pfeiffer, F. [Paul Scherrer Institut, CH-5232 Villigen (Switzerland); Ecole Polytechnique Federale de Lausanne, CH-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)], E-mail: d.a.bradley@surrey.ac.uk

2010-04-15

Synovial joints articulate in a lubricating environment, the system providing for smooth articulation. The articular cartilage overlying the bone consists of a network of collagen fibres. This network is essential to cartilage integrity, suffering damage in degenerative joint disease such as osteoarthritis. At Surrey and also in work conducted by this group at the Paul Scherrer Institute (PSI) synchrotron site we have been applying a number of techniques to study the bone-cartilage interface and of changes occurring in this with disease. One of the techniques attracting particular interest is X-ray phase contrast imaging, yielding information on anatomical features that manifest from the large scale organisation of collagen and the mineralised phase contained within the collagen fibres in the deep cartilage zone. This work briefly reviews some of the basic supporting physics of X-ray phase contrast imaging and then shows example images of the articular surface and subchondral bone and other supporting results obtained to-date. Present results have been obtained on sections of bone not displaying evidence of an osteoarthritic lesion and can be used as a baseline against which diseased bone can be compared.

Effects of low molecular weight hyaluronan combined with carprofen on canine osteoarthritis articular chondrocytes and cartilage explants in vitro.

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Euppayo, Thippaporn; Siengdee, Puntita; Buddhachat, Kittisak; Pradit, Waranee; Viriyakhasem, Nawarat; Chomdej, Siriwadee; Ongchai, Siriwan; Harada, Yasuji; Nganvongpanit, Korakot

2015-09-01

Intra-articular injection with non-steroidal anti-inflammatory drugs (NSAIDs) is used to treat inflammatory joint disease, but the side effects of NSAIDs include chondrotoxicity. Hyaluronan has shown positive effects on chondrocytes by reducing apoptosis and increasing proteoglycan synthesis. The purposes of this study were to evaluate the effects of low molecular weight hyaluronan (low MW HA), carprofen 25 mg/ml, carprofen 12.5 mg/ml, and a combination of HA and carprofen on canine osteoarthritis (OA) articular chondrocytes and a cartilage explant model in terms of cell viability, extracellular matrix remaining, and gene expression after exposure. In chondrocyte culture, MTT assay was used to evaluate the chondrotoxicity of IC50 and IC80 of carprofen with HA. In cartilage explant culture, two kinds of extracellular matrix (uronic acid and collagen) remaining in cartilage were used to evaluate cartilage damage for 14 d after treatment. Expression of COL2A1, AGG, and MMP3 was used to evaluate the synthesis and degradation of the matrix for 7 d after treatment. In chondrocyte culture, low MW HA could preserve OA chondrocyte viability but could not reduce the chondrotoxicity level of carprofen (P carprofen caused less destruction of uronic acid and collagen structure when compared with the control (P carprofen resulted in higher COL2A1 and AGG expression levels than carprofen alone.

Analysis of the cartilage proteome from three different mouse models of genetic skeletal diseases reveals common and discrete disease signatures

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Peter A. Bell

2013-06-01

Pseudoachondroplasia and multiple epiphyseal dysplasia are genetic skeletal diseases resulting from mutations in cartilage structural proteins. Electron microscopy and immunohistochemistry previously showed that the appearance of the cartilage extracellular matrix (ECM in targeted mouse models of these diseases is disrupted; however, the precise changes in ECM organization and the pathological consequences remain unknown. Our aim was to determine the effects of matrilin-3 and COMP mutations on the composition and extractability of ECM components to inform how these detrimental changes might influence cartilage organization and degeneration. Cartilage was sequentially extracted using increasing denaturants and the extraction profiles of specific proteins determined using SDS-PAGE/Western blotting. Furthermore, the relative composition of protein pools was determined using mass spectrometry for a non-biased semi-quantitative analysis. Western blotting revealed changes in the extraction of matrilins, COMP and collagen IX in mutant cartilage. Mass spectrometry confirmed quantitative changes in the extraction of structural and non-structural ECM proteins, including proteins with roles in cellular processes such as protein folding and trafficking. In particular, genotype-specific differences in the extraction of collagens XII and XIV and tenascins C and X were identified; interestingly, increased expression of several of these genes has recently been implicated in susceptibility and/or progression of murine osteoarthritis. We demonstrated that mutation of matrilin-3 and COMP caused changes in the extractability of other cartilage proteins and that proteomic analyses of Matn3 V194D, Comp T585M and Comp DelD469 mouse models revealed both common and discrete disease signatures that provide novel insight into skeletal disease mechanisms and cartilage degradation.

A novel nano-structured porous polycaprolactone scaffold improves hyaline cartilage repair in a rabbit model compared to a collagen type I/III scaffold: in vitro and in vivo studies.

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Christensen, Bjørn Borsøe; Foldager, Casper Bindzus; Hansen, Ole Møller; Kristiansen, Asger Albæk; Le, Dang Quang Svend; Nielsen, Agnete Desirée; Nygaard, Jens Vinge; Bünger, Cody Erik; Lind, Martin

2012-06-01

To develop a nano-structured porous polycaprolactone (NSP-PCL) scaffold and compare the articular cartilage repair potential with that of a commercially available collagen type I/III (Chondro-Gide) scaffold. By combining rapid prototyping and thermally induced phase separation, the NSP-PCL scaffold was produced for matrix-assisted autologous chondrocyte implantation. Lyophilizing a water-dioxane-PCL solution created micro and nano-pores. In vitro: The scaffolds were seeded with rabbit chondrocytes and cultured in hypoxia for 6 days. qRT-PCR was performed using primers for sox9, aggrecan, collagen type 1 and 2. In vivo: 15 New Zealand White Rabbits received bilateral osteochondral defects in the femoral intercondylar grooves. Autologous chondrocytes were harvested 4 weeks prior to surgery. There were 3 treatment groups: (1) NSP-PCL scaffold without cells. (2) The Chondro-Gide scaffold with autologous chondrocytes and (3) NSP-PCL scaffold with autologous chondrocytes. Observation period was 13 weeks. Histological evaluation was made using the O'Driscoll score. In vitro: The expressions of sox9 and aggrecan were higher in the NSP-PCL scaffold, while expression of collagen 1 was lower compared to the Chondro-Gide scaffold. In vivo: Both NSP-PCL scaffolds with and without cells scored significantly higher than the Chondro-Gide scaffold when looking at the structural integrity and the surface regularity of the repair tissue. No differences were found between the NSP-PCL scaffold with and without cells. The NSP-PCL scaffold demonstrated higher in vitro expression of chondrogenic markers and had higher in vivo histological scores compared to the Chondro-Gide scaffold. The improved chondrocytic differentiation can potentially produce more hyaline cartilage during clinical cartilage repair. It appears to be a suitable cell-free implant for hyaline cartilage repair and could provide a less costly and more effective treatment option than the Chondro-Gide scaffold with cells.

The formation of human auricular cartilage from microtic tissue: An in vivo study.

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Ishak, Mohamad Fikeri bin; See, Goh Bee; Hui, Chua Kien; Abdullah, Asma bt; Saim, Lokman bin; Saim, Aminuddin bin; Idrus, Ruszymah bt Haji

2015-10-01

This study aimed to isolate, culture-expand and characterize the chondrocytes isolated from microtic cartilage and evaluate its potential as a cell source for ear cartilage reconstruction. Specific attention was to construct the auricular cartilage tissue by using fibrin as scaffold. Cell culture experiment with the use of microtic chondrocytes. Cell culture experiment with the use of microtic chondrocytes. After ear reconstructive surgery at the Universiti Kebangsaan Malaysia Medical Center, chondrocytes were isolated from microtic cartilage. Chondrocytes isolated from the tissue were cultured expanded until passage 4 (P4). Upon confluency at P4, chondrocytes were harvested and tissue engineered constructs were made with human plasma polymerized to fibrin. Constructs formed later is implanted at the dorsal part of nude mice for 8 weeks, followed by post-implantation evaluation with histology staining (Hematoxylin and Eosin (H&E) and Safranin O), immunohistochemistry and RT-PCR for chondrogenic associated genes expression level. Under gross assessment, the construct after 8 weeks of implantation showed similar physical characteristics that of cartilage. Histological staining showed abundant lacunae cells embedded in extracellular matrix similar to that of native cartilage. Safranin O staining showed positive staining which indicates the presence of proteoglycan-rich matrix. Immunohistochemistry analysis showed the strong positive staining for collagen type II, the specific collagen type in the cartilage. Gene expression quantification showed no significant differences in the expression of chondrogenic gene used which is collagen type I, collagen type II, aggrecan core protein (ACP), elastin and sox9 genes when compared to construct formed from normal auricular tissue. Chondrocytes isolated from microtia cartilage has the potential to be used as an alternative cell source for external ear reconstruction in future clinical application. Copyright © 2015 Elsevier

Oral administration of undenatured native chicken type II collagen (UC-II) diminished deterioration of articular cartilage in a rat model of osteoarthritis (OA).

Science.gov (United States)

Bagi, C M; Berryman, E R; Teo, S; Lane, N E

2017-12-01

The aim of this study was to determine the ability of undenatured native chicken type II collagen (UC-II) to prevent excessive articular cartilage deterioration in a rat model of osteoarthritis (OA). Twenty male rats were subjected to partial medial meniscectomy tear (PMMT) surgery to induce OA. Immediately after the surgery 10 rats received vehicle and another 10 rats oral daily dose of UC-II at 0.66 mg/kg for a period of 8 weeks. In addition 10 naïve rats were used as an intact control and another 10 rats received sham surgery. Study endpoints included a weight-bearing capacity of front and hind legs, serum biomarkers of bone and cartilage metabolism, analyses of subchondral and cancellous bone at the tibial epiphysis and metaphysis, and cartilage pathology at the medial tibial plateau using histological methods. PMMT surgery produced moderate OA at the medial tibial plateau. Specifically, the deterioration of articular cartilage negatively impacted the weight bearing capacity of the operated limb. Immediate treatment with the UC-II preserved the weight-bearing capacity of the injured leg, preserved integrity of the cancellous bone at tibial metaphysis and limited the excessive osteophyte formation and deterioration of articular cartilage. Study results demonstrate that a clinically relevant daily dose of UC-II when applied immediately after injury can improve the mechanical function of the injured knee and prevent excessive deterioration of articular cartilage. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Reviewing subchondral cartilage surgery: considerations for standardised and outcome predictable cartilage remodelling: a technical note.

Science.gov (United States)

Benthien, Jan P; Behrens, Peter

2013-11-01

The potential of subchondral mesenchymal stem cell stimulation (MSS) for cartilage repair has led to the widespread use of microfracture as a first line treatment for full thickness articular cartilage defects. Recent focus on the effects of subchondral bone during cartilage injury and repair has expanded the understanding of the strengths and limitations in MSS and opened new pathways for potential improvement. Comparative studies have shown that bone marrow access has positive implications for pluripotential cell recruitment, repair quality and quantity, i.e. deeper channels elicited better cartilage fill, more hyaline cartilage character with higher type II collagen content and lower type I collagen content compared to shallow marrow access. A subchondral needling procedure using standardised and thin subchondral perforations deep into the subarticular bone marrow making the MSS more consistent with the latest developments in subchondral cartilage remodelling is proposed. As this is a novel method clinical studies have been initiated to evaluate the procedure especially compared to microfracturing. However, the first case studies and follow-ups indicate that specific drills facilitate reaching the subchondral bone marrow while the needle size makes perforation of the subchondral bone easier and more predictable. Clinical results of the first group of patients seem to compare well to microfracturing. The authors suggest a new method for a standardised procedure using a new perforating device. Advances in MSS by subchondral bone marrow perforation are discussed. It remains to be determined by clinical studies how this method compares to microfracturing. The subchondral needling offers the surgeon and the investigator a method that facilitates comparison studies because of its defined depth of subchondral penetration and needle size.

Increasing the Dose of Autologous Chondrocytes Improves Articular Cartilage Repair: Histological and Molecular Study in the Sheep Animal Model.

Science.gov (United States)

Guillén-García, Pedro; Rodríguez-Iñigo, Elena; Guillén-Vicente, Isabel; Caballero-Santos, Rosa; Guillén-Vicente, Marta; Abelow, Stephen; Giménez-Gallego, Guillermo; López-Alcorocho, Juan Manuel

2014-04-01

We hypothesized that implanting cells in a chondral defect at a density more similar to that of the intact cartilage could induce them to synthesize matrix with the features more similar to that of the uninjured one. We compared the implantation of different doses of chondrocytes: 1 million (n = 5), 5 million (n = 5), or 5 million mesenchymal cells (n = 5) in the femoral condyle of 15 sheep. Tissue generated by microfracture at the trochlea, and normal cartilage from a nearby region, processed as the tissues resulting from the implantation, were used as references. Histological and molecular (expression of type I and II collagens and aggrecan) studies were performed. The features of the cartilage generated by implantation of mesenchymal cells and elicited by microfractures were similar and typical of a poor repair of the articular cartilage (presence of fibrocartilage, high expression of type I collagen and a low mRNA levels of type II collagen and aggrecan). Nevertheless, in the samples obtained from tissues generated by implantation of chondrocytes, hyaline-like cartilage, cell organization, low expression rates of type I collagen and high levels of mRNA corresponding to type II collagen and aggrecan were observed. These histological features, show less variability and are more similar to those of the normal cartilage used as control in the case of 5 million cells implantation than when 1 million cells were used. The implantation of autologous chondrocytes in type I/III collagen membranes at high density could be a promising tool to repair articular cartilage.

IL-1ß and BMPs - Interactive players of cartilage matrix degradation and regeneration

Directory of Open Access Journals (Sweden)

T Aigner

2006-10-01

Full Text Available Intact human adult articular cartilage is central for the functioning of the articulating joints. This largely depends on the integrity of its extracellular matrix, given the high loading forces during movements in particular in the weight-bearing joints. Unlike the first impression of a more or less static tissue, articular cartilage shows - albeit in the adult organism a slow - tissue turnover. Thus, one of the most important questions in osteoarthritis research is to understand the balance of catabolic and anabolic factors in articular cartilage as this is the key to understand the biology of cartilage maintenance and degeneration. Anabolic and catabolic pathways are very much intermingled in articular cartilage. The balance between anabolism and catabolism is titrated on numerous levels, starting from the mediator-synthesizing cells which express either catabolic or anabolic factors. Also, on the level of the effector cells (i.e. chondrocytes anabolic and catabolic gene expression compete for a balance of matrix homeostasis, namely the synthesis of matrix components and the expression and activation of matrix-degrading proteases. Also, there are multiple layers of intracellular cross-talks in between the anabolic and catabolic signalling pathways. Maybe the most important lesson from this overview is the notion that the anabolic-catabolic balance as such counts and not so much sufficient net anabolism or limited catabolism alone. Thus, it might be neither the aim of osteoarthritis therapy to foster anabolism nor to knock down catabolism, but the balance of anabolic-catabolic activities as a total might need proper titration and balancing.

Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants

NARCIS (Netherlands)

Creemers, L. B.; Hoeben, K. A.; Jansen, D. C.; Buttle, D. J.; Beertsen, W.; Everts, V.

1998-01-01

The involvement of cysteine proteinases in the degradation of soft connective tissue collagen was studied in cultured periosteal explants. Using cysteine proteinase inhibitors that were active intracellularly or extracellularly (Ep453 and Ep475, respectively), it was shown that over-all collagen

Inhibition of cathepsin K reduces cartilage degeneration in the anterior cruciate ligament transection rabbit and murine models of osteoarthritis.

Science.gov (United States)

Hayami, Tadashi; Zhuo, Ya; Wesolowski, Gregg A; Pickarski, Maureen; Duong, Le T

2012-06-01

To investigate the disease modifying effects of cathepsin K (CatK) inhibitor L-006235 compared to alendronate (ALN) in two preclinical models of osteoarthritis (OA). Skeletally mature rabbits underwent sham or anterior cruciate ligament transection (ACLT)-surgery and were treated with L-006235 (L-235, 10 mg/kg or 50 mg/kg, p.o., daily) or ALN (0.6 mg/kg, s.c., weekly) for 8-weeks. ACLT joint instability was also induced in CatK(-/-) versus wild type (wt) mice and treated for 16-weeks. Changes in cartilage degeneration, subchondral bone volume and osteophyte area were determined by histology and μ-CT. Collagen type I helical peptide (HP-I), a bone resorption marker and collagen type II C-telopeptide (CTX-II), a cartilage degradation marker were measured. L-235 (50 mg/kg) and ALN treatment resulted in significant chondroprotective effects, reducing CTX-II by 60% and the histological Mankin score for cartilage damage by 46% in the ACLT-rabbits. Both doses of L-235 were more potent than ALN in protecting against focal subchondral bone loss, and reducing HP-I by 70% compared to vehicle. L-235 (50 mg/kg) and ALN significantly reduced osteophyte formation in histomorphometric analysis by 55%. The Mankin score in ACLT-CatK(-/-) mice was ~2.5-fold lower than the ACLT-wt mice and was not different from sham-CatK(-/-). Osteophyte development was not different among the groups. Inhibition of CatK provides significant benefits in ACLT-model of OA, including: 1) protection of subchondral bone integrity, 2) protection against cartilage degradation and 3) reduced osteophytosis. Preclinical evidence supports the role of CatK as a potential therapeutic target for the treatment of OA. Copyright © 2012 Elsevier Inc. All rights reserved.

Can photobiomodulation associated with implantation of mesenchymal adipose-derived stem cells attenuate the expression of MMPs and decrease degradation of type II collagen in an experimental model of osteoarthritis?

Science.gov (United States)

Stancker, Tatiane Garcia; Vieira, Stella Souza; Serra, Andrey Jorge; do Nascimento Lima, Rafael; Dos Santos Feliciano, Regiane; Silva, José Antônio; Dos Santos, Solange Almeida; Dos Santos Vieira, Marcia Ataize; Simões, Maíra Cecília Brandão; Leal-Junior, Ernesto Cesar; de Tarso Camillo de Carvalho, Paulo

2018-03-08

This study aimed to determine whether photobiomodulation therapy (PBMT) could improve the bioavailability and chondroprotective benefits of mesenchymal stem cells injected into the knees of rats used as an experimental model of osteoarthritis (OA) as well as reduce the expression of matrix metalloproteinases (MMPs) and degradation of type II collagen (COL2-1) in the cartilage. Adipose-derived stem/stromal cells (ADSCs) were collected from three male Fischer 344 rats and characterized by flow cytometry. Fifty female Fischer 344 rats were distributed into five groups of 10 animals each. These groups were as follows: control, OA, OA PBMT, OA ADSC, and OA ADSC PBMT. OA was induced in the animals using a 4% papain solution. Animals from the OA ADSC and OA ADSC PBMT groups received an intra-articular injection of 10 × 10 6 ADSCs and were treated with PBMT by irradiation (wavelength: 808 nm, power: 50 mW, energy: 42 J, energy density: 71.2 J/cm 2 , spot size: 0.028). Euthanasia was performed 7 days after the first treatment. The use of PBMT alone and the injection of ADSCs resulted in downregulation of pro-inflammatory cytokines and MPs in cartilage compared to the OA group. PBMT and ADSCs caused upregulation of tissue inhibitors of MPs 1 and 2 and mRNA and protein expression of COL2-1 in cartilage compared to the OA group. The intra-articular injection of ADSCs and PBMT prevented joint degeneration resulting from COL2-1 degradation and modulated inflammation by downregulating cytokines and MMPs in the OA group.

Rheological, biocompatibility and osteogenesis assessment of fish collagen scaffold for bone tissue engineering.

Science.gov (United States)

Elango, Jeevithan; Zhang, Jingyi; Bao, Bin; Palaniyandi, Krishnamoorthy; Wang, Shujun; Wenhui, Wu; Robinson, Jeya Shakila

2016-10-01

In the present investigation, an attempt was made to find an alternative to mammalian collagen with better osteogenesis ability. Three types of collagen scaffolds - collagen, collagen-chitosan (CCH), and collagen-hydroxyapatite (CHA) - were prepared from the cartilage of Blue shark and investigated for their physico-functional and mechanical properties in relation to biocompatibility and osteogenesis. CCH scaffold was superior with pH 4.5-4.9 and viscosity 9.7-10.9cP. Notably, addition of chitosan and HA (hydroxyapatite) improved the stiffness (11-23MPa) and degradation rate but lowered the water binding capacity and porosity of the scaffold. Interestingly, CCH scaffolds remained for 3days before complete in-vitro biodegradation. The decreased amount of viable T-cells and higher level of FAS/APO-1 were substantiated the biocompatibility properties of prepared collagen scaffolds. Osteogenesis study revealed that the addition of CH and HA in both fish and mammalian collagen scaffolds could efficiently promote osteoblast cell formation. The ALP activity was significantly high in CHA scaffold-treated osteoblast cells, which suggests an enhanced bone-healing process. Therefore, the present study concludes that the composite scaffolds prepared from fish collagen with higher stiffness, lower biodegradation rate, better biocompatible, and osteogenesis properties were suitable biomaterial for a bone tissue engineering application as an alternative to mammalian collagen scaffolds. Copyright © 2016 Elsevier B.V. All rights reserved.

Cartilage oligomeric matrix protein enhances matrix assembly during chondrogenesis of human mesenchymal stem cells.

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

2012-04-01

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.

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

The properties of bioengineered chondrocyte sheets for cartilage regeneration

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Ota Naoshi

2009-03-01

Full Text Available Abstract Background Although the clinical results of autologous chondrocyte implantation for articular cartilage defects have recently improved as a result of advanced techniques based on tissue engineering procedures, problems with cell handling and scaffold imperfections remain to be solved. A new cell-sheet technique has been developed, and is potentially able to overcome these obstacles. Chondrocyte sheets applicable to cartilage regeneration can be prepared with this cell-sheet technique using temperature-responsive culture dishes. However, for clinical application, it is necessary to evaluate the characteristics of the cells in these sheets and to identify their similarities to naive cartilage. Results The expression of SOX 9, collagen type 2, 27, integrin α10, and fibronectin genes in triple-layered chondrocyte sheets was significantly increased in comparison to those in conventional monolayer culture and in a single chondrocyte sheet, implying a nature similar to ordinary cartilage. In addition, immunohistochemistry demonstrated that collagen type II, fibronectin, and integrin α10 were present in the triple-layered chondrocyte sheets. Conclusion The results of this study indicate that these chondrocyte sheets with a consistent cartilaginous phenotype and adhesive properties may lead to a new strategy for cartilage regeneration.

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.

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

International Nuclear Information System (INIS)

Kaabar, W.; Laklouk, A.; Bunk, O.; Baily, M.; Farquharson, M.J.; Bradley, David

2010-01-01

Micro-proton-induced X-ray emission (μ-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.

Production of hyaline-like cartilage by bone marrow mesenchymal stem cells in a self-assembly model.

Science.gov (United States)

Elder, Steven H; Cooley, Avery J; Borazjani, Ali; Sowell, Brittany L; To, Harrison; Tran, Scott C

2009-10-01

A scaffoldless or self-assembly approach to cartilage tissue engineering has been used to produce hyaline cartilage from bone marrow-derived mesenchymal stem cells (bMSCs), but the mechanical properties of such engineered cartilage and the effects the transforming growth factor (TGF) isoform have not been fully explored. This study employs a cell culture insert model to produce tissue-engineered cartilage using bMSCs. Neonatal pig bMSCs were isolated by plastic adherence and expanded in monolayer before being seeded into porous transwell inserts and cultured for 4 or 8 weeks in defined chondrogenic media containing either TGF-beta1 or TGF-beta3. Following biomechanical evaluation in confined compression, colorimetric dimethyl methylene blue and Sircol dye-binding assays were used to analyze glycosaminoglycan (GAG) and collagen contents, respectively. Histological sections were stained with toluidine blue for proteoglycans and with picrosirius red to reveal collagen orientation, and immunostained for detection of collagen types I and II. Neocartilage increased in thickness, collagen, and GAG content between 4 and 8 weeks. Proteoglycan concentration increased with depth from the top surface. The tissue contained much more collagen type II than type I, and there was a consistent pattern of collagen alignment. TGF-beta1-treated and TGF-beta3-treated constructs were similar at 4 weeks, but 8-week TGF-beta1 constructs had a higher aggregate modulus and GAG content compared to TGF-beta3. These results demonstrate that bMSCs can generate functional hyaline-like cartilage through a self-assembling process.

Running a marathon induces changes in adipokine levels and in markers of cartilage degradation--novel role for resistin.

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Katriina Vuolteenaho

Full Text Available Running a marathon causes strenuous joint loading and increased energy expenditure. Adipokines regulate energy metabolism, but recent studies have indicated that they also exert a role in cartilage degradation in arthritis. Our aim was to investigate the effects of running a marathon on the levels of adipokines and indices of cartilage metabolism. Blood samples were obtained from 46 male marathoners before and after a marathon run. We measured levels of matrix metalloproteinase-3 (MMP-3, cartilage oligomeric protein (COMP and chitinase 3-like protein 1 (YKL-40 as biomarkers of cartilage turnover and/or damage and plasma concentrations of adipokines adiponectin, leptin and resistin. Mean marathon time was 3:30:46±0:02:46 (h:min:sec. The exertion more than doubled MMP-3 levels and this change correlated negatively with the marathon time (r = -0.448, p = 0.002. YKL-40 levels increased by 56% and the effect on COMP release was variable. Running a marathon increased the levels of resistin and adiponectin, while leptin levels remained unchanged. The marathon-induced changes in resistin levels were positively associated with the changes in MMP-3 (r = 0.382, p = 0.009 and YKL-40 (r = 0.588, p<0.001 and the pre-marathon resistin levels correlated positively with the marathon induced change in YKL-40 (r = 0.386, p = 0.008. The present results show the impact of running a marathon, and possible load frequency, on cartilage metabolism: the faster the marathon was run, the greater was the increase in MMP-3 levels. Further, the results introduce pro-inflammatory adipocytokine resistin as a novel factor, which enhances during marathon race and associates with markers of cartilage degradation.

Cartilage Integration: Evaluation of the reasons for failure of integration during cartilage repair. A review

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IM Khan

2008-09-01

Full Text Available Articular cartilage is a challenging tissue to reconstruct or replace principally because of its avascular nature; large chondral lesions in the tissue do not spontaneously heal. Where lesions do penetrate the bony subchondral plate, formation of hematomas and the migration of mesenchymal stem cells provide an inferior and transient fibrocartilagenous replacement for hyaline cartilage. To circumvent the poor intrinsic reparative response of articular cartilage several surgical techniques based on tissue transplantation have emerged. One characteristic shared by intrinsic reparative processes and the new surgical therapies is an apparent lack of lateral integration of repair or graft tissue with the host cartilage that can lead to poor prognosis. Many factors have been cited as impeding cartilage:cartilage integration including; chondrocyte cell death, chondrocyte dedifferentiation, the nature of the collagenous and proteoglycan networks that constitute the extracellular matrix, the type of biomaterial scaffold employed in repair and the origin of the cells used to repopulate the defect or lesion. This review addresses the principal intrinsic and extrinsic factors that impede integration and describe how manipulation of these factors using a host of strategies can positively influence cartilage integration.

Effect of a Rapidly Degrading Presolidified 10 kDa Chitosan/Blood Implant and Subchondral Marrow Stimulation Surgical Approach on Cartilage Resurfacing in a Sheep Model

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Bell, Angela D.; Hurtig, Mark B.; Quenneville, Eric; Rivard, Georges-Étienne; Hoemann, Caroline D.

2016-01-01

Objective This study tested the hypothesis that presolidified chitosan-blood implants are retained in subchondral bone channels perforated in critical-size sheep cartilage defects, and promote bone repair and hyaline-like cartilage resurfacing versus blood implant. Design Cartilage defects (10 × 10 mm) with 3 bone channels (1 drill, 2 Jamshidi biopsy, 2 mm diameter), and 6 small microfracture holes were created bilaterally in n = 11 sheep knee medial condyles. In one knee, 10 kDa chitosan–NaCl/blood implant (presolidified using recombinant factor VIIa or tissue factor), was inserted into each drill and Jamshidi hole. Contralateral knee defects received presolidified whole blood clot. Repair tissues were assessed histologically, biochemically, biomechanically, and by micro–computed tomography after 1 day (n = 1) and 6 months (n = 10). Results Day 1 defects showed a 60% loss of subchondral bone plate volume fraction along with extensive subchondral hematoma. Chitosan implant was resident at day 1, but had no effect on any subsequent repair parameter compared with blood implant controls. At 6 months, bone defects exhibited remodeling and hypomineralized bone repair and were partly resurfaced with tissues containing collagen type II and scant collagen type I, 2-fold lower glycosaminoglycan and fibril modulus, and 4.5-fold higher permeability compared with intact cartilage. Microdrill holes elicited higher histological ICRS-II overall assessment scores than Jamshidi holes (50% vs. 30%, P = 0.041). Jamshidi biopsy holes provoked sporadic osteonecrosis in n = 3 debrided condyles. Conclusions Ten kilodalton chitosan was insufficient to improve repair. Microdrilling is a feasible subchondral marrow stimulation surgical approach with the potential to elicit poroelastic tissues with at least half the compressive modulus as intact articular cartilage. PMID:28934884

Effect of a Rapidly Degrading Presolidified 10 kDa Chitosan/Blood Implant and Subchondral Marrow Stimulation Surgical Approach on Cartilage Resurfacing in a Sheep Model.

Science.gov (United States)

Bell, Angela D; Hurtig, Mark B; Quenneville, Eric; Rivard, Georges-Étienne; Hoemann, Caroline D

2017-10-01

Objective This study tested the hypothesis that presolidified chitosan-blood implants are retained in subchondral bone channels perforated in critical-size sheep cartilage defects, and promote bone repair and hyaline-like cartilage resurfacing versus blood implant. Design Cartilage defects (10 × 10 mm) with 3 bone channels (1 drill, 2 Jamshidi biopsy, 2 mm diameter), and 6 small microfracture holes were created bilaterally in n = 11 sheep knee medial condyles. In one knee, 10 kDa chitosan-NaCl/blood implant (presolidified using recombinant factor VIIa or tissue factor), was inserted into each drill and Jamshidi hole. Contralateral knee defects received presolidified whole blood clot. Repair tissues were assessed histologically, biochemically, biomechanically, and by micro-computed tomography after 1 day ( n = 1) and 6 months ( n = 10). Results Day 1 defects showed a 60% loss of subchondral bone plate volume fraction along with extensive subchondral hematoma. Chitosan implant was resident at day 1, but had no effect on any subsequent repair parameter compared with blood implant controls. At 6 months, bone defects exhibited remodeling and hypomineralized bone repair and were partly resurfaced with tissues containing collagen type II and scant collagen type I, 2-fold lower glycosaminoglycan and fibril modulus, and 4.5-fold higher permeability compared with intact cartilage. Microdrill holes elicited higher histological ICRS-II overall assessment scores than Jamshidi holes (50% vs. 30%, P = 0.041). Jamshidi biopsy holes provoked sporadic osteonecrosis in n = 3 debrided condyles. Conclusions Ten kilodalton chitosan was insufficient to improve repair. Microdrilling is a feasible subchondral marrow stimulation surgical approach with the potential to elicit poroelastic tissues with at least half the compressive modulus as intact articular cartilage.

Accumulation of advanced glycation end products decreases collagen turnover by bovine chondrocytes

NARCIS (Netherlands)

Groot, J. de; Verzijl, N.; Budde, M.; Bijlsma, J.W.J.; Lafeber, F.P.J.G.; TeKoppele, J.M.

2001-01-01

The integrity of the collagen network is essential for articular cartilage to fulfill its function in load support and distribution. Damage to the collagen network is one of the first characteristics of osteoarthritis. Since extensive collagen damage is considered irreversible, it is crucial that

Assessment of proteolytic degradation of the basement membrane: a fragment of type IV collagen as a biochemical marker for liver fibrosis

DEFF Research Database (Denmark)

Veidal, Sanne S.; Karsdal, Morten A.; Nawrocki, Arkadiusz

2011-01-01

Collagen deposition and an altered matrix metalloproteinase (MMP) expression profile are hallmarks of fibrosis. Type IV collagen is the most abundant structural basement membrane component of tissue, which increases 14-fold during fibrogenesis in the liver. Proteolytic degradation of collagens...

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)

International Nuclear Information System (INIS)

Kretzschmar, M.; Hainc, N.; Studler, U.; Bieri, O.; Miska, M.; Wiewiorski, M.; Valderrabano, V.

2015-01-01

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 2 /ms) was significantly higher compared to normal cartilage (1.46 μm 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.)

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.)

Cartilage Repair Surgery: Outcome Evaluation by Using Noninvasive Cartilage Biomarkers Based on Quantitative MRI Techniques?

Science.gov (United States)

Jungmann, Pia M.; Baum, Thomas; Bauer, Jan S.; Karampinos, Dimitrios C.; Link, Thomas M.; Li, Xiaojuan; Trattnig, Siegfried; Rummeny, Ernst J.; Woertler, Klaus; Welsch, Goetz H.

2014-01-01

Background. New quantitative magnetic resonance imaging (MRI) techniques are increasingly applied as outcome measures after cartilage repair. Objective. To review the current literature on the use of quantitative MRI biomarkers for evaluation of cartilage repair at the knee and ankle. Methods. Using PubMed literature research, studies on biochemical, quantitative MR imaging of cartilage repair were identified and reviewed. Results. Quantitative MR biomarkers detect early degeneration of articular cartilage, mainly represented by an increasing water content, collagen disruption, and proteoglycan loss. Recently, feasibility of biochemical MR imaging of cartilage repair tissue and surrounding cartilage was demonstrated. Ultrastructural properties of the tissue after different repair procedures resulted in differences in imaging characteristics. T2 mapping, T1rho mapping, delayed gadolinium-enhanced MRI of cartilage (dGEMRIC), and diffusion weighted imaging (DWI) are applicable on most clinical 1.5 T and 3 T MR scanners. Currently, a standard of reference is difficult to define and knowledge is limited concerning correlation of clinical and MR findings. The lack of histological correlations complicates the identification of the exact tissue composition. Conclusions. A multimodal approach combining several quantitative MRI techniques in addition to morphological and clinical evaluation might be promising. Further investigations are required to demonstrate the potential for outcome evaluation after cartilage repair. PMID:24877139

Cartilage Repair Surgery: Outcome Evaluation by Using Noninvasive Cartilage Biomarkers Based on Quantitative MRI Techniques?

Directory of Open Access Journals (Sweden)

Pia M. Jungmann

2014-01-01

Full Text Available Background. New quantitative magnetic resonance imaging (MRI techniques are increasingly applied as outcome measures after cartilage repair. Objective. To review the current literature on the use of quantitative MRI biomarkers for evaluation of cartilage repair at the knee and ankle. Methods. Using PubMed literature research, studies on biochemical, quantitative MR imaging of cartilage repair were identified and reviewed. Results. Quantitative MR biomarkers detect early degeneration of articular cartilage, mainly represented by an increasing water content, collagen disruption, and proteoglycan loss. Recently, feasibility of biochemical MR imaging of cartilage repair tissue and surrounding cartilage was demonstrated. Ultrastructural properties of the tissue after different repair procedures resulted in differences in imaging characteristics. T2 mapping, T1rho mapping, delayed gadolinium-enhanced MRI of cartilage (dGEMRIC, and diffusion weighted imaging (DWI are applicable on most clinical 1.5 T and 3 T MR scanners. Currently, a standard of reference is difficult to define and knowledge is limited concerning correlation of clinical and MR findings. The lack of histological correlations complicates the identification of the exact tissue composition. Conclusions. A multimodal approach combining several quantitative MRI techniques in addition to morphological and clinical evaluation might be promising. Further investigations are required to demonstrate the potential for outcome evaluation after cartilage repair.

Hyaluronan supplementation as a mechanical regulator of cartilage tissue development under joint-kinematic-mimicking loading.

Science.gov (United States)

Wu, Yabin; Stoddart, Martin J; Wuertz-Kozak, Karin; Grad, Sibylle; Alini, Mauro; Ferguson, Stephen J

2017-08-01

Articular cartilage plays an essential role in joint lubrication and impact absorption. Through this, the mechanical signals are coupled to the tissue's physiological response. Healthy synovial fluid has been shown to reduce and homogenize the shear stress acting on the cartilage surfaces due to its unique shear-thinning viscosity. As cartilage tissues are sensitive to mechanical changes in articulation, it was hypothesized that replacing the traditional culture medium with a healthy non-Newtonian lubricant could enhance tissue development in a cartilage engineering model, where joint-kinematic-mimicking mechanical loading is applied. Different amounts of hyaluronic acid were added to the culture medium to replicate the viscosities of synovial fluid at different health states. Hyaluronic acid supplementation, especially at a physiologically healthy concentration (2.0 mg ml -1 ), promoted a better preservation of chondrocyte phenotype. The ratio of collagen II to collagen I mRNA was 4.5 times that of the control group, implying better tissue development (however, with no significant difference of measured collagen II content), with a good retention of collagen II and proteoglycan in the mechanically active region. Simulating synovial fluid properties by hyaluronic acid supplementation created a favourable mechanical environment for mechanically loaded constructs. These findings may help in understanding the influence of joint articulation on tissue homeostasis, and moreover, improve methods for functional cartilage tissue engineering. © 2017 The Author(s).

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

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Lee, Won Hee [School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia (United States); Warrington, Junie P.; Sonntag, William E. [Reynolds Oklahoma Center on Aging, Department of Geriatric Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma (United States); Lee, Yong Woo, E-mail: ywlee@vt.edu [School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia (United States); Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia (United States)

2012-04-01

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

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

International Nuclear Information System (INIS)

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

2012-01-01

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

Cartilage Protective and Chondrogenic Capacity of WIN-34B, a New Herbal Agent, in the Collagenase-Induced Osteoarthritis Rabbit Model and in Progenitor Cells from Subchondral Bone

Directory of Open Access Journals (Sweden)

Jeong-Eun Huh

2013-01-01

Full Text Available We sought to determine the cartilage repair capacity of WIN-34B in the collagenase-induced osteoarthritis rabbit model and in progenitor cells from subchondral bone. The cartilage protective effect of WIN-34B was measured by clinical and histological scores, cartilage area, and proteoglycan and collagen contents in the collagenase-induced osteoarthritis rabbit model. The efficacy of chondrogenic differentiation of WIN-34B was assessed by expression of CD105, CD73, type II collagen, and aggrecan in vivo and was analyzed by the surface markers of progenitor cells, the mRNA levels of chondrogenic marker genes, and the level of proteoglycan, GAG, and type II collagen in vitro. Oral administration of WIN-34B significantly increased cartilage area, and this was associated with the recovery of proteoglycan and collagen content. Moreover, WIN-34B at 200 mg/kg significantly increased the expression of CD105, CD73, type II collagen, and aggrecan compared to the vehicle group. WIN-34B markedly enhanced the chondrogenic differentiation of CD105 and type II collagen in the progenitor cells from subchondral bone. Also, we confirmed that treatment with WIN-34B strongly increased the number of SH-2(CD105 cells and expression type II collagen in subchondral progenitor cells. Moreover, WIN-34B significantly increased proteoglycan, as measured by alcian blue staining; the mRNA level of type II α1 collagen, cartilage link protein, and aggrecan; and the inhibition of cartilage matrix molecules, such as GAG and type II collagen, in IL-1β-treated progenitor cells. These findings suggest that WIN-34B could be a potential candidate for effective anti-osteoarthritic therapy with cartilage repair as well as cartilage protection via enhancement of chondrogenic differentiation in the collagenase-induced osteoarthritis rabbit model and progenitor cells from subchondral bone.

Characterization of cells from pannus-like tissue over articular cartilage of advanced osteoarthritis.

Science.gov (United States)

Yuan, G-H; Tanaka, M; Masuko-Hongo, K; Shibakawa, A; Kato, T; Nishioka, K; Nakamura, H

2004-01-01

To identify the characteristics of cells isolated from pannus-like soft tissue on osteoarthritic cartilage (OA pannus cells), and to evaluate the role of this tissue in osteoarthritis (OA). OA pannus cells were isolated from pannus-like tissues in five joints obtained during arthroplasty. The phenotypic features of the isolated cells were characterized by safranin-O staining and immunohistochemical studies. Expression of MMP-1, MMP-3 and MMP-13 was also assessed using reverse transcriptase-polymerase chain reactions (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry. Foci and plaque formation of pannus-like tissue over cartilage surface were found in 15 of 21 (71.4%) OA joints macroscopically, and among them, only five samples had enough tissue to be isolated. OA pannus cells were positive for type I collagen and vimentin, besides they also expressed type II collagen and aggrecan mRNA. Spontaneous expression of MMP-1, MMP-3 and MMP-13 was detected in OA pannus cells. Similar or higher levels of MMPs were detected in the supernatant of cultured OA pannus cells compared to OA chondrocytes, and among these MMP-3 levels were relatively higher in OA pannus cells. Immunohistochemically, MMP-3 positive cells located preferentially in pannus-like tissue on the border of original hyaline cartilage. Our results showed that OA pannus cells shared the property of mesenchymal cells and chondrocytes; however, their origin seemed different from chondrocytes or synoviocytes. The spontaneous expression of MMPs suggests that they are involved in the articular degradation in OA.

Downregulation of miR-221-3p contributes to IL-1β-induced cartilage degradation by directly targeting the SDF1/CXCR4 signaling pathway.

Science.gov (United States)

Zheng, Xin; Zhao, Feng-Chao; Pang, Yong; Li, Dong-Ya; Yao, Sheng-Cheng; Sun, Shao-Song; Guo, Kai-Jin

2017-06-01

Osteoarthritis (OA) is characterized by degradation of chondrocyte extracellular matrix (ECM). Accumulating evidence suggests that microRNAs (miRNAs) are associated with OA, but little is known of their function in chondrocyte ECM degradation. The objective of this study was to investigate the expression and function of miRNAs in OA. miRNA expression profile was determined in OA cartilage tissues and controls, employing Solexa sequencing and reverse transcription quantitative PCR (RT-qPCR). According to a modified Mankin scale, cartilage degradation was evaluated. Functional analysis of the miRNAs on chondrocyte ECM degradation was performed after miRNA transfection and IL-1β treatment. Luciferase reporter assays and western blotting were employed to determine miRNA targets. Expression of miR-221-3p was downregulated in OA cartilage tissues, which was significantly correlated with a modified Mankin scale. Through gain-of-function and loss-of-function studies, miR-221-3p was shown to significantly affect matrix synthesis gene expression and chondrocyte proliferation and apoptosis. Using SW1353 and C28I2 cells, SDF1 was identified as a target of miR-221-3p. SDF1 overexpression resulted in increased expression of catabolic genes such as MMP-13 and ADAMTS-5 in response to IL-1β, but these effects were moderated by miR-221-3p. SDF1 treatment antagonized this effect, while knockdown of SDF1 by shSDF1 induced inhibitory effects on the expression of CXCR4 and its main target genes, similar to miR-221-3p. The results indicate that upregulation of miR-221-3p could prevent IL-1β-induced ECM degradation in chondrocytes. Targeting the SDF1/CXCR4 signaling pathway may be used as a therapeutic approach for OA. miR-221-3p is downregulated in human cartilage tissues. miR-221-3p levels are associated with cartilage degeneration grade. miR-221-3p upregulation prevents IL-1β-induced ECM degradation in chondrocytes. Protection of ECM degradation by miR-223-3p occurs via SDF1/CXCR4

Monitoring the effect of mechanical stress on mesenchymal stem cell collagen production by multiphoton microscopy

Science.gov (United States)

Chen, Wei-Liang; Chang, Chia-Cheng; Chiou, Ling-Ling; Li, Tsung-Hsien; Liu, Yuan; Lee, Hsuan-Shu; Dong, Chen-Yuan

2008-02-01

Tissue engineering is emerging as a promising method for repairing damaged tissues. Due to cartilage's common wear and injury, in vitro production of cartilage replacements have been an active area of research. Finding the optimal condition for the generation of the collagen matrix is crucial in reproducing cartilages that closely match those found in human. Using multiphoton autofluorescence and second-harmonic generation (SHG) microscopy we monitored the effect of mechanical stress on mesenchymal stem cell collagen production. Bone marrow mesenchymal stem cells in the form of pellets were cultured and periodically placed under different mechanical stress by centrifugation over a period of four weeks. The differently stressed samples were imaged several times during the four week period, and the collagen production under different mechanical stress is characterized.

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

In vitro degradation of dermal sheep collagen cross-linked using a water-soluble carbodiimide

NARCIS (Netherlands)

Damink, LHHO; Dijkstra, PJ; vanLuyn, MJA; vanWachem, PB; Nieuwenhuis, P; Feijen, J

Bacterial collagenase was used to study the susceptibility of dermal sheep collagen (DSC) cross-inked with a mixture of the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (EIN-DSC) towards enzymatic degradation. Contrary to

Optical characterization of porcine articular cartilage using a polarimetry technique with differential Mueller matrix formulism.

Science.gov (United States)

Chang, Ching-Min; Lo, Yu-Lung; Tran, Nghia-Khanh; Chang, Yu-Jen

2018-03-20

A method is proposed for characterizing the optical properties of articular cartilage sliced from a pig's thighbone using a Stokes-Mueller polarimetry technique. The principal axis angle, phase retardance, optical rotation angle, circular diattenuation, diattenuation axis angle, linear diattenuation, and depolarization index properties of the cartilage sample are all decoupled in the proposed analytical model. Consequently, the accuracy and robustness of the extracted results are improved. The glucose concentration, collagen distribution, and scattering properties of samples from various depths of the articular cartilage are systematically explored via an inspection of the related parameters. The results show that the glucose concentration and scattering effect are both enhanced in the superficial region of the cartilage. By contrast, the collagen density increases with an increasing sample depth.

BMP-2, hypoxia, and COL1A1/HtrA1 siRNAs favor neo-cartilage hyaline matrix formation in chondrocytes.

Science.gov (United States)

Ollitrault, David; Legendre, Florence; Drougard, Carole; Briand, Mélanie; Benateau, Hervé; Goux, Didier; Chajra, Hanane; Poulain, Laurent; Hartmann, Daniel; Vivien, Denis; Shridhar, Vijayalakshmi; Baldi, Alfonso; Mallein-Gerin, Frédéric; Boumediene, Karim; Demoor, Magali; Galera, Philippe

2015-02-01

Osteoarthritis (OA) is an irreversible pathology that causes a decrease in articular cartilage thickness, leading finally to the complete degradation of the affected joint. The low spontaneous repair capacity of cartilage prevents any restoration of the joint surface, making OA a major public health issue. Here, we developed an innovative combination of treatment conditions to improve the human chondrocyte phenotype before autologous chondrocyte implantation. First, we seeded human dedifferentiated chondrocytes into a collagen sponge as a scaffold, cultured them in hypoxia in the presence of a bone morphogenetic protein (BMP), BMP-2, and transfected them with small interfering RNAs targeting two markers overexpressed in OA dedifferentiated chondrocytes, that is, type I collagen and/or HtrA1 serine protease. This strategy significantly decreased mRNA and protein expression of type I collagen and HtrA1, and led to an improvement in the chondrocyte phenotype index of differentiation. The effectiveness of our in vitro culture process was also demonstrated in the nude mouse model in vivo after subcutaneous implantation. We, thus, provide here a new protocol able to favor human hyaline chondrocyte phenotype in primarily dedifferentiated cells, both in vitro and in vivo. Our study also offers an innovative strategy for chondrocyte redifferentiation and opens new opportunities for developing therapeutic targets.

A study of repair cartilage from osteochondrotic humeral condyles of swine: preliminary report.

OpenAIRE

Nakano, T; Aherne, F X

1992-01-01

A total of 16 animals, including 12 lame and four normal boars, were used. All lame boars had severe osteochondrotic humeral condyles in which repair cartilage tissues originating from subchondral bone were observed. Quantitative chemical studies of repair cartilage and normal cartilage were carried out using humeral condyles from four selected animals (two lame and two normal boars, respectively). The repair cartilage contained a higher concentration of collagen and lower concentration of pr...

A Comparison of Bone Marrow and Cord Blood Mesenchymal Stem Cells for Cartilage Self-Assembly.

Science.gov (United States)

White, Jamie L; Walker, Naomi J; Hu, Jerry C; Borjesson, Dori L; Athanasiou, Kyriacos A

2018-04-02

Joint injury is a common cause of premature retirement for the human and equine athlete alike. Implantation of engineered cartilage offers the potential to increase the success rate of surgical intervention and hasten recovery times. Mesenchymal stem cells (MSCs) are a particularly attractive cell source for cartilage engineering. While bone marrow-derived MSCs (BM-MSCs) have been most extensively characterized for musculoskeletal tissue engineering, studies suggest that cord blood MSCs (CB-MSCs) may elicit a more robust chondrogenic phenotype. The objective of this study was to determine a superior equine MSC source for cartilage engineering. MSCs derived from bone marrow or cord blood were stimulated to undergo chondrogenesis through aggregate redifferentiation and used to generate cartilage through the self-assembling process. The resulting neocartilage produced from either BM-MSCs or CB-MSCs was compared by measuring mechanical, biochemical, and histological properties. We found that while BM constructs possessed higher tensile properties and collagen content, CB constructs had superior compressive properties comparable to that of native tissue and higher GAG content. Moreover, CB constructs had alkaline phosphatase activity, collagen type X, and collagen type II on par with native tissue suggesting a more hyaline cartilage-like phenotype. In conclusion, while both BM-MSCs and CB-MSCs were able to form neocartilage, CB-MSCs resulted in tissue more closely resembling native equine articular cartilage as determined by a quantitative functionality index. Therefore, CB-MSCs are deemed a superior source for the purpose of articular cartilage self-assembly.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of a specific MMP-9 mediated degradation fragment of type III collagen--A novel biomarker of atherosclerotic plaque remodeling

DEFF Research Database (Denmark)

Barascuk, Natasha; Vassiliadis, Efstathios; Larsen, Lise

2011-01-01

Degradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine.......Degradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine....

Elemental and structural studies at the bone-cartilage interface

International Nuclear Information System (INIS)

Kaabar, W.; Daar, E.; Bunk, O.; Farquharson, M.J.; Laklouk, A.; Bailey, M.; Jeynes, C.; Gundogdu, O.; Bradley, D.A.

2011-01-01

Micro-Proton Induced X-ray Emission (μ-PIXE) and Proton Induced Gamma-ray Emission (PIGE) techniques were employed in the investigation of trace and essential elements distribution in normal and diseased human femoral head sections affected by osteoarthritis (OA). PIGE was exploited in the determination of elements of low atomic number z 15 viz Ca, Z, P and S were determined by PIXE. Accumulations of key elements in the bone and cartilage sections were observed, significant S and Na concentrations being found in the cartilage region particularly in normal tissues. Zn showed enhanced concentrations at the bone-cartilage interface. At a synchrotron facility, small angle X-ray scattering (SAXS) was utilized on a decalcified human femoral head section affected by OA, direct measurements being made of spatial alterations of collagen fibres. The SAXS results showed a slight decrease in the axial periodicity between normal collagen type I and that in diseased tissue in various sites, in contrast with the findings of others.

Protective effect of exogenous chondroitin 4,6-sulfate in the acute degradation of articular cartilage in the rabbit.

Science.gov (United States)

Uebelhart, D; Thonar, E J; Zhang, J; Williams, J M

1998-05-01

The injection of 2.0 mg chymopapain into the adolescent rabbit knee causes severe loss of articular cartilage proteoglycans (PG). Although chondrocytes attempt to restore lost PG, failure to repair ensues. Pure chondroitin 4,6-sulfate (Condrosulf, IBSA Lugano, Switzerland) has been used in clinical studies of human osteoarthritis (OA) as a slow-acting drug for OA (SYSADOA). Using our model of articular cartilage injury, we examined the effects of oral and intramuscular administration of Condrosulf after chymopapain-induced cartilage injury. In this study, animals received an injection of 2.0 mg chymopapain (Chymodiactin, Boots Pharmaceuticals) into the left knee and were sacrificed after 84 days. The contralateral right knee served as a noninjected control. Some animals received oral Condrosulf while others received intramuscular injections of Condrosulf. Serum keratan sulfate (KS) levels were monitored to ensure degradation of the cartilage PG. Those animals not exhibiting at least a 100% increase of serum KS following chymopapain injection were excluded from the study. At sacrifice, cartilage PG contents were markedly reduced in animals receiving an injection of 2.0 mg chymopapain with no further treatment. In contrast, oral administration of Condrosulf beginning 11 days prior to chymopapain injury resulted in significantly higher (P = 0.0036) cartilage PG contents. Intramuscular administration of Condrosulf resulted in higher, but less significantly so (P = 0.0457), cartilage PG contents. These results suggest that daily Condrosulf treatment prior to and continuing after chymopapain injury may have a protective effect on the damaged cartilage, allowing it to continue to re-synthesize matrix PG after the treatment is discontinued.

Use of the second harmonic generation microscopy to evaluate chondrogenic differentiation of mesenchymal stem cells for cartilage repair

Science.gov (United States)

Bordeaux-Rego, P.; Baratti, M. O.; Duarte, A. S. S.; Ribeiro, T. B.; Andreoli-Risso, M. F.; Vidal, B.; Miranda, J. B.; Adur, J.; de Thomaz, A. A.; Pelegati, V. B.; Costa, F. F.; Carvalho, H. F.; Cesar, C. L.; Luzo, A.; Olalla Saad, S. T.

2012-03-01

Articular cartilage injury remains one of the major concerns in orthopedic surgery. Mesenchymal stem cell (MSC) transplantation has been introduced to avoid some of the side effects and complications of current techniques.. With the aim to evaluate chondrogenic differentiation of mesenchymal stem cells, we used Second Harmonic Generation (SHG) microscopy to analyze the aggregation and orientation of collagen fibrils in the hyaline cartilage of rabbit knees. The experiment was performed using implants with type II collagen hydrogel (a biomaterial that mimics the microenvironment of the cartilage), one implant containing MSC and one other without MSC (control). After 10 weeks, the rabbit knees were dissected and fibril collagen distribution and spatial organization in the extracellular matrix of the lesions were verified by SHG. The result showed significant differences, whereas in histological sections of the cartilaginous lesions with MSC the collagen fibers are organized and regular; in the control sections the collagen fibers are more irregular, with absence of cells. A macroscopic analysis of the lesions confirmed this difference, showing a greater percentage of lesions filling in knees treated with MSC than in the knees used as controls. This study demonstrates that SHG microscopy will be an excellent tool to help in the evaluation of the effectiveness of MSC-based cell therapy for cartilage repair.

Fourier transform infrared spectroscopy to quantify collagen and elastin in an in vitro model of extracellular matrix degradation in aorta.

Science.gov (United States)

Cheheltani, Rabee; McGoverin, Cushla M; Rao, Jayashree; Vorp, David A; Kiani, Mohammad F; Pleshko, Nancy

2014-06-21

Extracellular matrix (ECM) is a key component and regulator of many biological tissues including aorta. Several aortic pathologies are associated with significant changes in the composition of the matrix, especially in the content, quality and type of aortic structural proteins, collagen and elastin. The purpose of this study was to develop an infrared spectroscopic methodology that is comparable to biochemical assays to quantify collagen and elastin in aorta. Enzymatically degraded porcine aorta samples were used as a model of ECM degradation in abdominal aortic aneurysm (AAA). After enzymatic treatment, Fourier transform infrared (FTIR) spectra of the aortic tissue were acquired by an infrared fiber optic probe (IFOP) and FTIR imaging spectroscopy (FT-IRIS). Collagen and elastin content were quantified biochemically and partial least squares (PLS) models were developed to predict collagen and elastin content in aorta based on FTIR spectra. PLS models developed from FT-IRIS spectra were able to predict elastin and collagen content of the samples with strong correlations (RMSE of validation = 8.4% and 11.1% of the range respectively), and IFOP spectra were successfully used to predict elastin content (RMSE = 11.3% of the range). The PLS regression coefficients from the FT-IRIS models were used to map collagen and elastin in tissue sections of degraded porcine aortic tissue as well as a human AAA biopsy tissue, creating a similar map of each component compared to histology. These results support further application of FTIR spectroscopic techniques for evaluation of AAA tissues.

Fourier Transform Infrared Spectroscopy to Quantify Collagen and Elastin in an In Vitro Model of Extracellular Matrix Degradation in Aorta

Science.gov (United States)

Cheheltani, Rabee; McGoverin, Cushla M.; Rao, Jayashree; Vorp, David A.; Kiani, Mohammad F.; Pleshko, N.

2014-01-01

Extracellular matrix (ECM) is a key component and regulator of many biological tissues including aorta. Several aortic pathologies are associated with significant changes in the composition of the matrix, especially in the content, quality and type of aortic structural proteins, collagen and elastin. The purpose of this study was to develop an infrared spectroscopic methodology that is comparable to biochemical assays to quantify collagen and elastin in aorta. Enzymatically degraded porcine aorta samples were used as a model of ECM degradation in abdominal aortic aneurysm (AAA). After enzymatic treatment, Fourier transform infrared (FTIR) spectra of the aortic tissue were acquired by an infrared fiber optic probe (IFOP) and FTIR imaging spectroscopy (FT-IRIS). Collagen and elastin content were quantified biochemically and partial least squares (PLS) models were developed to predict collagen and elastin content in aorta based on FTIR spectra. PLS models developed from FT-IRIS spectra were able to predict elastin and collagen content of the samples with strong correlations (RMSE of validation = 8.4% and 11.1% of the range respectively), and IFOP spectra were successfully used to predict elastin content (RMSE = 11.3% of the range). The PLS regression coefficients from the FT-IRIS models were used to map collagen and elastin in tissue sections of degraded porcine aortic tissue as well as a human AAA biopsy tissue, creating a similar map of each component compared to histology. These results support further application of FTIR spectroscopic techniques for evaluation of AAA tissues. PMID:24761431

Injectable perlecan domain 1-hyaluronan microgels potentiate the cartilage repair effect of BMP2 in a murine model of early osteoarthritis

International Nuclear Information System (INIS)

Srinivasan, Padma P; McCoy, Sarah Y; Yang Weidong; Farach-Carson, Mary C; Kirn-Safran, Catherine B; Jha, Amit K; Jia Xinqiao

2012-01-01

The goal of this study was to use bioengineered injectable microgels to enhance the action of bone morphogenetic protein 2 (BMP2) and stimulate cartilage matrix repair in a reversible animal model of osteoarthritis (OA). A module of perlecan (PlnD1) bearing heparan sulfate (HS) chains was covalently immobilized to hyaluronic acid (HA) microgels for the controlled release of BMP2 in vivo. Articular cartilage damage was induced in mice using a reversible model of experimental OA and was treated by intra-articular injection of PlnD1-HA particles with BMP2 bound to HS. Control injections consisted of BMP2-free PlnD1-HA particles, HA particles, free BMP2 or saline. Knees dissected following these injections were analyzed using histological, immunostaining and gene expression approaches. Our results show that knees treated with PlnD1-HA/BMP2 had lesser OA-like damage compared to control knees. In addition, the PlnD1-HA/BMP2-treated knees had higher mRNA levels encoding for type II collagen, proteoglycans and xylosyltransferase 1, a rate-limiting anabolic enzyme involved in the biosynthesis of glycosaminoglycan chains, relative to control knees (PlnD1-HA). This finding was paralleled by enhanced levels of aggrecan in the articular cartilage of PlnD1-HA/BMP2-treated knees. Additionally, decreases in the mRNA levels encoding for cartilage-degrading enzymes and type X collagen were seen relative to controls. In conclusion, PlnD1-HA microgels constitute a formulation improvement compared to HA for efficient in vivo delivery and stimulation of proteoglycan and cartilage matrix synthesis in mouse articular cartilage. Ultimately, PlnD1-HA/BMP2 may serve as an injectable therapeutic agent for slowing or inhibiting the onset of OA after knee injury.

The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells.

Science.gov (United States)

Levato, Riccardo; Webb, William R; Otto, Iris A; Mensinga, Anneloes; Zhang, Yadan; van Rijen, Mattie; van Weeren, René; Khan, Ilyas M; Malda, Jos

2017-10-01

Cell-laden hydrogels are the primary building blocks for bioprinting, and, also termed bioinks, are the foundations for creating structures that can potentially recapitulate the architecture of articular cartilage. To be functional, hydrogel constructs need to unlock the regenerative capacity of encapsulated cells. The recent identification of multipotent articular cartilage-resident chondroprogenitor cells (ACPCs), which share important traits with adult stem cells, represents a new opportunity for cartilage regeneration. However, little is known about the suitability of ACPCs for tissue engineering, especially in combination with biomaterials. This study aimed to investigate the potential of ACPCs in hydrogels for cartilage regeneration and biofabrication, and to evaluate their ability for zone-specific matrix production. Gelatin methacryloyl (gelMA)-based hydrogels were used to culture ACPCs, bone marrow mesenchymal stromal cells (MSCs) and chondrocytes, and as bioinks for printing. Our data shows ACPCs outperformed chondrocytes in terms of neo-cartilage production and unlike MSCs, ACPCs had the lowest gene expression levels of hypertrophy marker collagen type X, and the highest expression of PRG4, a key factor in joint lubrication. Co-cultures of the cell types in multi-compartment hydrogels allowed generating constructs with a layered distribution of collagens and glycosaminoglycans. By combining ACPC- and MSC-laden bioinks, a bioprinted model of articular cartilage was generated, consisting of defined superficial and deep regions, each with distinct cellular and extracellular matrix composition. Taken together, these results provide important information for the use of ACPC-laden hydrogels in regenerative medicine, and pave the way to the biofabrication of 3D constructs with multiple cell types for cartilage regeneration or in vitro tissue models. Despite its limited ability to repair, articular cartilage harbors an endogenous population of progenitor cells

Suppression of elevated cartilage turnover in postmenopausal women and in ovariectomized rats by estrogen and a selective estrogen-receptor modulator (SERM).

Science.gov (United States)

Christgau, Stephan; Tankó, László B; Cloos, Paul A C; Mouritzen, Ulrik; Christiansen, Claus; Delaissé, Jean-Marie; Høegh-Andersen, Pernille

2004-01-01

Several observational studies indicate that estrogen deficiency increases the incidence of osteoarthritis in postmenopausal women. To validate this observation, we investigated the effects of ovariectomy (OVX) on cartilage erosion in rats using histology and an established bio-assay of cartilage-specific collagen type II degradation products (CTX-II). Furthermore, we investigated whether estrogen and levormeloxifene, a selective estrogen-receptor modulator (SERM), can prevent the OVX-induced changes in cartilage degradation. The clinical relevance was assessed in postmenopausal women by measuring the changes in CTX-II during 12-month treatment with levormeloxifene versus placebo. Sixty 6-month-old rats were divided in five groups. One group was subjected to sham and the others to OVX, followed by treatment with vehicle alone, estradiol or 0.2 mg/kg/day or 5 mg/kg/day of levormeloxifene. The rats were treated for 9 weeks with biweekly blood and urine sampling for measurement of bone resorption and cartilage turnover. After study termination, hind knees were removed for histological analysis of erosions. The effect of levormeloxifene in post-menopausal women was assessed by measuring CTX-II in samples from 301 women who were participating in a phase II study of this SERM. OVX rats showed significant increases in the urinary excretion of CTX-II. After 9 weeks this was manifested as increased surface erosion of knee articular cartilage compared with sham-operated rats. Treatment with estrogen or levormeloxifene prevented the OVX-induced changes. There was a significant correlation between the 4-week changes in CTX-II and cartilage erosion at week 9 (r = 0.64, P women treated with levormeloxifene, the urinary excretion of CTX-II was decreased by approximately 50% and restored CTX-II levels to the premenopausal range. This study is the first to demonstrate that a SERM suppresses cartilage degradation in both rodents and humans, suggesting potential therapeutical benefits

Regional polarization sensitivity of articular cartilage by using polarization sensitive optical coherence tomography

Science.gov (United States)

Xie, Tuqiang; Guo, Shuguang; Chen, Zhongping; Peavy, George M.

2007-02-01

In this study, PS-OCT is used to image fresh bovine joints to investigate the orientation of collagen fibrils in relation to optical phase retardation to better understand the distribution of normal matrix orientation and articular cartilage birefringence in different regions of a whole joint. Understanding and mapping variations in matrix organization and orientation within the normal joint is an important issue in potential applications of PS-OCT for evaluation and diagnosis of degenerative joint disease (DJD). The experimental results demonstrate that articular cartilage is not polarization sensitive on the edge of the medial, but polarization sensitive on the lateral edge of the tibial plateau. The collagen orientation on the edge of the joint is different from the central areas of the joint. Normal articular cartilage demonstrates regional polarization sensitivity within joints that is important to understand in order to accurately assess cartilage health by PS-OCT.

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

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

International Nuclear Information System (INIS)

Moussa, Mayssam; Lajeunesse, Daniel; Hilal, George; El Atat, Oula; Haykal, Gaby; Serhal, Rim; Chalhoub, Antonio; Khalil, Charbel; Alaaeddine, Nada

2017-01-01

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

Glucosamine but not ibuprofen alters cartilage turnover in osteoarthritis patients in response to physical training

DEFF Research Database (Denmark)

Petersen, Susanne Germann; Saxne, T; Heinegard, D

2010-01-01

OBJECTIVE: To investigate changes in levels of serum cartilage oligomeric matrix protein (COMP) and urine c-telopeptide of type-2 collagen (CTX-II) as markers for cartilage turnover in patients with osteoarthritis (OA) of the knee, in response to muscle strength training in combination with treat......OBJECTIVE: To investigate changes in levels of serum cartilage oligomeric matrix protein (COMP) and urine c-telopeptide of type-2 collagen (CTX-II) as markers for cartilage turnover in patients with osteoarthritis (OA) of the knee, in response to muscle strength training in combination......). RESULTS: All three groups increased their muscle strength following 12 weeks of strength training (Preduced in the glucosamine-treated group after the training period (P=0.012), whereas they did not change in the two other groups. Glucosamine reduced COMP statistically...

Omentin-1 prevents cartilage matrix destruction by regulating matrix metalloproteinases.

Science.gov (United States)

Li, Zhigang; Liu, Baoyi; Zhao, Dewei; Wang, BenJie; Liu, Yupeng; Zhang, Yao; Li, Borui; Tian, Fengde

2017-08-01

Matrix metalloproteinases (MMPs) play a crucial role in the degradation of the extracellular matrix and pathological progression of osteoarthritis (OA). Omentin-1 is a newly identified anti-inflammatory adipokine. Little information regarding the protective effects of omentin-1 in OA has been reported before. In the current study, our results indicated that omentin-1 suppressed expression of MMP-1, MMP-3, and MMP-13 induced by the proinflammatory cytokine interleukin-1β (IL-1β) at both the mRNA and protein levels in human chondrocytes. Importantly, administration of omentin-1 abolished IL-1β-induced degradation of type II collagen (Col II) and aggrecan, the two major extracellular matrix components in articular cartilage, in a dose-dependent manner. Mechanistically, omentin-1 ameliorated the expression of interferon regulatory factor 1 (IRF-1) by blocking the JAK-2/STAT3 pathway. Our results indicate that omentin-1 may have a potential chondroprotective therapeutic capacity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Gene expression profile of the cartilage tissue spontaneously regenerated in vivo by using a novel double-network gel: Comparisons with the normal articular cartilage

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Kurokawa Takayuki

2011-09-01

Full Text Available Abstract Background We have recently found a phenomenon that spontaneous regeneration of a hyaline cartilage-like tissue can be induced in a large osteochondral defect by implanting a double-network (DN hydrogel plug, which was composed of poly-(2-Acrylamido-2-methylpropanesulfonic acid and poly-(N, N'-Dimetyl acrylamide, at the bottom of the defect. The purpose of this study was to clarify gene expression profile of the regenerated tissue in comparison with that of the normal articular cartilage. Methods We created a cylindrical osteochondral defect in the rabbit femoral grooves. Then, we implanted the DN gel plug at the bottom of the defect. At 2 and 4 weeks after surgery, the regenerated tissue was analyzed using DNA microarray and immunohistochemical examinations. Results The gene expression profiles of the regenerated tissues were macroscopically similar to the normal cartilage, but showed some minor differences. The expression degree of COL2A1, COL1A2, COL10A1, DCN, FMOD, SPARC, FLOD2, CHAD, CTGF, and COMP genes was greater in the regenerated tissue than in the normal cartilage. The top 30 genes that expressed 5 times or more in the regenerated tissue as compared with the normal cartilage included type-2 collagen, type-10 collagen, FN, vimentin, COMP, EF1alpha, TFCP2, and GAPDH genes. Conclusions The tissue regenerated by using the DN gel was genetically similar but not completely identical to articular cartilage. The genetic data shown in this study are useful for future studies to identify specific genes involved in spontaneous cartilage regeneration.

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...

In vitro degradation of porous nano-hydroxyapatite/collagen/PLLA scaffold reinforced by chitin fibres

International Nuclear Information System (INIS)

Li Xiaoming; Feng Qingling; Cui Fuzhai

2006-01-01

In this paper, a novel porous scaffold for bone tissue engineering was prepared with nano-hydroxyapatite/collagen/Poly-L-lactic acid (PLLA) composite reinforced by chitin fibres. To enhance the strength of the scaffold further, PLLA was linked with chitin fibres by Dicyclohexylcarbodimide (DCC). The structures of the reinforced scaffold with and without linking were characterized by Scanning Electron Microscopy (SEM). The chemical characteristics of the chitin fibres with and without linking were evaluated by Fourier-transformed infrared (FTIR) spectroscopy. The mechanical performance during degradation in vitro was investigated. The results indicated that the nano-hydroxyapatite/collagen/PLLA composite reinforced by chitin fibres with linking kept better mechanical properties than that of the composite without linking. These results denoted that the stronger interfacial bonding strength of the scaffold with linking could decrease the degradation rate in vitro. The reinforced composite with the link-treatment can be severed as a scaffold for bone tissue engineering

A tissue adaptation model based on strain-dependent collagen degradation and contact-guided cell traction.

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Heck, T A M; Wilson, W; Foolen, J; Cilingir, A C; Ito, K; van Donkelaar, C C

2015-03-18

Soft biological tissues adapt their collagen network to the mechanical environment. Collagen remodeling and cell traction are both involved in this process. The present study presents a collagen adaptation model which includes strain-dependent collagen degradation and contact-guided cell traction. Cell traction is determined by the prevailing collagen structure and is assumed to strive for tensional homeostasis. In addition, collagen is assumed to mechanically fail if it is over-strained. Care is taken to use principally measurable and physiologically meaningful relationships. This model is implemented in a fibril-reinforced biphasic finite element model for soft hydrated tissues. The versatility and limitations of the model are demonstrated by corroborating the predicted transient and equilibrium collagen adaptation under distinct mechanical constraints against experimental observations from the literature. These experiments include overloading of pericardium explants until failure, static uniaxial and biaxial loading of cell-seeded gels in vitro and shortening of periosteum explants. In addition, remodeling under hypothetical conditions is explored to demonstrate how collagen might adapt to small differences in constraints. Typical aspects of all essentially different experimental conditions are captured quantitatively or qualitatively. Differences between predictions and experiments as well as new insights that emerge from the present simulations are discussed. This model is anticipated to evolve into a mechanistic description of collagen adaptation, which may assist in developing load-regimes for functional tissue engineered constructs, or may be employed to improve our understanding of the mechanisms behind physiological and pathological collagen remodeling. Copyright © 2014 Elsevier Ltd. All rights reserved.

Aging histological changes in the cartilages of the cricoarytenoid joint

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Dedivitis Rogério Aparecido

2004-01-01

Full Text Available PURPOSE: Analysis of ossification, bone marrow formation, perichondrium thickness, muscle fibers, collagen fibers and elastic fibers quantities of cricoid and arytenoid cartilages. Design: Correlation morphologic study. METHODS: Twenty-four cricoarytenoid joints were obtained from Caucasian male fresh cadavers divided into three groups with eight specimens in each: group I - adolescents, from 15 to 20; group II - adults, from 25 to 35; and group III - elderly, from 60 to 75. The specimens were stained with H-E; trichrome; Picrosirius; and elastic stain. Histometry was performed for quantitative analysis. Bonferroni Test, Fisher Test and the Variance Analysis were used. RESULTS: At the microscopic analysis, the group I specimens presented typical hyaline cartilage, thin perichondrium, bulky muscle fibers and were surrounded by collagen fibers. In group II, there were ossification in small well defined central areas of four specimens, with lamellar bone tissue. In two of these cases there were central bone cavity full of fat tissue. The other parameters were similar to group I. In group III, most part of hyaline cartilage was replaced by typical lamellar bone tissue with poorly outlined haversian systems. Hematopoietic tissue was noted in six cases and fat tissue in the other two. Perichondrium was thicker. Small muscle fibers were smaller and surrounded by collagen in great quantity. Elastic fibers were present in small quantity in the outer portion of perichondrium in all the groups. CONCLUSIONS: In spite of its lack in adolescence, ossification occurs in cricoid and arytenoid cartilages during adulthood and intensifies with age; bone marrow is formed in ossification tissue with hematopoietic tissue in group III; perichondrium becomes thicker in group III; muscle tissue atrophies in group III and is replaced by collagen fibers; these fibers thicken with age; and elastic fibers is always present in the perichondrium in low quantity.

Cartilage T2 assessment: differentiation of normal hyaline cartilage and reparative tissue after arthroscopic cartilage repair in equine subjects.

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White, Lawrence M; Sussman, Marshall S; Hurtig, Mark; Probyn, Linda; Tomlinson, George; Kandel, Rita

2006-11-01

To prospectively assess T2 mapping characteristics of normal articular cartilage and of cartilage at sites of arthroscopic repair, including comparison with histologic results and collagen organization assessed at polarized light microscopy (PLM). Study protocol was compliant with the Canadian Council on Animal Care Guidelines and approved by the institutional animal care committee. Arthroscopic osteochondral autograft transplantation (OAT) and microfracture arthroplasty (MFx) were performed in knees of 10 equine subjects (seven female, three male; age range, 3-5 years). A site of arthroscopically normal cartilage was documented in each joint as a control site. Joints were harvested at 12 (n = 5) and 24 (n = 5) weeks postoperatively and were imaged at 1.5-T magnetic resonance (MR) with a 10-echo sagittal fast spin-echo acquisition. T2 maps of each site (21 OAT harvest, 10 MFx, 12 OAT plug, and 10 control sites) were calculated with linear least-squares curve fitting. Cartilage T2 maps were qualitatively graded as "organized" (normal transition of low-to-high T2 signal from deep to superficial cartilage zones) or "disorganized." Quantitative mean T2 values were calculated for deep, middle, and superficial cartilage at each location. Results were compared with histologic and PLM assessments by using kappa analysis. T2 maps were qualitatively graded as organized at 20 of 53 sites and as disorganized at 33 sites. Perfect agreement was seen between organized T2 and histologic findings of hyaline cartilage and between disorganized T2 and histologic findings of fibrous reparative tissue (kappa = 1.0). Strong agreement was seen between organized T2 and normal PLM findings and between disorganized T2 and abnormal PLM findings (kappa = .92). Quantitative assessment of the deep, middle, and superficial cartilage, respectively, showed mean T2 values of 53.3, 58.6, and 54.9 msec at reparative fibrous tissue sites and 40.7, 53.6, and 61.6 msec at hyaline cartilage sites. A

Joint immobilization inhibits spontaneous hyaline cartilage regeneration induced by a novel double-network gel implantation.

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Arakaki, Kazunobu; Kitamura, Nobuto; Kurokawa, Takayuki; Onodera, Shin; Kanaya, Fuminori; Gong, Jian-Ping; Yasuda, Kazunori

2011-02-01

We have recently discovered that spontaneous hyaline cartilage regeneration can be induced in an osteochondral defect in the rabbit, when we implant a novel double-network (DN) gel plug at the bottom of the defect. To clarify whether joint immobilization inhibits the spontaneous hyaline cartilage regeneration, we conducted this study with 20 rabbits. At 4 or 12 weeks after surgery, the defect in the mobile knees was filled with a sufficient volume of the hyaline cartilage tissue rich in proteoglycan and type-2 collagen, while no cartilage tissues were observed in the defect in the immobilized knees. Type-2 collagen, Aggrecan, and SOX9 mRNAs were expressed only in the mobile knees at each period. This study demonstrated that joint immobilization significantly inhibits the spontaneous hyaline cartilage regeneration induced by the DN gel implantation. This fact suggested that the mechanical environment is one of the significant factors to induce this phenomenon.

Neo-Epitopes—Fragments of Cartilage and Connective Tissue Degradation in Early Rheumatoid Arthritis and Unclassified Arthritis

DEFF Research Database (Denmark)

Maijer, Karen I; Gudmann, Natasja Stæhr; Karsdal, Morten Asser

2016-01-01

Objective: Tissue destruction in rheumatoid arthritis (RA) is predominantly mediated by matrix metalloproteinases (MMPs), thereby generating protein fragments. Previous studies have revealed that these fragments include MMP-mediated collagen type I, II, and III degradation, citrullinated and MMP...

Nasal chondrocyte-based engineered autologous cartilage tissue for repair of articular cartilage defects: an observational first-in-human trial.

Science.gov (United States)

Mumme, Marcus; Barbero, Andrea; Miot, Sylvie; Wixmerten, Anke; Feliciano, Sandra; Wolf, Francine; Asnaghi, Adelaide M; Baumhoer, Daniel; Bieri, Oliver; Kretzschmar, Martin; Pagenstert, Geert; Haug, Martin; Schaefer, Dirk J; Martin, Ivan; Jakob, Marcel

2016-10-22

Articular cartilage injuries have poor repair capacity, leading to progressive joint damage, and cannot be restored predictably by either conventional treatments or advanced therapies based on implantation of articular chondrocytes. Compared with articular chondrocytes, chondrocytes derived from the nasal septum have superior and more reproducible capacity to generate hyaline-like cartilage tissues, with the plasticity to adapt to a joint environment. We aimed to assess whether engineered autologous nasal chondrocyte-based cartilage grafts allow safe and functional restoration of knee cartilage defects. In a first-in-human trial, ten patients with symptomatic, post-traumatic, full-thickness cartilage lesions (2-6 cm 2 ) on the femoral condyle or trochlea were treated at University Hospital Basel in Switzerland. Chondrocytes isolated from a 6 mm nasal septum biopsy specimen were expanded and cultured onto collagen membranes to engineer cartilage grafts (30 × 40 × 2 mm). The engineered tissues were implanted into the femoral defects via mini-arthrotomy and assessed up to 24 months after surgery. Primary outcomes were feasibility and safety of the procedure. Secondary outcomes included self-assessed clinical scores and MRI-based estimation of morphological and compositional quality of the repair tissue. This study is registered with ClinicalTrials.gov, number NCT01605201. The study is ongoing, with an approved extension to 25 patients. For every patient, it was feasible to manufacture cartilaginous grafts with nasal chondrocytes embedded in an extracellular matrix rich in glycosaminoglycan and type II collagen. Engineered tissues were stable through handling with forceps and could be secured in the injured joints. No adverse reactions were recorded and self-assessed clinical scores for pain, knee function, and quality of life were improved significantly from before surgery to 24 months after surgery. Radiological assessments indicated variable degrees of

Overexpression of hsa-miR-148a promotes cartilage production and inhibits cartilage degradation by osteoarthritic chondrocytes

NARCIS (Netherlands)

Vonk, L A; Kragten, A H M; Dhert, W J A; Saris, D B F; Creemers, L B

OBJECTIVE: Hsa-miR-148a expression is decreased in Osteoarthritis (OA) cartilage, but its functional role in cartilage has never been studied. Therefore, our aim was to investigate the effects of overexpressing hsa-miR-148a on cartilage metabolism of OA chondrocytes. DESIGN: OA chondrocytes were

Inhibition of cartilage degradation and suppression of PGE2 and MMPs expression by pomegranate fruit extract in a model of posttraumatic osteoarthritis.

Science.gov (United States)

Akhtar, Nahid; Khan, Nazir M; Ashruf, Omer S; Haqqi, Tariq M

2017-01-01

Osteoarthritis (OA) is characterized by cartilage degradation in the affected joints. Pomegranate fruit extract (PFE) inhibits cartilage degradation in vitro. The aim of this study was to determine whether oral consumption of PFE inhibits disease progression in rabbits with surgically induced OA. OA was surgically induced in the tibiofemoral joints of adult New Zealand White rabbits. In one group, animals were fed PFE in water for 8 wk postsurgery. In the second group, animals were fed PFE for 2 wk before surgery and for 8 wk postsurgery. Histologic assessment and scoring of the cartilage was per Osteoarthritis Research Society International guidelines. Gene expression and matrix metalloproteinases (MMP) activity were determined using quantitative reverse transcriptase polymerase chain reaction and fluorometric assay, respectively. Interleukin (IL)-1 β, MMP-13, IL-6, prostaglandin (PG)E 2 , and type II collagen (COL2A1) levels in synovial fluid/plasma/culture media were quantified using enzyme-linked immunosorbent assay. Expression of active caspase-3 and poly (ADP-ribose) polymerase p85 was determined by immunohistochemistry. Effect of PFE and inhibitors of MMP-13, mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB was studied in IL-1 β-stimulated rabbit articular chondrocytes. Safranin-O-staining and chondrocyte cluster formation was significantly reduced in the anterior cruciate ligament transaction plus PFE fed groups. Expression of MMP-3, MMP-9, and MMP-13 mRNA was higher in the cartilage of rabbits given water alone but was significantly lower in the animals fed PFE. PFE-fed rabbits had lower IL-6, MMP-13, and PGE 2 levels in the synovial fluid and plasma, respectively, and showed higher expression of aggrecan and COL2A1 mRNA. Significantly higher numbers of chondrocytes were positive for markers of apoptosis in the joints of rabbits with OA given water only compared with those in the PFE-fed groups. PFE pretreatment significantly

Overexpression of hsa-miR-148a promotes cartilage production and inhibits cartilage degradation by osteoarthritic chondrocytes

NARCIS (Netherlands)

Vonk, Lucienne A.; Kragten, Angela H.M.; Dhert, Wouter J.; Saris, Daniël B.F.; Creemers, Laura B.

2014-01-01

Objective Hsa-miR-148a expression is decreased in OA cartilage, but its functional role in cartilage has never been studied. Therefore, our aim was to investigate the effects of overexpressing hsa-miR-148a on cartilage metabolism of OA chondrocytes. Design OA chondrocytes were transfected with a

Compositional studies at the Bone-Cartilage interface using PIXE, RBS and cSAXS techniques

International Nuclear Information System (INIS)

Kaabar, W.; Gundogdu, O.; Bradley, D.A.; Bunk, O.; Pfeiffer, F.; Pfeiffer, F.; Farquharson, M.J.; Webb, M.; Jeynes, C.

2009-01-01

Micro Proton Induced X-ray Emission (μ-PIXE) analysis has been employed herein in investigating and quantifying the distribution of a number of essential cations in two thin slices of normal and diseased human articular cartilage, the latter being affected by osteoarthritis (OA). The elemental distribution maps for Ca, P, K, S and Zn in the normal and diseased slices showed similar patterns with marked increases in elemental concentrations in the bone-cartilage interface. The S concentration was significantly lower in bone than in cartilage. Conversely, the Ca and P concentrations were higher in bone. The Ca/P ratio (2.22) of the diseased slice was determined by employing the Rutherford backscattering technique (RBS). The RBS figures of this investigation agree with values previously reported by others. Structural and organisational changes of collagen networks were investigated by coherent Small-Angle X-ray Scattering (SAXS) using beamline facilities at the Swiss Light Source (SLS) for a decalcified diseased human articular cartilage slice. The SAXS findings showed a gradual reorientation of collagen type II fibres of cartilage from parallel to the surface of the joint to normal to the bone-cartilage interface. Similar patterns of orientation were observed at the subchondral bone to bone-cartilage interface

Elemental and structural studies at the bone-cartilage interface

Energy Technology Data Exchange (ETDEWEB)

Kaabar, W., E-mail: w.kaabar@surrey.ac.uk [Department of Physics, University of Surrey, Guildford GU2 7XH (United Kingdom); Daar, E. [Department of Physics, University of Surrey, Guildford GU2 7XH (United Kingdom); Bunk, O. [Swiss Light Source, Paul Scherrer Institute, 5232 Villigen (Switzerland); Farquharson, M.J. [Department of Medical Physics and Applied Radiation Sciences, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1 (Canada); Laklouk, A. [Al-Fateh University, Tripoli (Libya); Bailey, M.; Jeynes, C. [Surrey Ion Beam Centre, University of Surrey, Guildford GU2 7XH (United Kingdom); Gundogdu, O. [Umuttepe Campus, University of Kocaeli, 41380 Kocaeli (Turkey); Bradley, D.A. [Department of Physics, University of Surrey, Guildford GU2 7XH (United Kingdom)

2011-10-01

Micro-Proton Induced X-ray Emission ({mu}-PIXE) and Proton Induced Gamma-ray Emission (PIGE) techniques were employed in the investigation of trace and essential elements distribution in normal and diseased human femoral head sections affected by osteoarthritis (OA). PIGE was exploited in the determination of elements of low atomic number z<15 such as Na and F whereas elements with z>15 viz Ca, Z, P and S were determined by PIXE. Accumulations of key elements in the bone and cartilage sections were observed, significant S and Na concentrations being found in the cartilage region particularly in normal tissues. Zn showed enhanced concentrations at the bone-cartilage interface. At a synchrotron facility, small angle X-ray scattering (SAXS) was utilized on a decalcified human femoral head section affected by OA, direct measurements being made of spatial alterations of collagen fibres. The SAXS results showed a slight decrease in the axial periodicity between normal collagen type I and that in diseased tissue in various sites, in contrast with the findings of others.

Matrix metalloproteinase-9-mediated type III collagen degradation as a novel serological biochemical marker for liver fibrogenesis

DEFF Research Database (Denmark)

Veidal, Sanne S; Vassiliadis, Efstathios; Barascuk, Natasha

2010-01-01

During fibrogenesis in the liver, in which excessive remodelling of the extracellular matrix (ECM) occurs, both the quantity of type III collagen (CO3) and levels of matrix metalloproteinases (MMPs), including MMP-9, increase significantly. MMPs play major roles in ECM remodelling, via...... their activity in the proteolytic degradation of extracellular macromolecules such as collagens, resulting in the generation of specific cleavage fragments. These neo-epitopes may be used as markers of fibrosis....

Toward understanding the role of cartilage particulates in synovial inflammation.

Science.gov (United States)

Silverstein, A M; Stefani, R M; Sobczak, E; Tong, E L; Attur, M G; Shah, R P; Bulinski, J C; Ateshian, G A; Hung, C T

2017-08-01

Arthroscopy with lavage and synovectomy can remove tissue debris from the joint space and the synovial lining to provide pain relief to patients with osteoarthritis (OA). Here, we developed an in vitro model to study the interaction of cartilage wear particles with fibroblast-like synoviocytes (FLS) to better understand the interplay of cartilage particulates with cytokines on cells of the synovium. In this study sub-10 μm cartilage particles or 1 μm latex particles were co-cultured with FLS ±10 ng/mL interleukin-1α (IL-1α) or tumor necrosis factor-α (TNF-α). Samples were analyzed for DNA, glycosaminoglycan (GAG), and collagen, and media samples were analyzed for media GAG, nitric oxide (NO) and prostaglandin-E2 (PGE2). The nature of the physical interaction between the particles and FLS was determined by microscopy. Both latex and cartilage particles could be phagocytosed by FLS. Cartilage particles were internalized and attached to the surface of both dense monolayers and individual cells. Co-culture of FLS with cartilage particulates resulted in a significant increase in cell sheet DNA and collagen content as well as NO and PGE2 synthesis compared to control and latex treated groups. The proliferative response of FLS to cartilage wear particles resulted in an overall increase in extracellular matrix (ECM) content, analogous to the thickening of the synovial lining observed in OA patients. Understanding how cartilage particles interface with the synovium may provide insight into how this interaction contributes to OA progression and may guide the role of lavage and synovectomy for degenerative disease. Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

[Injectable hydrogel functionalised with thrombocyte-rich solution and microparticles for accelerated cartilage regeneration].

Science.gov (United States)

Rampichová, M; Buzgo, M; Křížková, B; Prosecká, E; Pouzar, M; Štrajtová, L

2013-01-01

Articular cartilage defects arise due to injury or osteochondral disease such as osteonecrosis or osteochondritis dissecans. In adult patients cartilage has minimal ability to repair itself and the lesions develop into degenerative arthritis. Overcoming the low regenerative capacity of the cartilage cells and the Hayflick limit poses a challenge for the therapy of osteochondral defects. Composite scaffolds with appropriate biomechanical properties combined with a suitable blend of proliferation and differentiation factors could be a solution. The aim of this in vitro study was to develop a novel functionalised hydrogel with an integrated drug delivery system stimulating articular cartilage regeneration. Injectable collagen/ hyaluronic acid/fibrin composite hydrogel was mixed with nanofibre-based microparticles. These were loaded with ascorbic acid and dexamethasone. In addition, the effect of thrombocyte-rich solution (TRS) was studied. The gels seeded with mesenchymal stem cells (MSCs) were cultivated for 14 days. The viability, proliferation and morphology of the cells were evaluated using molecular and microscopic methods. Scaffold degradation was also assessed. The cultivation study showed that MSCs remained viable in all experimental groups, which indicated good biocompatibility of the gel. However, the number of cells in the groups enriched with microparticles was lower than in the other groups. On the other hand, confocal microscopy showed higher cell viability and rounded morphology of the cells, which can be associated with chodrogenic differentiation. The scaffolds containing microparticles showed significantly higher stability during the 14-day experiment. Our results suggest that the addition of microparticles to the scaffold improved cell differentiation into the chondrogenic lineage, resulting in a lower proliferation rate. Cell viability was better in the groups enriched with microparticles that served as an efficient drug delivery system. In

Injectable glycosaminoglycan-protein nano-complex in semi-interpenetrating networks: A biphasic hydrogel for hyaline cartilage regeneration.

Science.gov (United States)

Radhakrishnan, Janani; Subramanian, Anuradha; Sethuraman, Swaminathan

2017-11-01

Articular hyaline cartilage regeneration remains challenging due to its less intrinsic reparability. The study develops injectable biphasic semi-interpenetrating polymer networks (SIPN) hydrogel impregnated with chondroitin sulfate (ChS) nanoparticles for functional cartilage restoration. ChS loaded zein nanoparticles (∼150nm) prepared by polyelectrolyte-protein complexation were interspersed into injectable SIPNs developed by blending alginate with poly(vinyl alcohol) and calcium crosslinking. The hydrogel exhibited interconnected porous microstructure (39.9±5.8μm pore diameter, 57.7±5.9% porosity), 92% swellability and >350Pa elastic modulus. Primary chondrocytes compatibility, chondrocyte-matrix interaction with cell-cell clustering and spheroidal morphology was demonstrated in ChS loaded hydrogel and long-term (42days) proliferation was also determined. Higher fold expression of cartilage-specific genes sox9, aggrecan and collagen-II was observed in ChS loaded hydrogel while exhibiting poor expression of collagen-I. Immunoblotting of aggregan and collagen II demonstrate favorable positive influence of ChS on chondrocytes. Thus, the injectable biphasic SIPNs could be promising composition-mimetic substitute for cartilage restoration at irregular defects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Semiquantitative analysis of ECM molecules in the different cartilage layers in early and advanced osteoarthritis of the knee joint.

Science.gov (United States)

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

2012-05-01

The study was conducted to examine the expression of collagen type I and II in the different cartilage layers in relation to other ECM molecules during the progression of early osteoarthritic degeneration in human articular cartilage (AC). Quantitative real-time (RT)-PCR and colorimetrical techniques were used for calibration of Photoshop-based image analysis in detecting such lesions. Immunohistochemistry and histology were performed with 40 cartilage tissue samples showing mild (ICRS grade 1b) respectively moderate/advanced (ICRS grade 3a or 3b) (20 each) osteoarthritis compared with 15 healthy biopsies. Furthermore, we quantified our results on the gene expression of collagen type I and II and aggrecan with the help of real-time (RT)-PCR. Proteoglycan content was measured colorimetrically. The digitized images of histology and immunohistochemistry stains were analyzed with Photoshop software. T-test and Spearman correlation analysis were used for statistical analysis. In the earliest stages of AC deterioration the loss of collagen type II was associated with the appearance of collagen type I, shown by increasing amounts of collagen type I mRNA. During subsequent stages, a progressive loss of structural integrity was associated with increasing deposition of collagen type I as part of a natural healing response. A decrease of collagen type II is visible especially in the upper fibrillated area of the advanced osteoarthritic samples, which then leads to an overall decrease. Analysis of proteoglycan showed losses of the overall content and a loss of the classical zonal formation. Correlation analysis of the proteoglycan Photoshop measurements with the RT-PCR revealed strong correlation for Safranin O and collagen type I, medium for collagen type II, alcian blue and glycoprotein but weak correlation with PCR aggrecan results. Photoshop based image analysis might become a valuable supplement for well known histopathological grading systems of lesioned articular

Degradation of polyvinyl chloride (PVC) / hydrolyzed collagen (HC) blends active sludge test.

Science.gov (United States)

Agafiţei, Gabriela-Elena; Pascu, Mihaela; Cazacu, Georgeta; Vasile, Cornelia

2008-01-01

Biodegradable polymers represent a solution for the environment protection: they decrease the landfill space, by declining the petrochemical sources, and offer also an alternative solution for the recycling. The behavior during degradation in the presence of active sludge of some polyvinyl chloride (PVC) based blends with variable content of hydrolyzed collagen (HC) has been followed. Some samples were subjected to UV irradiation, for 30 hours. The modifications induced in the environment by the polymer systems (pH variation, bacterial composition), as well as the changes of the properties of the blends (weight losses, aspect etc.) were studied. During the first moments of degradation in active sludge, all the samples absorbed water, behavior which favored the biodegradation. The bacteriological analysis of the sludge indicates the presence of some microbiological species. Generally, the populations of microorganisms decrease, excepting the sulphito-reducing anaerobic bacteria, the actinomycetes and other anaerobic bacteria. PVC/HC blends are degraded with a significant rate in active sewage sludge. More susceptible for the degradation are the UV irradiated blends. After the migration of the components with a small molecular mass in the environment, the natural polymer is degraded. The degradation effect increases with the content in the natural polymer.

Molecular modulation of articular cartilage degradation

NARCIS (Netherlands)

Landman, Ellie

2013-01-01

Cartilage homeostasis is maintained due to a balance between anabolic and catabolic processes, that are regulated by a complex network of signaling pathways. Disturbance of one or more of these pathways disrupts this balance, resulting in excessive breakdown of the extracellular matrix and

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.

Properties of Cartilage on Micro- and Nanolevel

Directory of Open Access Journals (Sweden)

Sergei A. Chizhik

2010-01-01

Full Text Available Results of investigation of the elastic modulus for cartilage tissue using a technique of micro- and nanoindentation performed with help of an atomic force microscope are presented. SEM and AFM methods were applied to visualize a topography of surface layers of the entire cartilage and as well as its slices and thus to reveal features of the collagen fibers orientation. The technique used for a quantitative evaluation of the elastic modulus under compression against a ball microindenter (curvature radius - 350 micron and a nanoindenter (30 nm is described. It was shown that the cartilage behavior is highly stabile under the load if the entire composite structure of cartilage tissue is engaged into the deformation process. Tribological characteristics were investigated using the ball indenter oscillated by a tuning fork. Dependence of the friction coefficient from applied loads was obtained that revealed strong influence of an interstitial fluid on friction properties. Friction coefficient of a rat cartilage tissue as 0.08 was obtained using a developed plant prototype for tribological measurements based on the AFM construction.

Evaluation of influence of proteoglycans on hydration of articular cartilage with the use of ultrasound

Directory of Open Access Journals (Sweden)

Yi-yi YANG

2015-04-01

Full Text Available Objective　To monitor the changes in hydration behaviour of articular cartilage induced by degradation of proteoglycans, and to explore the effect of proteoglycans on hydration behaviour of articular cartilage by using high-frequency ultrasound. Methods　Twelve porcine patellae with smooth cartilage surface were prepared and equally divided into two groups: normal group without any enzyme treatment, and trypsin group they were treated with 0.25% trypsin for 8h to digest proteoglycan in the cartilage. The hydration behaviour of the cartilage tissue was scanned by high-frequency ultrasound system with a central frequency of 25MHz. Parameters including cartilage hydration strain and cartilage thickness were measured. The histopathological changes in the articular cartilage were observed under a light microscope. Results　It took approximately 20min to reach equilibrium during the hydration process in the normal cartilages, while proteoglycan-degraded cartilage took only about 5min to achieve equilibrium. The equilibrium strain of normal cartilage was 3.5%±0.5%. The degradation of proteoglycans induced a significant decrease in equilibrium strain (1.8%±0.2%, P0.05. Conclusion　Proteoglycans play an important role in hydration behaviour of articular cartilage. The degradation of proteoglycans could induce degeneration of cartilage structure and decrease in hydration behaviour after dehydration. DOI: 10.11855/j.issn.0577-7402.2015.03.03

Raman microspectrometry of laser-reshaped rabbit auricular cartilage: preliminary study on laser-induced cartilage mineralization

Science.gov (United States)

Heger, Michal; Mordon, Serge R.; Leroy, Gérard; Fleurisse, Laurence; Creusy, Collette

2006-03-01

Laser-assisted cartilage reshaping (LACR) is a relatively novel technique designed to noninvasively and permanently restructure cartilaginous tissue. It is believed that heat-induced stress relaxation, in which a temperature-mediated disruption of H2O binding is associated with conformational alterations in the proteoglycan and collagen-rich matrix, constitutes the underlying mechanism of LACR. Several reports have suggested that laser-mediated cartilage mineralization may contribute to the permanent shape change of laser-reshaped cartilage. In an effort to validate these results in the context of Er:glass LACR, we performed a preliminary Raman microspectrometric study to characterize the crystal deposits in laser-irradiated chondrocytes and extracellular matrix. For the first time, we identified intracellular calcium sulfate deposits and extracellular calcium phosphate (apatite) crystals in laser-reshaped rabbit auricular cartilage. Calcium carbonate deposits are localized in both irradiated and nonirradiated samples, suggesting that this mineral plays no role in conformational retention. In our discussion, we elaborate on the possible molecular and cellular mechanisms responsible for intra- and extracellular crystallization, and propose a novel hypothesis on the formation of apatite, inasmuch as the biological function of this mineral (providing structure and rigidity in bones and dental enamel) may be extrapolated to the permanent shape change of laser-irradiated cartilage.

[Water-exchange processes in hyaline cartilage and its basic components in a normal state and in osteoarthritis].

Science.gov (United States)

Nikolaeva, S S; Chkhol, K Z; Bykov, V A; Roshchina, A A; Iakovleva, L V; Koroleva, O A; Omel'ianenko, N P; Rebrov, L B

2000-01-01

The content of different forms of tissue water was studied in the normal articular cartilage and osteoarthrosis cartilage and its structural components: collagen, potassium hyaluronate, sodium chondroitinsulphate and its complexes. In the components of cartilage matrix a few of fractions of bound water different in the strength of binding are present. At the maximal humidity, all water in collagen binds with the active groups of biopolymers and in the glycosaminoglycans, in addition to bound water, are present, two crystal forms of freezing water (free water) at least. The quantity of free water in the collagen-chondroitin sulphat membrane, is increased with the increase of chondroitin sulphate. In the collagen-hyaluronate complex, fraction of free water is found only at the low concentration of hyaluronate kalium. It was shown that in the hyalin cartilage, in different from the other connective tissue (skin, achilles tendon), the most part of water is free water and its quantity is increased in the osteoarthrosis. It is supposed that the rearrangement of binding and free-water fractions in the osteoarthrosis is the result of deficiency of hyaluronic acid and therefore this may be regarded in the improvement of methods of treatment. This scientific and methodical approach allow to receive information on the forms and binding energy of water in the biological tissues, which is absorbed from fluids and steam phase and determine characters of the pathological changes.

Histological comparison of the alar nasal cartilages in unilateral cleft lip

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Modolin Miguel

2002-01-01

Full Text Available Patients with unilateral cleft lip display characteristic nasal changes that are independent of the degree of deformity. Defenders of the intrinsic theory consider these deformities to be due to embryogenic alterations of the alar nasal cartilages. Those that propose the extrinsic theory defend the thesis that the deformity is due to disorganization of the perioral muscles deformed by the cleft. The purpose of this study is to contribute histological evidence to help clarify the issue. PATIENTS AND METHODS: Specimens of the lateral portion of both the healthy and the cleft side of the alar cartilages were obtained from 18 patients. These uniformly cut specimens were stained by hematoxylin and eosin. Samples from 2 patients were excluded due to imperfections. The same pathologist examined all the slides. He was unaware of the origins of the specimens; he counted the number of chondrocytes and quantified the cartilage matrixes. RESULTS: All data was analyzed statistically, and no significant statistical differences were apparent, either in the number of chondrocytes or the cartilage matrix between the healthy side and the cleft side. DISCUSSION: These results apparently support the group that defend the extrinsic theory; nevertheless, the doubt about the composition of the cartilage matrix remains, not only concerning the glycosaminoglycans that compose them, but also regarding elastin and collagen and its linkages that can cause different degrees of collagen consistency.

Chemical changes demonstrated in cartilage by synchrotron infrared microspectroscopy in an antibody-induced murine model of rheumatoid arthritis

Science.gov (United States)

Croxford, Allyson M.; Selva Nandakumar, Kutty; Holmdahl, Rikard; Tobin, Mark J.; McNaughton, Don; Rowley, Merrill J.

2011-06-01

Collagen antibody-induced arthritis develops in mice following passive transfer of monoclonal antibodies (mAbs) to type II collagen (CII) and is attributed to effects of proinflammatory immune complexes, but transferred mAbs may react directly and damagingly with CII. To determine whether such mAbs cause cartilage damage in vivo in the absence of inflammation, mice lacking complement factor 5 that do not develop joint inflammation were injected intravenously with two arthritogenic mAbs to CII, M2139 and CIIC1. Paws were collected at day 3, decalcified, paraffin embedded, and 5-μm sections were examined using standard histology and synchrotron Fourier-transform infrared microspectroscopy (FTIRM). None of the mice injected with mAb showed visual or histological evidence of inflammation but there were histological changes in the articular cartilage including loss of proteoglycan and altered chondrocyte morphology. Findings using FTIRM at high lateral resolution revealed loss of collagen and the appearance of a new peak at 1635 cm-1 at the surface of the cartilage interpreted as cellular activation. Thus, we demonstrate the utility of synchrotron FTIRM for examining chemical changes in diseased cartilage at the microscopic level and establish that arthritogenic mAbs to CII do cause cartilage damage in vivo in the absence of inflammation.

Stem Cells and Gene Therapy for Cartilage Repair

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Umile Giuseppe Longo

2012-01-01

Full Text Available Cartilage defects represent a common problem in orthopaedic practice. Predisposing factors include traumas, inflammatory conditions, and biomechanics alterations. Conservative management of cartilage defects often fails, and patients with this lesions may need surgical intervention. Several treatment strategies have been proposed, although only surgery has been proved to be predictably effective. Usually, in focal cartilage defects without a stable fibrocartilaginous repair tissue formed, surgeons try to promote a natural fibrocartilaginous response by using marrow stimulating techniques, such as microfracture, abrasion arthroplasty, and Pridie drilling, with the aim of reducing swelling and pain and improving joint function of the patients. These procedures have demonstrated to be clinically useful and are usually considered as first-line treatment for focal cartilage defects. However, fibrocartilage presents inferior mechanical and biochemical properties compared to normal hyaline articular cartilage, characterized by poor organization, significant amounts of collagen type I, and an increased susceptibility to injury, which ultimately leads to premature osteoarthritis (OA. Therefore, the aim of future therapeutic strategies for articular cartilage regeneration is to obtain a hyaline-like cartilage repair tissue by transplantation of tissues or cells. Further studies are required to clarify the role of gene therapy and mesenchimal stem cells for management of cartilage lesions.

Formation of Hyaline Cartilage Tissue by Passaged Human Osteoarthritic Chondrocytes.

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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.

Magnetic resonance imaging of articular cartilage: ex vivo study on normal cartilage correlated with magnetic resonance microscopy

International Nuclear Information System (INIS)

Cova, M.; Frezza, F.; Pozzi-Mucelli, R.S.; Dalla-Palma, L.; Toffanin, R.; Pozzi-Mucelli, M.; Mlynarik, V.; Vittur, F.

1998-01-01

The aims of this study were (a) to compare the MR appearance of normal articular cartilage in ex vivo MR imaging (MRI) and MR microscopy (MRM) images of disarticulated human femoral heads, (b) to evaluate by MRM the topographic variations in articular cartilage of disarticulated human femoral heads, and subsequently, (c) to compare MRM images with histology. Ten disarticulated femoral heads were examined. Magnetic resonance images were obtained using spin-echo (SE) and gradient-echo (GE) sequences. Microimages were acquired on cartilage-bone cylindrical plugs excised from four regions (superior, inferior, anterior, posterior) of one femoral head, using a modified SE sequence. Both MRI and MRM images were obtained before and after a 90 rotation of the specimen, around the axis perpendicular to the examined cartilage surface. Finally, MRM images were correlated with histology. A trilaminar appearance of articular cartilage was observed with MRI and with a greater detail with MRM. A good correlation between MRI and MRM features was demonstrated. Both MRI and MRM showed a loss of the trilaminar cartilage appearance after specimen rotation, with greater evidence on MRM images. Cartilage excised from the four regions of the femoral head showed a different thickness, being thickest in the samples excised from the superior site. The MRM technique confirms the trilaminar MRI appearance of human articular cartilage, showing good correlation with histology. The loss of the trilaminar appearance of articular cartilage induced by specimen rotation suggests that this feature is partially related to the collagen-fiber orientation within the different layers. The MRM technique also shows topographic variations in thickness of human articular cartilage. (orig.)

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.

* Human Amniotic Mesenchymal Stromal Cells as Favorable Source for Cartilage Repair.

Science.gov (United States)

Muiños-López, Emma; Hermida-Gómez, Tamara; Fuentes-Boquete, Isaac; de Toro-Santos, Javier; Blanco, Francisco Javier; Díaz-Prado, Silvia María

2017-09-01

Localized trauma-derived breakdown of the hyaline articular cartilage may progress toward osteoarthritis, a degenerative condition characterized by total loss of articular cartilage and joint function. Tissue engineering technologies encompass several promising approaches with high therapeutic potential for the treatment of these focal defects. However, most of the research in tissue engineering is focused on potential materials and structural cues, while little attention is directed to the most appropriate source of cells endowing these materials. In this study, using human amniotic membrane (HAM) as scaffold, we defined a novel static in vitro model for cartilage repair. In combination with HAM, four different cell types, human chondrocytes, human bone marrow-derived mesenchymal stromal cells (hBMSCs), human amniotic epithelial cells, and human amniotic mesenchymal stromal cells (hAMSCs) were assessed determining their therapeutic potential. A chondral lesion was drilled in human cartilage biopsies simulating a focal defect. A pellet of different cell types was implanted inside the lesion and covered with HAM. The biopsies were maintained for 8 weeks in culture. Chondrogenic differentiation in the defect was analyzed by histology and immunohistochemistry. HAM scaffold showed good integration and adhesion to the native cartilage in all groups. Although all cell types showed the capacity of filling the focal defect, hBMSCs and hAMSCs demonstrated higher levels of new matrix synthesis. However, only the hAMSCs-containing group presented a significant cytoplasmic content of type II collagen when compared with chondrocytes. More collagen type I was identified in the new synthesized tissue of hBMSCs. In accordance, hBMSCs and hAMSCs showed better International Cartilage Research Society scoring although without statistical significance. HAM is a useful material for articular cartilage repair in vitro when used as scaffold. In combination with hAMSCs, HAM showed better

Cartilage regeneration by chondrogenic induced adult stem cells in osteoarthritic sheep model.

Science.gov (United States)

Ude, Chinedu C; Sulaiman, Shamsul B; Min-Hwei, Ng; Hui-Cheng, Chen; Ahmad, Johan; Yahaya, Norhamdan M; Saim, Aminuddin B; Idrus, Ruszymah B H

2014-01-01

In this study, Adipose stem cells (ADSC) and bone marrow stem cells (BMSC), multipotent adult cells with the potentials for cartilage regenerations were induced to chondrogenic lineage and used for cartilage regenerations in surgically induced osteoarthritis in sheep model. Osteoarthritis was induced at the right knee of sheep by complete resection of the anterior cruciate ligament and medial meniscus following a 3-weeks exercise regimen. Stem cells from experimental sheep were culture expanded and induced to chondrogenic lineage. Test sheep received a single dose of 2 × 10(7) autologous PKH26-labelled, chondrogenically induced ADSCs or BMSCs as 5 mls injection, while controls received 5 mls culture medium. The proliferation rate of ADSCs 34.4 ± 1.6 hr was significantly higher than that of the BMSCs 48.8 ± 5.3 hr (P = 0.008). Chondrogenic induced BMSCs had significantly higher expressions of chondrogenic specific genes (Collagen II, SOX9 and Aggrecan) compared to chondrogenic ADSCs (P = 0.031, 0.010 and 0.013). Grossly, the treated knee joints showed regenerated de novo cartilages within 6 weeks post-treatment. On the International Cartilage Repair Society grade scores, chondrogenically induced ADSCs and BMSCs groups had significantly lower scores than controls (P = 0.0001 and 0.0001). Fluorescence of the tracking dye (PKH26) in the injected cells showed that they had populated the damaged area of cartilage. Histological staining revealed loosely packed matrixes of de novo cartilages and immunostaining demonstrated the presence of cartilage specific proteins, Collagen II and SOX9. Autologous chondrogenically induced ADSCs and BMSCs could be promising cell sources for cartilage regeneration in osteoarthritis.

Cartilage regeneration by chondrogenic induced adult stem cells in osteoarthritic sheep model.

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Chinedu C Ude

Full Text Available OBJECTIVES: In this study, Adipose stem cells (ADSC and bone marrow stem cells (BMSC, multipotent adult cells with the potentials for cartilage regenerations were induced to chondrogenic lineage and used for cartilage regenerations in surgically induced osteoarthritis in sheep model. METHODS: Osteoarthritis was induced at the right knee of sheep by complete resection of the anterior cruciate ligament and medial meniscus following a 3-weeks exercise regimen. Stem cells from experimental sheep were culture expanded and induced to chondrogenic lineage. Test sheep received a single dose of 2 × 10(7 autologous PKH26-labelled, chondrogenically induced ADSCs or BMSCs as 5 mls injection, while controls received 5 mls culture medium. RESULTS: The proliferation rate of ADSCs 34.4 ± 1.6 hr was significantly higher than that of the BMSCs 48.8 ± 5.3 hr (P = 0.008. Chondrogenic induced BMSCs had significantly higher expressions of chondrogenic specific genes (Collagen II, SOX9 and Aggrecan compared to chondrogenic ADSCs (P = 0.031, 0.010 and 0.013. Grossly, the treated knee joints showed regenerated de novo cartilages within 6 weeks post-treatment. On the International Cartilage Repair Society grade scores, chondrogenically induced ADSCs and BMSCs groups had significantly lower scores than controls (P = 0.0001 and 0.0001. Fluorescence of the tracking dye (PKH26 in the injected cells showed that they had populated the damaged area of cartilage. Histological staining revealed loosely packed matrixes of de novo cartilages and immunostaining demonstrated the presence of cartilage specific proteins, Collagen II and SOX9. CONCLUSION: Autologous chondrogenically induced ADSCs and BMSCs could be promising cell sources for cartilage regeneration in osteoarthritis.

Study on nano-structured hydroxyapatite/zirconia stabilized yttria on healing of articular cartilage defect in rabbit

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Amir Sotoudeh

2013-05-01

Full Text Available PURPOSE: Articular Cartilage has limited potential for self-repair and tissue engineering approaches attempt to repair articular cartilage by scaffolds. We hypothesized that the combined hydroxyapatite and zirconia stabilized yttria would enhance the quality of cartilage healing. METHODS: In ten New Zealand white rabbits bilateral full-thickness osteochondral defect, 4 mm in diameter and 3 mm depth, was created on the articular cartilage of the patellar groove of the distal femur. In group I the scaffold was implanted into the right stifle and the same defect was created in the left stifle without any transplant (group II. Specimens were harvested at 12 weeks after implantation, examined histologically for morphologic features, and stained immunohistochemically for type-II collagen. RESULTS: In group I the defect was filled with a white translucent cartilage tissue In contrast, the defects in the group II remained almost empty. In the group I, the defects were mostly filled with hyaline-like cartilage evidenced but defects in group II were filled with fibrous tissue with surface irregularities. Positive immunohistochemical staining of type-II collagen was observed in group I and it was absent in the control group. CONCLUSION: The hydroxyapatite/yttria stabilized zirconia scaffold would be an effective scaffold for cartilage tissue engineering.

Differentiation of human mesenchymal stromal cells cultured on collagen sponges for cartilage repair.

Science.gov (United States)

Sanjurjo-Rodríguez, Clara; Martínez-Sánchez, Adela Helvia; Hermida-Gómez, Tamara; Fuentes-Boquete, Isaac; Díaz-Prado, Silvia; Blanco, Francisco J

2016-11-01

The aim of this study was to evaluate proliferation and chondrogenic differentiation of human bone-marrow mesenchymal stromal cells (hBMSCs) cultured on collagen biomaterials. hBMSCs were seeded on five different collagen (Col) sponges: C1C2 (types I and II Col), C1C2HS (types I and II Col plus heparan sulphate (HS)), C1C2CHS (types I and II Col plus chondroitin sulphate (CHS)), C1-OLH3 (type I Col plus low molecular weight heparin) and C1CHS (type I Col plus CHS). The resulting constructs were analyzed by histological and immunohistochemical staining, molecular biology and electron microscopy. Col released into culture media was measured by a dye-binding method Results: hBMSCs on biomaterials C1C2, C1C2HS and C1C2CHS had more capacity to attach, proliferate and synthesize Col II and proteoglycans in the extracellular matrix (ECM) than on C1-OLH3 and C1CHS. The presence of aggrecan was detected only at the gene level. Total Col liberated by the cells in the supernatants in all scaffold cultures was detected. The level of Col I in the ECM was lower in C1-OLH3 and that of Col II was highest in C1C2 and C1C2HS. Electron microscopy showed differently shaped cells, from rounded to flattened, in all constructs. Col fibers in bundles were observed in C1C2CHS by transmission electron microscopy. The results show that Col I and Col II (C1C2, C1C2HS and C1C2CHS) biomaterials allowed cell proliferation and chondrogenic-like differentiation of hBMSCs at an early stage. Constructs cultured on C1C2HS and C1C2CHS showed better cartilage-like phenotype than the other ones.

Cartilage oligomeric matrix protein specific antibodies are pathogenic

DEFF Research Database (Denmark)

Geng, Hui; Nandakumar, Kutty Selva; Pramhed, Anna

2012-01-01

-specific monoclonal antibodies (mAbs). METHODS: B cell immunodominant regions on the COMP molecule were measured with a novel enzyme-linked immunosorbent assay using mammalian expressed full-length mouse COMP as well as a panel of recombinant mouse COMP fragments. 18 mAbs specific to COMP were generated......ABSTRACT: INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP...

A Simple and Efficient Method to Improve Mechanical Properties of Collagen Scaffolds by UV Irradiation

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F. Khayyatan

2010-12-01

Full Text Available Collagen is the major protein component of cartilage, bone, skin and connective tissue and constitutes the major part of the extracellular matrix. Collagen type I has complex structural hierarchy, which consists of treepolypeptide α-chains wound together in a rod-like helical structure. Collagen is an important biomaterial, finding many applications in the field of tissue engineering. It has been processed into various shapes, such as, gel, film, sponge and fiber. It is commonly used as the scaffolding material for tissue engineering due to its many superior properties including low antigenicity and high growth promotion. Unfortunately, poor mechanical properties and rapid degradation rates of collagen scaffolds can cause instability and difficulty in handling. By crosslinking, the structural stability of the collagen and its rate of resorption can be adapted with respect to its demanding requirements. The strength, resorption rate, and biocompatibility of collagenous biomaterials are profoundly influenced by the method and extent of crosslinking. In thisstudy, the effect of UV irradiation on collagen scaffolds has been carried out.Collagen scaffolds were fabricated using freeze drying method with freezing temperature of -80oC, then exposed to UV irradiation. Mean pore size of the scaffolds was obtained as 98.52±14.51 μm using scanning electron microscopy. Collagen scaffolds exposed to UV Irradiation (254 nm for 15 min showed the highest tensile strain (17.37±0.98 %, modulus (1.67±0.15 MPa and maximum load (24.47±2.38 cN values. As partial loss of the native collagen structure may influence attachment, migration, and proliferation of cells on collagen scaffolds, we detected no intact α-chains after SDS-Page chromatography. We demonstrate that UV irradiation is a rapid and easily controlled means of increasing the mechanical strength of collagen scaffolds without any molecular fracture.

Enhanced mechanical properties of thermosensitive chitosan hydrogel by silk fibers for cartilage tissue engineering

International Nuclear Information System (INIS)

Mirahmadi, Fereshteh; Tafazzoli-Shadpour, Mohammad; Shokrgozar, Mohammad Ali; Bonakdar, Shahin

2013-01-01

Articular cartilage has limited repair capability following traumatic injuries and current methods of treatment remain inefficient. Reconstructing cartilage provides a new way for cartilage repair and natural polymers are often used as scaffold because of their biocompatibility and biofunctionality. In this study, we added degummed chopped silk fibers and electrospun silk fibers to the thermosensitive chitosan/glycerophosphate hydrogels to reinforce two hydrogel constructs which were used as scaffold for hyaline cartilage regeneration. The gelation temperature and gelation time of hydrogel were analyzed by the rheometer and vial tilting method. Mechanical characterization was measured by uniaxial compression, indentation and dynamic mechanical analysis assay. Chondrocytes were then harvested from the knee joint of the New Zealand white rabbits and cultured in constructs. The cell proliferation, viability, production of glycosaminoglycans and collagen type II were assessed. The results showed that mechanical properties of the hydrogel were significantly enhanced when a hybrid with two layers of electrospun silk fibers was made. The results of GAG and collagen type II in cell-seeded scaffolds indicate support of the chondrogenic phenotype for chondrocytes with a significant increase in degummed silk fiber–hydrogel composite for GAG content and in two-layer electrospun fiber–hydrogel composite for Col II. It was concluded that these two modified scaffolds could be employed for cartilage tissue engineering. - Highlights: • Chitosan hydrogel composites fabricated by two forms of silk fiber • Silk fibers provide structural support for the hydrogel matrix. • The mechanical properties of hydrogel significantly improved by associating with silk. • Production of GAG and collagen type II was demonstrated within the scaffolds

Enhanced mechanical properties of thermosensitive chitosan hydrogel by silk fibers for cartilage tissue engineering

Energy Technology Data Exchange (ETDEWEB)

Mirahmadi, Fereshteh [Faculty of Biomedical Engineering, Amirkabir University of Technology, Tehran (Iran, Islamic Republic of); National Cell Bank of Iran, Pasteur Institute of Iran, Tehran (Iran, Islamic Republic of); Tafazzoli-Shadpour, Mohammad, E-mail: Tafazoli@aut.ac.ir [Faculty of Biomedical Engineering, Amirkabir University of Technology, Tehran (Iran, Islamic Republic of); Shokrgozar, Mohammad Ali, E-mail: mashokrgozar@pasteur.ac.ir [National Cell Bank of Iran, Pasteur Institute of Iran, Tehran (Iran, Islamic Republic of); Bonakdar, Shahin [National Cell Bank of Iran, Pasteur Institute of Iran, Tehran (Iran, Islamic Republic of)

2013-12-01

Articular cartilage has limited repair capability following traumatic injuries and current methods of treatment remain inefficient. Reconstructing cartilage provides a new way for cartilage repair and natural polymers are often used as scaffold because of their biocompatibility and biofunctionality. In this study, we added degummed chopped silk fibers and electrospun silk fibers to the thermosensitive chitosan/glycerophosphate hydrogels to reinforce two hydrogel constructs which were used as scaffold for hyaline cartilage regeneration. The gelation temperature and gelation time of hydrogel were analyzed by the rheometer and vial tilting method. Mechanical characterization was measured by uniaxial compression, indentation and dynamic mechanical analysis assay. Chondrocytes were then harvested from the knee joint of the New Zealand white rabbits and cultured in constructs. The cell proliferation, viability, production of glycosaminoglycans and collagen type II were assessed. The results showed that mechanical properties of the hydrogel were significantly enhanced when a hybrid with two layers of electrospun silk fibers was made. The results of GAG and collagen type II in cell-seeded scaffolds indicate support of the chondrogenic phenotype for chondrocytes with a significant increase in degummed silk fiber–hydrogel composite for GAG content and in two-layer electrospun fiber–hydrogel composite for Col II. It was concluded that these two modified scaffolds could be employed for cartilage tissue engineering. - Highlights: • Chitosan hydrogel composites fabricated by two forms of silk fiber • Silk fibers provide structural support for the hydrogel matrix. • The mechanical properties of hydrogel significantly improved by associating with silk. • Production of GAG and collagen type II was demonstrated within the scaffolds.

Assessing the extent of bone degradation using glutamine deamidation in collagen.

Science.gov (United States)

Wilson, Julie; van Doorn, Nienke L; Collins, Matthew J

2012-11-06

Collagen peptides are analyzed using a low-cost, high-throughput method for assessing deamidation using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). For each chosen peptide, the theoretical distribution is calculated and the measured distribution for each sample compared with this to determine the extent of glutamine deamidation. The deamidation of glutamine (Q) to glutamic acid (E) results in a mass shift of +0.984 Da. Thus, from the resolution of our data, the second peak in the isotope distribution for a peptide containing one glutamine residue coincides with the first peak of the isotope distribution for the peptide in which the residue is deamidated. A genetic algorithm is used to determine the extent of deamidation that gives the best fit to the measured distribution. The method can be extended to peptides containing more than one glutamine residue. The extent of protein degradation assessed in this way could be used, for example, to assess the damage of collagen, and screen samples for radiocarbon dating and DNA analysis.

Polymers in Cartilage Defect Repair of the Knee: Current Status and Future Prospects

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Ralph M. Jeuken

2016-06-01

Full Text Available Cartilage defects in the knee are often seen in young and active patients. There is a need for effective joint preserving treatments in patients suffering from cartilage defects, as untreated defects often lead to osteoarthritis. Within the last two decades, tissue engineering based techniques using a wide variety of polymers, cell sources, and signaling molecules have been evaluated. We start this review with basic background information on cartilage structure, its intrinsic repair, and an overview of the cartilage repair treatments from a historical perspective. Next, we thoroughly discuss polymer construct components and their current use in commercially available constructs. Finally, we provide an in-depth discussion about construct considerations such as degradation rates, cell sources, mechanical properties, joint homeostasis, and non-degradable/hybrid resurfacing techniques. As future prospects in cartilage repair, we foresee developments in three areas: first, further optimization of degradable scaffolds towards more biomimetic grafts and improved joint environment. Second, we predict that patient-specific non-degradable resurfacing implants will become increasingly applied and will provide a feasible treatment for older patients or failed regenerative treatments. Third, we foresee an increase of interest in hybrid construct, which combines degradable with non-degradable materials.

Low-field one-dimensional and direction-dependent relaxation imaging of bovine articular cartilage

Science.gov (United States)

Rössler, Erik; Mattea, Carlos; Mollova, Ayret; Stapf, Siegfried

2011-12-01

The structure of articular cartilage is separated into three layers of differently oriented collagen fibers, which is accompanied by a gradient of increasing glycosaminoglycan (GAG) and decreasing water concentration from the top layer towards the bone interface. The combined effect of these structural variations results in a change of the longitudinal and transverse relaxation times as a function of the distance from the cartilage surface. In this paper, this dependence is investigated at a magnetic field strength of 0.27 T with a one-dimensional depth resolution of 50 μm on bovine hip and stifle joint articular cartilage. By employing this method, advantage is taken of the increasing contrast of the longitudinal relaxation rate found at lower magnetic field strengths. Furthermore, evidence for an orientational dependence of relaxation times with respect to an axis normal to the surface plane is given, an observation that has recently been reported using high-field MRI and that was explained by preferential orientations of collagen bundles in each of the three cartilage zones. In order to quantify the extent of a further contrast mechanism and to estimate spatially dependent glycosaminoglycan concentrations, the data are supplemented by proton relaxation times that were acquired in bovine articular cartilage that was soaked in a 0.8 mM aqueous Gd ++ solution.

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

Science.gov (United States)

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

2015-06-16

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

In vitro and in vivo evaluation of chitosan–gelatin scaffolds for cartilage tissue engineering

Energy Technology Data Exchange (ETDEWEB)

Whu, Shu Wen [Department of Orthopaedic Surgery, Chang Gung Memorial Hospital at Keelung, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Hung, Kun-Che; Hsieh, Kuo-Huang [Institute of Polymer Science and Engineering, National Taiwan University, Taipei, Taiwan (China); Chen, Chih-Hwa [Department of Orthopaedic Surgery, Chang Gung Memorial Hospital at Keelung, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Tsai, Ching-Lin, E-mail: tsaicl@ntuh.gov.tw [Department of Orthopaedics, National Taiwan University Hospital, Taipei, Taiwan (China); Hsu, Shan-hui, E-mail: shhsu@ntu.edu.tw [Institute of Polymer Science and Engineering, National Taiwan University, Taipei, Taiwan (China)

2013-07-01

Chitosan–gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan–gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1{sub WSC}) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1{sub WSC} promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1{sub WSC} constructs (0.187 ± 0.095 μg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329 ± 0.660 μg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1{sub WSC} constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1{sub WSC} scaffolds may enhance the cartilage regeneration in vitro and in vivo. - Highlights: • We developed a chitosan–gelatin scaffold crosslinked with carbodiimide. • Neocartilage formation was more evident in crosslinked vs. non-crosslinked scaffolds. • Histological features of tide line and lacunae were observed in vivo at 6.5 months. • Compressive

On the detection of early osteoarthritis by quantitative microscopic imaging

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Mittelstaedt, Daniel John

Articular cartilage is a thin layer of connective tissue that protects the ends of bones in diarthroidal joints. Cartilage distributes mechanical forces during daily movement throughout its unique depth-dependent structure. The extracellular matrix (ECM) of cartilage primarily contains water, collagen, and glycosaminoglycan (GAG). The collagen fibers are intertwined with negatively charged GAG and surround the cells (i.e. chondrocytes) in cartilage. Degradation to the ECM reduces the load bearing properties of cartilage which can be initiated by injury (e.g. anterior cruciate ligament (ACL) rupture) or disease (e.g. osteoarthritis (OA)). Magnetic resonance imaging (MRI) and x-ray computed tomography (CT) are noninvasive imaging techniques that are increasingly being used in the clinical detection of cartilage degradation. The aim of the first project in this dissertation was to quantify and compare the depth-dependent GAG concentration from healthy and biochemically degraded humeral ex vivo articular cartilage using quantitative contrast enhanced micro-computed tomography (qCECT) at high resolution. The second project in this dissertation was aimed to measure the topographical and depth-dependent GAG concentration using qCECT and delayed gadolinium enhanced magnetic resonance imaging of cartilage (dGEMRIC) from the medial tibia cartilage three weeks after unilateral ACL transection which is an animal model of OA (i.e. modified Pond-Nuki model). These GAG measurements were correlated with a biochemical method, inductively couple plasma optical emission spectrometry, to compare the degradation on the medial tibia between the OA and contralateral cartilage. The third project in this dissertation used the same cartilage specimens as in project two to investigate the change in T2 due to OA and the effect on T2 from a contrast agent. Furthermore, the change in T2 relaxation was investigated from static unconfined compression with correlations by biomechanical

Application of multiphysics models to efficient design of experiments of solute transport across articular cartilage.

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Pouran, Behdad; Arbabi, Vahid; Weinans, Harrie; Zadpoor, Amir A

2016-11-01

Transport of solutes helps to regulate normal physiology and proper function of cartilage in diarthrodial joints. Multiple studies have shown the effects of characteristic parameters such as concentration of proteoglycans and collagens and the orientation of collagen fibrils on the diffusion process. However, not much quantitative information and accurate models are available to help understand how the characteristics of the fluid surrounding articular cartilage influence the diffusion process. In this study, we used a combination of micro-computed tomography experiments and biphasic-solute finite element models to study the effects of three parameters of the overlying bath on the diffusion of neutral solutes across cartilage zones. Those parameters include bath size, degree of stirring of the bath, and the size and concentration of the stagnant layer that forms at the interface of cartilage and bath. Parametric studies determined the minimum of the finite bath size for which the diffusion behavior reduces to that of an infinite bath. Stirring of the bath proved to remarkably influence neutral solute transport across cartilage zones. The well-stirred condition was achieved only when the ratio of the diffusivity of bath to that of cartilage was greater than ≈1000. While the thickness of the stagnant layer at the cartilage-bath interface did not significantly influence the diffusion behavior, increase in its concentration substantially elevated solute concentration in cartilage. Sufficient stirring attenuated the effects of the stagnant layer. Our findings could be used for efficient design of experimental protocols aimed at understanding the transport of molecules across articular cartilage. Copyright © 2016 Elsevier Ltd. All rights reserved.

The signs of differentiation of chondrocytes in the formation of early cartilage lesions in the elderly

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Elena Vasil'evna Chetina

2011-01-01

Results. The activity of collagen type II cleavage was shown to be increased in the area of age-related OA-like cartilage lesions. This was accompanied by the high expression of collagenases of metalloproteinases (MMP 1, 14 (MT1-MMP, aggrecanases - desintegrin and MMP with thrombospondin type 1 motif (ADAMTS 5, the cytokines of interleukins (IL 1α/β and tumor necrosis factor-α (TNF-α, as well as the genes associated with chondrocyte hypertrophy of type X collagen (C0L10A1, MMP 13 and 9, Indian hedgehog (Ihh and cas-pase 3 in the immediate vicinity of a lesion area. At the same time, there was a high expression of growth factors associated with the proliferation phase of chondrocytes, namely: parathyroid hormone-related peptide (PTHrP, fibroblast growth factor-2 (FGF-2, transforming growth factor β1/2 (TGF-β1/2, as well as macromolecules of matrix of type II collagen (C0L2A1 and aggrecan in both the areas adjacent to the lesions and at a considerable distance from their center. However, these areas showed no higher collagen cleavage activity. Nether higher collagen cleavage, nor excess expression of the genes examined were observed in the absolutely intact cartilage areas. Conclusion. Our studies have indicated that the area of very early age-related OA-like focal cartilage lesions exhibits enhanced type II collagen cleavage that is attended by the expression of the genes associated with chondrocyte differentiation in the embryonic growth plate.

Magnetic resonance imaging reflects cartilage proteoglycan degradation in the rabbit knee

International Nuclear Information System (INIS)

Paul, P.K.; O'Byrne, E.; Blancuzzi, V.; Wilson, D.; Gunson, D.; Douglas, F.L.; Wang Jinzhao; Mezrich, R.S.

1991-01-01

Cartilage degeneration in osteoarthritis is initiated by a loss of proteoglycan. Intra-articular injection of papain causes a reversible loss of proteoglycan in rabbit knees. Rabbits were scanned with magnetic resonance imaging (MRI), using a 1.5T Signa superconducting magnet with 3 inch surface coil. Spin echo sequences were performed in the coronal and sagittal planes at 0, 24, 48, and 72 h after intra-articular injection of papain to abtain T 1 , proton density, and T 2 -weighted images. Cartilage proteoglycan content was measured biochemically and histochemically. Reduced articular cartilage thickness in the MR images of papain-treated knees corresponded to changes in cartilage proteoglycan content. (orig.)

Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model.

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Goodrich, L R; Hidaka, C; Robbins, P D; Evans, C H; Nixon, A J

2007-05-01

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral joint received 2 x 10(7) naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and

A Dual Flow Bioreactor for Cartilage Tissue Engineering

NARCIS (Netherlands)

Spitters, Tim

2014-01-01

Preventing the onset of a degenerative disease like osteoarthritis by restoring tissue function before cartilage degradation occurs will decrease health costs, reduce socio-economic burdens of patients and preserve quality of life. However, producing ex vivo cartilage implants of clinically relevant

Cationic Contrast Agent Diffusion Differs Between Cartilage and Meniscus.

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Honkanen, Juuso T J; Turunen, Mikael J; Freedman, Jonathan D; Saarakkala, Simo; Grinstaff, Mark W; Ylärinne, Janne H; Jurvelin, Jukka S; Töyräs, Juha

2016-10-01

Contrast enhanced computed tomography (CECT) is a non-destructive imaging technique used for the assessment of composition and structure of articular cartilage and meniscus. Due to structural and compositional differences between these tissues, diffusion and distribution of contrast agents may differ in cartilage and meniscus. The aim of this study is to determine the diffusion kinematics of a novel iodine based cationic contrast agent (CA(2+)) in cartilage and meniscus. Cylindrical cartilage and meniscus samples (d = 6 mm, h ≈ 2 mm) were harvested from healthy bovine knee joints (n = 10), immersed in isotonic cationic contrast agent (20 mgI/mL), and imaged using a micro-CT scanner at 26 time points up to 48 h. Subsequently, normalized X-ray attenuation and contrast agent diffusion flux, as well as water, collagen and proteoglycan (PG) contents in the tissues were determined. The contrast agent distributions within cartilage and meniscus were different. In addition, the normalized attenuation and diffusion flux were higher (p < 0.05) in cartilage. Based on these results, diffusion kinematics vary between cartilage and meniscus. These tissue specific variations can affect the interpretation of CECT images and should be considered when cartilage and meniscus are assessed simultaneously.

Autologous Cartilage Chip Transplantation Improves Repair Tissue Composition Compared With Marrow Stimulation.

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Christensen, Bjørn Borsøe; Olesen, Morten Lykke; Lind, Martin; Foldager, Casper Bindzus

2017-06-01

Repair of chondral injuries by use of cartilage chips has recently demonstrated clinical feasibility. To investigate in vivo cartilage repair outcome of autologous cartilage chips compared with marrow stimulation in full-thickness cartilage defects in a minipig model. Controlled laboratory study. Six Göttingen minipigs received two 6-mm chondral defects in the medial and lateral trochlea of each knee. The two treatment groups were (1) autologous cartilage chips embedded in fibrin glue (ACC) (n = 12) and (2) marrow stimulation (MST) (n = 12). The animals were euthanized after 6 months, and the composition of repair tissue was quantitatively determined using histomorphometry. Semiquantitative evaluation was performed by means of the International Cartilage Repair Society (ICRS) II score. Collagen type II staining was used to further evaluate the repair tissue composition. Significantly more hyaline cartilage was found in the ACC (17.1%) compared with MST (2.9%) group ( P cartilage repair tissue compared with MST at 6 months postoperatively. Further studies are needed to investigate ACC as a possible alternative first-line treatment for focal cartilage injuries in the knee.

Degradability of injectable calcium sulfate/mineralized collagen-based bone repair material and its effect on bone tissue regeneration

International Nuclear Information System (INIS)

Chen, Zonggang; Kang, Lingzhi; Meng, Qing-Yuan; Liu, Huanye; Wang, Zhaoliang; Guo, Zhongwu; Cui, Fu-Zhai

2014-01-01

The nHAC/CSH composite is an injectable bone repair material with controllable injectability and self-setting properties prepared by introducing calcium sulfate hemihydrate (CSH) into mineralized collagen (nHAC). When mixed with water, the nHAC/CSH composites can be transformed into mineralized collagen/calcium sulfate dihydrate (nHAC/CSD) composites. The nHAC/CSD composites have good biocompatibility and osteogenic capability. Considering that the degradation behavior of bone repair material is another important factor for its clinical applications, the degradability of nHAC/CSD composites was studied. The results showed that the degradation ratio of the nHAC/CSD composites with lower nHAC content increased with the L/S ratio increase of injectable materials, but the variety of L/S ratio had no significant effect on the degradation ratio of the nHAC/CSD composites with higher nHAC content. Increasing nHAC content in the composites could slow down the degradation of nHAC/CSD composite. Setting accelerator had no significant effect on the degradability of nHAC/CSD composites. In vivo histological analysis suggests that the degradation rate of materials can match the growth rate of new mandibular bone tissues in the implanted site of rabbit. The regulable degradability of materials resulting from the special prescriptions of injectable nHAC/CSH composites will further improve the workability of nHAC/CSD composites. - Highlights: • The nHAC/CSH composite can be as an injectable bone repair material. • The L/S ratio and nHAC content have a significant effect on material degradability. • The degradability of bone materials can be regulated to match tissue repair. • The regulable degradability will further improve the workability of bone materials

Degradability of injectable calcium sulfate/mineralized collagen-based bone repair material and its effect on bone tissue regeneration

Energy Technology Data Exchange (ETDEWEB)

Chen, Zonggang, E-mail: chenzg@sdu.edu.cn [National Glycoengineering Research Center, Shandong University, Jinan 250100 (China); Department of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Kang, Lingzhi [National Glycoengineering Research Center, Shandong University, Jinan 250100 (China); Meng, Qing-Yuan [Department of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Liu, Huanye [Department of Prosthodontics, School of Stomatology, China Medical University, Shenyang 110001 (China); Wang, Zhaoliang [Jinan Military General Hospital of PLA, Jinan 250031 (China); Guo, Zhongwu, E-mail: zwguo@sdu.edu.cn [National Glycoengineering Research Center, Shandong University, Jinan 250100 (China); Cui, Fu-Zhai, E-mail: cuifz@mail.tsinghua.edu.cn [Department of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China)

2014-12-01

The nHAC/CSH composite is an injectable bone repair material with controllable injectability and self-setting properties prepared by introducing calcium sulfate hemihydrate (CSH) into mineralized collagen (nHAC). When mixed with water, the nHAC/CSH composites can be transformed into mineralized collagen/calcium sulfate dihydrate (nHAC/CSD) composites. The nHAC/CSD composites have good biocompatibility and osteogenic capability. Considering that the degradation behavior of bone repair material is another important factor for its clinical applications, the degradability of nHAC/CSD composites was studied. The results showed that the degradation ratio of the nHAC/CSD composites with lower nHAC content increased with the L/S ratio increase of injectable materials, but the variety of L/S ratio had no significant effect on the degradation ratio of the nHAC/CSD composites with higher nHAC content. Increasing nHAC content in the composites could slow down the degradation of nHAC/CSD composite. Setting accelerator had no significant effect on the degradability of nHAC/CSD composites. In vivo histological analysis suggests that the degradation rate of materials can match the growth rate of new mandibular bone tissues in the implanted site of rabbit. The regulable degradability of materials resulting from the special prescriptions of injectable nHAC/CSH composites will further improve the workability of nHAC/CSD composites. - Highlights: • The nHAC/CSH composite can be as an injectable bone repair material. • The L/S ratio and nHAC content have a significant effect on material degradability. • The degradability of bone materials can be regulated to match tissue repair. • The regulable degradability will further improve the workability of bone materials.

Single-photon absorption of isolated collagen mimetic peptides and triple-helix models in the VUV-X energy range

NARCIS (Netherlands)

Schwob, Lucas; Lalande, Mathieu; Rangama, Jimmy; Egorov, Dmitrii; Hoekstra, Ronnie; Pandey, Rahul; Eden, Samuel; Schlathölter, Thomas; Vizcaino, Violaine; Poully, Jean-Christophe

2017-01-01

Cartilage and tendons owe their special mechanical properties to the fibrous collagen structure. These strong fibrils are aggregates of a sub-unit consisting of three collagen proteins wound around each other in a triple helix. Even though collagen is the most abundant protein in the human body, the

Contribution of proteoglycan osmotic swelling pressure to the compressive properties of articular cartilage.

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Han, EunHee; Chen, Silvia S; Klisch, Stephen M; Sah, Robert L

2011-08-17

The negatively charged proteoglycans (PG) provide compressive resistance to articular cartilage by means of their fixed charge density (FCD) and high osmotic pressure (π(PG)), and the collagen network (CN) provides the restraining forces to counterbalance π(PG). Our objectives in this work were to: 1), account for collagen intrafibrillar water when transforming biochemical measurements into a FCD-π(PG) relationship; 2), compute π(PG) and CN contributions to the compressive behavior of full-thickness cartilage during bovine growth (fetal, calf, and adult) and human adult aging (young and old); and 3), predict the effect of depth from the articular surface on π(PG) in human aging. Extrafibrillar FCD (FCD(EF)) and π(PG) increased with bovine growth due to an increase in CN concentration, whereas PG concentration was steady. This maturation-related increase was amplified by compression. With normal human aging, FCD(EF) and π(PG) decreased. The π(PG)-values were close to equilibrium stress (σ(EQ)) in all bovine and young human cartilage, but were only approximately half of σ(EQ) in old human cartilage. Depth-related variations in the strain, FCD(EF), π(PG), and CN stress profiles in human cartilage suggested a functional deterioration of the superficial layer with aging. These results suggest the utility of the FCD-π(PG) relationship for elucidating the contribution of matrix macromolecules to the biomechanical properties of cartilage. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

[Research of repairing rabbit knee joint cartilage defect by compound material of fibrin glue and decalcified bone matrix (DBM) and chondrocytes].

Science.gov (United States)

He, Jie; Yang, Xiang; Yue, Peng-ju; Wang, Guan-yu; Guo, Ting; Zhao, Jian-ning

2009-07-01

To investigate the feasibility and effectivity of using compound material of fibrin glue and DBM as scaffolds for cartilage tissue engineering. Chondrocytes isolated from articular cartilage were seeded into prepared scaffolds, after incubation for 4 weeks in vitro. Chondrocytes and fibrin glue and DBM constructs were implanted in the joint cave of rabbit. The specimens were excised at the 4th, 8th, 12th week, examined grossly analyzed by haematoxylin cosine, toluidine blues staining and type II collagen immunohistochemistry reaction. Wakitani score was counted to evaluate the repairing effect. Grossly analysis showed some ivory tissue filled the caves after 4 weeks and the caves were full filled with smooth surface after 12 weeks. The microscope showed a good deal of chondrocytes appeared after 8 weeks and more type II collagen than 4 weeks. Twelve weeks later, cartilage lacuna could be observed. The cells arrangement and the amount of type II collagen both showed the same as the natural one. Complicated material of fibrin glue and DBM as scaffolds can be used as scaffolds for cartilage tissue engineering.

Discovery and development of the N-terminal procollagen type II (NPII) biomarker: a tool for measuring collagen type II synthesis.

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Nemirovskiy, O V; Sunyer, T; Aggarwal, P; Abrams, M; Hellio Le Graverand, M P; Mathews, W R

2008-12-01

Progression of joint damage in osteoarthritis (OA) is likely to result from an imbalance between cartilage degradation and synthesis processes. Markers reflecting these two components appear to be promising in predicting the rate of OA progression. Both N- and C-terminal propeptides of type II collagen reflect the rates of collagen type II synthesis. The ability to quantify the procollagen peptides in biological fluids would enable a better understanding of OA disease pathology and provide means for assessing the proof of mechanism of anabolic disease modifying OA drugs (DMOADs). A polyclonal antibody that recognizes the sequence GPKGQKGEPGDIKDI in the propeptide region of rat, dog, and human type II collagen was raised in chicken and peptide-affinity purified. The immunoaffinity liquid chromatography mass spectrometry (LC-MS/MS) was used to extensively characterize N-terminal procollagen type II (NPII) peptides found in biological fluids. The novel competition enzyme-linked immunosorbent assay (ELISA) assay was developed to quantitatively measure the NPII peptides. Several peptides ranging from 17 to 41 amino acids with various modifications including hydroxylations on proline and lysine residues, oxidation of lysines to allysines, and attachments of glucose and galactose moieties to hydroxylysines were identified in a simple system such as ex vivo cultures of human articular cartilage (HAC) explants as well as in more complex biological fluids such as human urine and plasma. A competitive ELISA assay has been developed and applied to urine, plasma, and synovial fluid matrices in human, rat and dog samples. A novel NPII assay has been developed and applied to OA and normal human subjects to understand the changes in collagen type II synthesis related to the pathology of OA.

Of mice, men and elephants: the relation between articular cartilage thickness and body mass.

Directory of Open Access Journals (Sweden)

Jos Malda

Full Text Available Mammalian articular cartilage serves diverse functions, including shock absorption, force transmission and enabling low-friction joint motion. These challenging requirements are met by the tissue's thickness combined with its highly specific extracellular matrix, consisting of a glycosaminoglycan-interspersed collagen fiber network that provides a unique combination of resilience and high compressive and shear resistance. It is unknown how this critical tissue deals with the challenges posed by increases in body mass. For this study, osteochondral cores were harvested post-mortem from the central sites of both medial and lateral femoral condyles of 58 different mammalian species ranging from 25 g (mouse to 4000 kg (African elephant. Joint size and cartilage thickness were measured and biochemical composition (glycosaminoclycan, collagen and DNA content and collagen cross-links densities were analyzed. Here, we show that cartilage thickness at the femoral condyle in the mammalian species investigated varies between 90 µm and 3000 µm and bears a negative allometric relationship to body mass, unlike the isometric scaling of the skeleton. Cellular density (as determined by DNA content decreases with increasing body mass, but gross biochemical composition is remarkably constant. This however need not affect life-long performance of the tissue in heavier mammals, due to relatively constant static compressive stresses, the zonal organization of the tissue and additional compensation by joint congruence, posture and activity pattern of larger mammals. These findings provide insight in the scaling of articular cartilage thickness with body weight, as well as in cartilage biochemical composition and cellularity across mammalian species. They underscore the need for the use of appropriate in vivo models in translational research aiming at human applications.

Of Mice, Men and Elephants: The Relation between Articular Cartilage Thickness and Body Mass

Science.gov (United States)

Malda, Jos; de Grauw, Janny C.; Benders, Kim E. M.; Kik, Marja J. L.; van de Lest, Chris H. A.; Creemers, Laura B.; Dhert, Wouter J. A.; van Weeren, P. René

2013-01-01

Mammalian articular cartilage serves diverse functions, including shock absorption, force transmission and enabling low-friction joint motion. These challenging requirements are met by the tissue’s thickness combined with its highly specific extracellular matrix, consisting of a glycosaminoglycan-interspersed collagen fiber network that provides a unique combination of resilience and high compressive and shear resistance. It is unknown how this critical tissue deals with the challenges posed by increases in body mass. For this study, osteochondral cores were harvested post-mortem from the central sites of both medial and lateral femoral condyles of 58 different mammalian species ranging from 25 g (mouse) to 4000 kg (African elephant). Joint size and cartilage thickness were measured and biochemical composition (glycosaminoclycan, collagen and DNA content) and collagen cross-links densities were analyzed. Here, we show that cartilage thickness at the femoral condyle in the mammalian species investigated varies between 90 µm and 3000 µm and bears a negative allometric relationship to body mass, unlike the isometric scaling of the skeleton. Cellular density (as determined by DNA content) decreases with increasing body mass, but gross biochemical composition is remarkably constant. This however need not affect life-long performance of the tissue in heavier mammals, due to relatively constant static compressive stresses, the zonal organization of the tissue and additional compensation by joint congruence, posture and activity pattern of larger mammals. These findings provide insight in the scaling of articular cartilage thickness with body weight, as well as in cartilage biochemical composition and cellularity across mammalian species. They underscore the need for the use of appropriate in vivo models in translational research aiming at human applications. PMID:23437402

Quantitative assessment of optical properties in healthy cartilage and repair tissue by optical coherence tomography and histology (Conference Presentation)

Science.gov (United States)

Jansen, Sanne M. A.; Cernohorsky, Paul; de Bruin, Daniel M.; van der Pol, Edwin; Savci-Heijink, Cemile D.; Strackee, Simon D.; Faber, Dirk J.; van Leeuwen, Ton G.

2016-02-01

Quantification of the OCT signal is an important step toward clinical implementation of a diagnostic tool in cartilage imaging. Discrimination of structural cartilage differences in patients with osteoarthritis is critical, yet challenging. This study assesses the variation in the optical attenuation coefficient (μOCT) between healthy cartilage, repair tissue, bone and layers within repair tissue in a controlled setting. OCT and histology was used to assess goat talus articular surfaces in which central osteochondral defects were created. Exact matches of OCT and histology were selected for research. μOCT measurements were taken from healthy cartilage, repair tissue and bone. Measured μOCT in healthy cartilage was higher compared to both repair tissue and bone tissue. Two possible mechanisms for the difference in attenuation were investigated. We studied morphological parameters in terms of nucleus count, nucleus size and inter-nucleus distance. Collagen content in healthy cartilage and repair tissue was assessed using polarization microscopy. Quantitative analysis of the nuclei did not demonstrate a difference in nucleus size and count between healthy cartilage and repair tissue. In healthy cartilage, cells were spaced farther apart and had a lower variation in local nuclear density compared to repair tissue. Polarization microscopy suggested higher collagen content in healthy cartilage compared to repair tissue. μOCT measurements can distinguish between healthy cartilage, repair tissue and bone. Results suggest that cartilage OCT attenuation measurements could be of great impact in clinical diagnostics of osteoarthritis.

Anomalous NMR Relaxation in Cartilage Matrix Components and Native Cartilage: Fractional-Order Models

Science.gov (United States)

Magin, Richard L.; Li, Weiguo; Velasco, M. Pilar; Trujillo, Juan; Reiter, David A.; Morgenstern, Ashley; Spencer, Richard G.

2011-01-01

We present a fractional-order extension of the Bloch equations to describe anomalous NMR relaxation phenomena (T1 and T2). The model has solutions in the form of Mittag-Leffler and stretched exponential functions that generalize conventional exponential relaxation. Such functions have been shown by others to be useful for describing dielectric and viscoelastic relaxation in complex, heterogeneous materials. Here, we apply these fractional-order T1 and T2 relaxation models to experiments performed at 9.4 and 11.7 Tesla on type I collagen gels, chondroitin sulfate mixtures, and to bovine nasal cartilage (BNC), a largely isotropic and homogeneous form of cartilage. The results show that the fractional-order analysis captures important features of NMR relaxation that are typically described by multi-exponential decay models. We find that the T2 relaxation of BNC can be described in a unique way by a single fractional-order parameter (α), in contrast to the lack of uniqueness of multi-exponential fits in the realistic setting of a finite signal-to-noise ratio. No anomalous behavior of T1 was observed in BNC. In the single-component gels, for T2 measurements, increasing the concentration of the largest components of cartilage matrix, collagen and chondroitin sulfate, results in a decrease in α, reflecting a more restricted aqueous environment. The quality of the curve fits obtained using Mittag-Leffler and stretched exponential functions are in some cases superior to those obtained using mono- and bi-exponential models. In both gels and BNC, α appears to account for microstructural complexity in the setting of an altered distribution of relaxation times. This work suggests the utility of fractional-order models to describe T2 NMR relaxation processes in biological tissues. PMID:21498095

Repair of articular cartilage defects by tissue-engineered cartilage constructed with adipose-derived stem cells and acellular cartilaginous matrix in rabbits.

Science.gov (United States)

Wang, Z J; An, R Z; Zhao, J Y; Zhang, Q; Yang, J; Wang, J B; Wen, G Y; Yuan, X H; Qi, X W; Li, S J; Ye, X C

2014-06-18

After injury, inflammation, or degeneration, articular cartilage has limited self-repair ability. We aimed to explore the feasibility of repair of articular cartilage defects with tissue-engineered cartilage constructed by acellular cartilage matrices (ACMs) seeded with adipose-derived stem cells (ADSCs). The ADSCs were isolated from 3-month-old New Zealand albino rabbit by using collagenase and cultured and amplified in vitro. Fresh cartilage isolated from adult New Zealand albino rabbit were freeze-dried for 12 h and treated with Triton X-100, DNase, and RNase to obtain ACMs. ADSCs were seeded in the acellular cartilaginous matrix at 2x10(7)/mL, and cultured in chondrogenic differentiation medium for 2 weeks to construct tissue-engineered cartilage. Twenty-four New Zealand white rabbits were randomly divided into A, B, and C groups. Engineered cartilage was transplanted into cartilage defect position of rabbits in group A, group B obtained ACMs, and group C did not receive any transplants. The rabbits were sacrificed in week 12. The restored tissue was evaluated using macroscopy, histology, immunohistochemistry, and transmission electron microscopy (TEM). In the tissue-engineered cartilage group (group A), articular cartilage defects of the rabbits were filled with chondrocyte-like tissue with smooth surface. Immunohistochemistry showed type II-collagen expression and Alcian blue staining was positive. TEM showed chondrocytes in the recesses, with plenty of secretary matrix particles. In the scaffold group (group B), the defect was filled with fibrous tissue. No repaired tissue was found in the blank group (group C). Tissue-engineered cartilage using ACM seeded with ADSCs can help repair articular cartilage defects in rabbits.

Amino acids supply in culture media is not a limiting factor in the matrix synthesis of engineered cartilage tissue

Science.gov (United States)

Ng, K. W.; DeFrancis, J. G.; Kugler, L. E.; Kelly, T.-A. N.; Ho, M. M.; O’Conor, C. J.; Ateshian, G. A.; Hung, C. T.

2013-01-01

Summary Increased amino acid supplementation (0.5×, 1.0×, and 5.0× recommended concentrations or additional proline) was hypothesized to increase the collagen content in engineered cartilage. No significant differences were found between groups in matrix content or dynamic modulus. Control constructs possessed the highest compressive Young’s modulus on day 42. On day 42, compared to controls, decreased type II collagen was found with 0.5×, 1.0×, and 5.0× supplementation and significantly increased DNA content found in 1.0× and 5.0×. No effects were observed on these measures with added proline. These results lead us to reject our hypothesis and indicate that the low collagen synthesis in engineered cartilage is not due to a limited supply of amino acids in media but may require a further stimulatory signal. The results of this study also highlight the impact that culture environment can play on the development of engineered cartilage. PMID:17713744

Photoactivated methods for enabling cartilage-to-cartilage tissue fixation

Science.gov (United States)

Sitterle, Valerie B.; Roberts, David W.

2003-06-01

The present study investigates whether photoactivated attachment of cartilage can provide a viable method for more effective repair of damaged articular surfaces by providing an alternative to sutures, barbs, or fibrin glues for initial fixation. Unlike artificial materials, biological constructs do not possess the initial strength for press-fitting and are instead sutured or pinned in place, typically inducing even more tissue trauma. A possible alternative involves the application of a photosensitive material, which is then photoactivated with a laser source to attach the implant and host tissues together in either a photothermal or photochemical process. The photothermal version of this method shows potential, but has been almost entirely applied to vascularized tissues. Cartilage, however, exhibits several characteristics that produce appreciable differences between applying and refining these techniques when compared to previous efforts involving vascularized tissues. Preliminary investigations involving photochemical photosensitizers based on singlet oxygen and electron transfer mechanisms are discussed, and characterization of the photodynamic effects on bulk collagen gels as a simplified model system using FTIR is performed. Previous efforts using photothermal welding applied to cartilaginous tissues are reviewed.

PEDF Is Associated with the Termination of Chondrocyte Phenotype and Catabolism of Cartilage Tissue.

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Klinger, P; Lukassen, S; Ferrazzi, F; Ekici, A B; Hotfiel, T; Swoboda, B; Aigner, T; Gelse, K

2017-01-01

Objective. To investigate the expression and target genes of pigment epithelium-derived factor (PEDF) in cartilage and chondrocytes, respectively. Methods. We analyzed the expression pattern of PEDF in different human cartilaginous tissues including articular cartilage, osteophytic cartilage, and fetal epiphyseal and growth plate cartilage, by immunohistochemistry and quantitative real-time (qRT) PCR. Transcriptome analysis after stimulation of human articular chondrocytes with rhPEDF was performed by RNA sequencing (RNA-Seq) and confirmed by qRT-PCR. Results. Immunohistochemically, PEDF could be detected in transient cartilaginous tissue that is prone to undergo endochondral ossification, including epiphyseal cartilage, growth plate cartilage, and osteophytic cartilage. In contrast, PEDF was hardly detected in healthy articular cartilage and in the superficial zone of epiphyses, regions that are characterized by a permanent stable chondrocyte phenotype. RNA-Seq analysis and qRT-PCR demonstrated that rhPEDF significantly induced the expression of a number of matrix-degrading factors including SAA1, MMP1, MMP3, and MMP13. Simultaneously, a number of cartilage-specific genes including COL2A1, COL9A2, COMP, and LECT were among the most significantly downregulated genes. Conclusions. PEDF represents a marker for transient cartilage during all neonatal and postnatal developmental stages and promotes the termination of cartilage tissue by upregulation of matrix-degrading factors and downregulation of cartilage-specific genes. These data provide the basis for novel strategies to stabilize the phenotype of articular cartilage and prevent its degradation.

Relaxin's induction of metalloproteinases is associated with the loss of collagen and glycosaminoglycans in synovial joint fibrocartilaginous explants

Science.gov (United States)

Naqvi, Tabassum; Duong, Trang T; Hashem, Gihan; Shiga, Momotoshi; Zhang, Qin; Kapila, Sunil

2005-01-01

Diseases of specific fibrocartilaginous joints are especially common in women of reproductive age, suggesting that female hormones contribute to their etiopathogenesis. Previously, we showed that relaxin dose-dependently induces matrix metalloproteinase (MMP) expression in isolated joint fibrocartilaginous cells. Here we determined the effects of relaxin with or without β-estradiol on the modulation of MMPs in joint fibrocartilaginous explants, and assessed the contribution of these proteinases to the loss of collagen and glycosaminoglycan (GAG) in this tissue. Fibrocartilaginous discs from temporomandibular joints of female rabbits were cultured in medium alone or in medium containing relaxin (0.1 ng/ml) or β-estradiol (20 ng/ml) or relaxin plus β-estradiol. Additional experiments were done in the presence of the MMP inhibitor GM6001 or its control analog. After 48 hours of culture, the medium was assayed for MMPs and the discs were analyzed for collagen and GAG concentrations. Relaxin and β-estradiol plus relaxin induced the MMPs collagenase-1 and stromelysin-1 in fibrocartilaginous explants – a finding similar to that which we observed in pubic symphysis fibrocartilage, but not in articular cartilage explants. The induction of these proteinases by relaxin or β-estradiol plus relaxin was accompanied by a loss of GAGs and collagen in joint fibrocartilage. None of the hormone treatments altered the synthesis of GAGs, suggesting that the loss of this matrix molecule probably resulted from increased matrix degradation. Indeed, fibrocartilaginous explants cultured in the presence of GM6001 showed an inhibition of relaxin-induced and β-estradiol plus relaxin-induced collagenase and stromelysin activities to control baseline levels that were accompanied by the maintenance of collagen or GAG content at control levels. These findings show for the first time that relaxin has degradative effects on non-reproductive synovial joint fibrocartilaginous tissue and

The non-phagocytic route of collagen uptake

DEFF Research Database (Denmark)

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

2011-01-01

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

Culture of bovine articular chondrocytes in funnel-like collagen-PLGA hybrid sponges

International Nuclear Information System (INIS)

Lu Hongxu; Ko, Young-Gwang; Kawazoe, Naoki; Chen Guoping

2011-01-01

Three-dimensional porous scaffolds play an important role in tissue engineering and regenerative medicine. Structurally, these porous scaffolds should have an open and interconnected porous architecture to facilitate a homogeneous cell distribution. Moreover, the scaffolds should be mechanically strong to support new tissue formation. We developed a novel type of funnel-like collagen sponge using embossing ice particulates as a template. The funnel-like collagen sponges could promote the homogeneous cell distribution, ECM production and chondrogenesis. However, the funnel-like collagen sponges deformed during cell culture due to their weak mechanical strength. To solve this problem, we reinforced the funnel-like collagen sponges with a knitted poly(D,L-lactic-co-glycolic acid) (PLGA) mesh by hybridizing these two types of materials. The hybrid scaffolds were used to culture bovine articular chondrocytes. The cell adhesion, distribution, proliferation and chondrogenesis were investigated. The funnel-like structure promoted the even cell distribution and homogeneous ECM production. The PLGA knitted mesh protected the scaffold from deformation during cell culture. Histological and immunohistochemical staining and cartilaginous gene expression analyses revealed the cartilage-like properties of the cell/scaffold constructs after in vivo implantation. The hybrid scaffold, composed of a funnel-like collagen sponge and PLGA mesh, would be a useful tool for cartilage tissue engineering.

Culture of bovine articular chondrocytes in funnel-like collagen-PLGA hybrid sponges

Energy Technology Data Exchange (ETDEWEB)

Lu Hongxu; Ko, Young-Gwang; Kawazoe, Naoki; Chen Guoping, E-mail: Guoping.Chen@nims.go.jp [Tissue Regeneration Materials Unit, International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044 (Japan)

2011-08-15

Three-dimensional porous scaffolds play an important role in tissue engineering and regenerative medicine. Structurally, these porous scaffolds should have an open and interconnected porous architecture to facilitate a homogeneous cell distribution. Moreover, the scaffolds should be mechanically strong to support new tissue formation. We developed a novel type of funnel-like collagen sponge using embossing ice particulates as a template. The funnel-like collagen sponges could promote the homogeneous cell distribution, ECM production and chondrogenesis. However, the funnel-like collagen sponges deformed during cell culture due to their weak mechanical strength. To solve this problem, we reinforced the funnel-like collagen sponges with a knitted poly(D,L-lactic-co-glycolic acid) (PLGA) mesh by hybridizing these two types of materials. The hybrid scaffolds were used to culture bovine articular chondrocytes. The cell adhesion, distribution, proliferation and chondrogenesis were investigated. The funnel-like structure promoted the even cell distribution and homogeneous ECM production. The PLGA knitted mesh protected the scaffold from deformation during cell culture. Histological and immunohistochemical staining and cartilaginous gene expression analyses revealed the cartilage-like properties of the cell/scaffold constructs after in vivo implantation. The hybrid scaffold, composed of a funnel-like collagen sponge and PLGA mesh, would be a useful tool for cartilage tissue engineering.

Progenitor cells in auricular cartilage demonstrate cartilage-forming capacity in 3D hydrogel culture

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IA Otto

2018-02-01

Full Text Available Paramount for the generation of auricular structures of clinically-relevant size is the acquisition of a large number of cells maintaining an elastic cartilage phenotype, which is the key in producing a tissue capable of withstanding forces subjected to the auricle. Current regenerative medicine strategies utilize chondrocytes from various locations or mesenchymal stromal cells (MSCs. However, the quality of neo-tissues resulting from these cell types is inadequate due to inefficient chondrogenic differentiation and endochondral ossification, respectively. Recently, a subpopulation of stem/progenitor cells has been identified within the auricular cartilage tissue, with similarities to MSCs in terms of proliferative capacity and cell surface biomarkers, but their potential for tissue engineering has not yet been explored. This study compared the in vitro cartilage-forming ability of equine auricular cartilage progenitor cells (AuCPCs, bone marrow-derived MSCs and auricular chondrocytes in gelatin methacryloyl (gelMA-based hydrogels over a period of 56 d, by assessing their ability to undergo chondrogenic differentiation. Neocartilage formation was assessed through gene expression profiling, compression testing, biochemical composition and histology. Similar to MSCs and chondrocytes, AuCPCs displayed a marked ability to generate cartilaginous matrix, although, under the applied culture conditions, MSCs outperformed both cartilage-derived cell types in terms of matrix production and mechanical properties. AuCPCs demonstrated upregulated mRNA expression of elastin, low expression of collagen type X and similar levels of proteoglycan production and mechanical properties as compared to chondrocytes. These results underscored the AuCPCs’ tissue-specific differentiation potential, making them an interesting cell source for the next generation of elastic cartilage tissue-engineered constructs.

CARS hyperspectral imaging of cartilage aiming for state discrimination of cell

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Shiozawa, Manabu; Shirai, Masataka; Izumisawa, Junko; Tanabe, Maiko; Watanabe, Koichi

2016-03-01

Non-invasive cell analyses are increasingly important for medical field. A CARS microscope is one of the non-invasive imaging equipments and enables to obtain images indicating molecular distribution. Some studies on discrimination of cell state by using CARS images of lipid are reported. However, due to low signal intensity, it is still challenging to obtain images of the fingerprint region (800~1800 cm-1), in which many spectrum peaks correspond to compositions of a cell. Here, to identify cell differentiation by using multiplex CARS, we investigated hyperspectral imaging of fingerprint region of living cells. To perform multiplex CARS, we used a prototype of a compact light source, which consists of a microchip laser, a single-mode fiber, and a photonic crystal fiber to generate supercontinuum light. Assuming application to regenerative medicine, we chose a cartilage cell, whose differentiation is difficult to be identified by change of the cell morphology. Because one of the major components of cartilage is collagen, we focused on distribution of proline, which accounts for approximately 20% of collagen in general. The spectrum quality was improved by optical adjustments about power branching ratio and divergence of broadband Stokes light. Hyperspectral images were successfully obtained by the improvement. Periphery of a cartilage cell was highlighted in CARS image of proline, and this result suggests correspondence with collagen generated as extracellular matrix. A possibility of cell analyses by using CARS hyperspectral imaging was indicated.

Sciatic nerve regeneration in rats by a promising electrospun collagen/poly(ε-caprolactone nerve conduit with tailored degradation rate

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Jiang Xinquan

2011-07-01

Full Text Available Abstract Background To cope with the limitations faced by autograft acquisitions particularly for multiple nerve injuries, artificial nerve conduit has been introduced by researchers as a substitute for autologous nerve graft for the easy specification and availability for mass production. In order to best mimic the structures and components of autologous nerve, great efforts have been made to improve the designation of nerve conduits either from materials or fabrication techniques. Electrospinning is an easy and versatile technique that has recently been used to fabricate fibrous tissue-engineered scaffolds which have great similarity to the extracellular matrix on fiber structure. Results In this study we fabricated a collagen/poly(ε-caprolactone (collagen/PCL fibrous scaffold by electrospinning and explored its application as nerve guide substrate or conduit in vitro and in vivo. Material characterizations showed this electrospun composite material which was made of submicron fibers possessed good hydrophilicity and flexibility. In vitro study indicated electrospun collagen/PCL fibrous meshes promoted Schwann cell adhesion, elongation and proliferation. In vivo test showed electrospun collagen/PCL porous nerve conduits successfully supported nerve regeneration through an 8 mm sciatic nerve gap in adult rats, achieving similar electrophysiological and muscle reinnervation results as autografts. Although regenerated nerve fibers were still in a pre-mature stage 4 months postoperatively, the implanted collagen/PCL nerve conduits facilitated more axons regenerating through the conduit lumen and gradually degraded which well matched the nerve regeneration rate. Conclusions All the results demonstrated this collagen/PCL nerve conduit with tailored degradation rate fabricated by electrospinning could be an efficient alternative to autograft for peripheral nerve regeneration research. Due to its advantage of high surface area for cell attachment, it

Biophysical Stimuli: A Review of Electrical and Mechanical Stimulation in Hyaline Cartilage.

Science.gov (United States)

Vaca-González, Juan J; Guevara, Johana M; Moncayo, Miguel A; Castro-Abril, Hector; Hata, Yoshie; Garzón-Alvarado, Diego A

2017-09-01

Objective Hyaline cartilage degenerative pathologies induce morphologic and biomechanical changes resulting in cartilage tissue damage. In pursuit of therapeutic options, electrical and mechanical stimulation have been proposed for improving tissue engineering approaches for cartilage repair. The purpose of this review was to highlight the effect of electrical stimulation and mechanical stimuli in chondrocyte behavior. Design Different information sources and the MEDLINE database were systematically revised to summarize the different contributions for the past 40 years. Results It has been shown that electric stimulation may increase cell proliferation and stimulate the synthesis of molecules associated with the extracellular matrix of the articular cartilage, such as collagen type II, aggrecan and glycosaminoglycans, while mechanical loads trigger anabolic and catabolic responses in chondrocytes. Conclusion The biophysical stimuli can increase cell proliferation and stimulate molecules associated with hyaline cartilage extracellular matrix maintenance.

Micrometer scale guidance of mesenchymal stem cells to form structurally oriented large-scale tissue engineered cartilage.

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Chou, Chih-Ling; Rivera, Alexander L; Williams, Valencia; Welter, Jean F; Mansour, Joseph M; Drazba, Judith A; Sakai, Takao; Baskaran, Harihara

2017-09-15

Current clinical methods to treat articular cartilage lesions provide temporary relief of the symptoms but fail to permanently restore the damaged tissue. Tissue engineering, using mesenchymal stem cells (MSCs) combined with scaffolds and bioactive factors, is viewed as a promising method for repairing cartilage injuries. However, current tissue engineered constructs display inferior mechanical properties compared to native articular cartilage, which could be attributed to the lack of structural organization of the extracellular matrix (ECM) of these engineered constructs in comparison to the highly oriented structure of articular cartilage ECM. We previously showed that we can guide MSCs undergoing chondrogenesis to align using microscale guidance channels on the surface of a two-dimensional (2-D) collagen scaffold, which resulted in the deposition of aligned ECM within the channels and enhanced mechanical properties of the constructs. In this study, we developed a technique to roll 2-D collagen scaffolds containing MSCs within guidance channels in order to produce a large-scale, three-dimensional (3-D) tissue engineered cartilage constructs with enhanced mechanical properties compared to current constructs. After rolling the MSC-scaffold constructs into a 3-D cylindrical structure, the constructs were cultured for 21days under chondrogenic culture conditions. The microstructure architecture and mechanical properties of the constructs were evaluated using imaging and compressive testing. Histology and immunohistochemistry of the constructs showed extensive glycosaminoglycan (GAG) and collagen type II deposition. Second harmonic generation imaging and Picrosirius red staining indicated alignment of neo-collagen fibers within the guidance channels of the constructs. Mechanical testing indicated that constructs containing the guidance channels displayed enhanced compressive properties compared to control constructs without these channels. In conclusion, using a novel

The collagen receptor uPARAP/Endo180

DEFF Research Database (Denmark)

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

2009-01-01

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

Does increased local bone resorption secondary to breast and prostate cancer result in increased cartilage degradation?

DEFF Research Database (Denmark)

Leeming, Diana J; Byrjalsen, Inger; Qvist, Per

2008-01-01

BACKGROUND: Breast and prostate cancer patients often develop lesions of locally high bone turnover, when the primary tumor metastasizes to the bone causing an abnormal high bone resorption at this site. The objective of the present study was to determine whether local increased bone turnover in ...... experiments revealed that osteoclasts released CTXI fragments but not CTXII from bone specimens. The same was observed for cathepsin K. CONCLUSION: Data suggest that an uncoupling between bone resorption and cartilage degradation occurs in breast and lung cancer patient....

Artificial cartilage bio-matrix formed of hyaluronic acid and Mg2+-polyphosphate

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X Wang

2016-11-01

Full Text Available Here we show that inorganic polyphosphate (polyP, a polyanionic metabolic regulator consisting of multiple phosphate residues linked by energy-rich phosphoanhydride bonds, is present in the synovial fluid. In a biomimetic approach, to enhance cartilage synthesis and regeneration, we prepared amorphous polyP microparticles with Mg2+ as counterions. The particles were characterised by X-ray diffraction (XRD, energy-dispersive X-ray (EDX and Fourier transformed infrared spectroscopic (FTIR analyses. Similar particles were obtained after addition of Mg2+ ions to a solution containing hyaluronic acid, as a major component of the synovial fluid, and soluble Na-polyP. The viscous paste-like material formed, composed of globular microparticles with diameter of 400 nm, strongly promoted the adhesion of chondrocytes and caused a significant upregulation of the expression of the genes encoding collagen type 3A1, as a marker for chondrocyte differentiation, and SOX9, a transcription factor that regulates chondrocyte differentiation and proliferation. The expression level of the collagen type 3A1 gene was also enhanced by exposure of chondrocytes to synovial fluid that was found to contain polyP with a size of about 80 phosphate residues. This stimulatory effect was abolished after pre-incubation of the synovial fluid with the polyP degrading alkaline phosphatase. We propose a strategy for treatment of joint dysfunctions caused by osteoarthritis based on the application of amorphous Mg2+-polyP microparticles thatprevent calcium crystal formation in the synovial fluid using scavenging Ca2+ ions (Mg2+/Ca2+ exchange and enhance chondrocyte function after binding of the Ca2+-polyP to hyaluronic acid at the cartilage surface.

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

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Petri Tanska

2013-01-01

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

PHAGOCYTOSIS AND REMODELING OF COLLAGEN MATRICES

OpenAIRE

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

2007-01-01

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

Influence of extremely low frequency, low energy electromagnetic fields and combined mechanical stimulation on chondrocytes in 3-D constructs for cartilage tissue engineering.

Science.gov (United States)

Hilz, Florian M; Ahrens, Philipp; Grad, Sibylle; Stoddart, Martin J; Dahmani, Chiheb; Wilken, Frauke L; Sauerschnig, Martin; Niemeyer, Philipp; Zwingmann, Jörn; Burgkart, Rainer; von Eisenhart-Rothe, Rüdiger; Südkamp, Norbert P; Weyh, Thomas; Imhoff, Andreas B; Alini, Mauro; Salzmann, Gian M

2014-02-01

Articular cartilage, once damaged, has very low regenerative potential. Various experimental approaches have been conducted to enhance chondrogenesis and cartilage maturation. Among those, non-invasive electromagnetic fields have shown their beneficial influence for cartilage regeneration and are widely used for the treatment of non-unions, fractures, avascular necrosis and osteoarthritis. One very well accepted way to promote cartilage maturation is physical stimulation through bioreactors. The aim of this study was the investigation of combined mechanical and electromagnetic stress affecting cartilage cells in vitro. Primary articular chondrocytes from bovine fetlock joints were seeded into three-dimensional (3-D) polyurethane scaffolds and distributed into seven stimulated experimental groups. They either underwent mechanical or electromagnetic stimulation (sinusoidal electromagnetic field of 1 mT, 2 mT, or 3 mT; 60 Hz) or both within a joint-specific bioreactor and a coil system. The scaffold-cell constructs were analyzed for glycosaminoglycan (GAG) and DNA content, histology, and gene expression of collagen-1, collagen-2, aggrecan, cartilage oligomeric matrix protein (COMP), Sox9, proteoglycan-4 (PRG-4), and matrix metalloproteinases (MMP-3 and -13). There were statistically significant differences in GAG/DNA content between the stimulated versus the control group with highest levels in the combined stimulation group. Gene expression was significantly higher for combined stimulation groups versus static control for collagen 2/collagen 1 ratio and lower for MMP-13. Amongst other genes, a more chondrogenic phenotype was noticed in expression patterns for the stimulated groups. To conclude, there is an effect of electromagnetic and mechanical stimulation on chondrocytes seeded in a 3-D scaffold, resulting in improved extracellular matrix production. © 2013 Wiley Periodicals, Inc.

DENTINE CARIES: ACID-TOLERANT MICROORGANISMS AND ASPECTS ON COLLAGEN DEGRADATION.

Science.gov (United States)

Lager, Anders Hedenbjörk

2014-01-01

. Key findings: Each investigated lesion harbored a unique microbiota in terms of both species composition and numbers of microorganisms. This indicates that various combinations of aciduric microorganisms can colonize, survive in and probably also propagate dentine carious lesions. We also found that solid pH-selective agars can be used successfully to select acid-tolerant microorganisms in caries lesions. This would preserve their phenotypic traits for further study. In Paper III, the relation between salivary levels of matrix metalloproteinase-8 (MMP-8), salivary levels of tissue inhibitor of MMP (TIMP-1), and the presence of manifest caries lesions in a large number of subjects was investigated. Saliva samples were collected and analyzed for concentrations of MMP-8, TIMP-1 and total protein using immunofluorometric assays, enzyme linked immunosorbent assays and Bradford assays, respectively. Key findings: Subjects with manifest caries lesions had significantly elevated levels of salivary MMP-8 compared to subjects without caries lesions. TIMP-1 was not significant in any case. In Paper IV, a new method for generating bioactive demineralized dentine matrix substrate (DDM) was developed using a dialysis system and two different demineralization approaches (acetic acid or EDTA). The generated DDM was subsequently analyzed for the presence of type 1 collagen, active MMP-8 and hydroxyproline (HYP) levels using SDS-PAGE, ELISA or immunofluorescence assay. Key findings: Both demineralization methods produced a substrate rich in collagen and with preserved MMP-8 activity. This report presents new knowledge on the composition of the acid tolerant dentine caries microbiota from three levels in dentine carious lesions and on the efficacy of operative caries removal on the numbers of viable microorganisms in the caries free cavity using two operative methods. Moreover, the basic mechanisms behind collagen degradation in the dentine caries process are studied from both a

Enhanced mechanical properties of thermosensitive chitosan hydrogel by silk fibers for cartilage tissue engineering.

Science.gov (United States)

Mirahmadi, Fereshteh; Tafazzoli-Shadpour, Mohammad; Shokrgozar, Mohammad Ali; Bonakdar, Shahin

2013-12-01

Articular cartilage has limited repair capability following traumatic injuries and current methods of treatment remain inefficient. Reconstructing cartilage provides a new way for cartilage repair and natural polymers are often used as scaffold because of their biocompatibility and biofunctionality. In this study, we added degummed chopped silk fibers and electrospun silk fibers to the thermosensitive chitosan/glycerophosphate hydrogels to reinforce two hydrogel constructs which were used as scaffold for hyaline cartilage regeneration. The gelation temperature and gelation time of hydrogel were analyzed by the rheometer and vial tilting method. Mechanical characterization was measured by uniaxial compression, indentation and dynamic mechanical analysis assay. Chondrocytes were then harvested from the knee joint of the New Zealand white rabbits and cultured in constructs. The cell proliferation, viability, production of glycosaminoglycans and collagen type II were assessed. The results showed that mechanical properties of the hydrogel were significantly enhanced when a hybrid with two layers of electrospun silk fibers was made. The results of GAG and collagen type II in cell-seeded scaffolds indicate support of the chondrogenic phenotype for chondrocytes with a significant increase in degummed silk fiber-hydrogel composite for GAG content and in two-layer electrospun fiber-hydrogel composite for Col II. It was concluded that these two modified scaffolds could be employed for cartilage tissue engineering. © 2013.

Tissue Engineering Based Therapy for Articular Cartilage Defects - A New Approach

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Abraham S

2007-01-01

Full Text Available Background: Articular cartilage, the load-bearing tissue in diarthrodial joints, when damaged due to trauma could lead to osteoarthritis. At present Autologous Cartilage Implantation is an established method in which patients own chondrocytes are isolated and then implanted after in vitro expansion over the affected area with bovine or porcine collagen matrix. This procedure results in more of Collagen Type I during in vitro expansion, which eventually becomes fibrocartilage. Also it requires growth factors. We have in this study tried growing human Chondrocytes without growth factors using synthetic scaffolds to grow more Collagen Type II Materials and Methods: Human cartilage specimens were harvested through arthroscopy from the non-weight bearing area of the knee joint from 13 patients who underwent surgical procedures of the knee joint after getting their informed consent. The tissues were transported in saline taking 1 hour to laboratory and subjected to digestion with Collagenase type II for 16~18 Hrs. The chondrocyte cells obtained after dissociation were divided into two groups for culture. Gr. I were embedded in a Thermogelation polymer (TGP and Gr. II in basal culture media (DMEM + Ascorbic Acid without using any growth factors. The Group II cells were viable only for 4 weeks and then started degenerating. The TGP-Chondrocytes scaffolds were grown for 16 weeks and the specimens were harvested at 4, 8, 12 and 16-week intervals and their morphology and molecular characteristics were studied by H&E staining, S-100 protein analysis and RT-PCR.Results: Human chondrocytes could be cultured in both TGP (group I and Basal culture media (group II. The Gr. I cells were viable upto the 16th week while the Group II chondrocytes started degenerating after the 4 week. Both the groups were proven positive for S-100 protein, a Chondrocyte specific marker protein; Gr. II specimens after 4 weeks, and Gr. I specimens after 4, 8, 12 and 16 weeks. RT

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

International Nuclear Information System (INIS)

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

1988-01-01

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

Transcriptomic profiling of cartilage ageing

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Mandy Jayne Peffers

2014-12-01

Full Text Available The musculoskeletal system is severely affected by the ageing process, with many tissues undergoing changes that lead to loss of function and frailty. Articular cartilage is susceptible to age related diseases, such as osteoarthritis. Applying RNA-Seq to young and old equine cartilage, we identified an over-representation of genes with reduced expression relating to extracellular matrix, degradative proteases, matrix synthetic enzymes, cytokines and growth factors in cartilage from older donors. Here we describe the contents and quality controls in detail for the gene expression and related results published by Peffers and colleagues in Arthritis Research and Therapy 2013 associated with the data uploaded to ArrayExpress (E-MTAB-1386.

MR imaging reflects cartilage proteoglycan degradation in the rabbit knee joint

International Nuclear Information System (INIS)

Paul, P.K.; O'Byrne, E.M.; Blancuzzi, V.; Wilson, D.; Douglas, F.L.; Mezrich, R.S.

1989-01-01

Depletion of proteoglycan (PG) from articular cartilage is an early feature of osteoarthritis (OA). Noninvasive assessment of joint morphology corresponding to changes in cartilage PG is crucial for early diagnosis of OA and for demonstration of efficacy of drugs for OA. Intraarticular injection of papain causes a reversible loss of cartilage PG in intact joints. Both knees of NZW rabbits were scanned with a 1.5-T Signa MR imager with a 3-inch surface coil. A spin-echo technique was used, and coronal and sagittal MR images were obtained at 0, 24, 48, and 72 hours after injection of 5 U papain. An 8-cm field of view, a 3-mm section thickness, and a 128 x 256 matrix was used to obtain T1-, proton density-, and T2-weighted images. Cartilage was dissected from the femur for measurement of PG with 1,9-dimethylmethylene blue. Results are presented

Arthrogenicity of type II collagen monoclonal antibodies associated with complement activation and antigen affinity

OpenAIRE

Koobkokkruad, Thongchai; Kadotani, Tatsuya; Hutamekalin, Pilaiwanwadee; Mizutani, Nobuaki; Yoshino, Shin

2011-01-01

Abstract Background The collagen antibody-induced arthritis (CAIA) model, which employs a cocktail of monoclonal antibodies (mAbs) to type II collagen (CII), has been widely used for studying the pathogenesis of autoimmune arthritis. In this model, not all mAbs to CII are capable of inducing arthritis because one of the initial events is the formation of collagen-antibody immune complexes on the cartilage surface or in the synovium, and subsequent activation of the complement by the complexes...

The cartilage-derived, C-type lectin (CLECSF1): structure of the gene and chromosomal location.

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Neame, P J; Tapp, H; Grimm, D R

1999-09-03

Cartilage is a tissue that is primarily extracellular matrix, the bulk of which consists of proteoglycan aggregates constrained within a collagen framework. Candidate components that organize the extracellular assembly of the matrix consist of collagens, proteoglycans and multimeric glycoproteins. We describe the human gene structure of a potential organizing factor, a cartilage-derived member of the C-type lectin superfamily (CLECSF1; C-type lectin superfamily) related to the serum protein, tetranectin. We show by Northern analysis that this protein is restricted to cartilage and locate the gene on chromosome 16q23. We have characterized 10.9 kb of sequence upstream of the first exon. Similarly to human tetranectin, there are three exons. The residues that are conserved between CLECSF1 and tetranectin suggest that the cartilage-derived protein forms a trimeric structure similar to that of tetranectin, with three N-terminal alpha-helical domains aggregating through hydrophobic faces. The globular, C-terminal domain that has been shown to bind carbohydrate in some members of the family and plasminogen in tetranectin, is likely to have a similar overall structure to that of tetranectin.

Type V Collagen is Persistently Altered after Inguinal Hernia Repair

DEFF Research Database (Denmark)

Lorentzen, L; Henriksen, N A; Juhl, P

2018-01-01

BACKGROUND AND AIMS: Hernia formation is associated with alterations of collagen metabolism. Collagen synthesis and degradation cause a systemic release of products, which are measurable in serum. Recently, we reported changes in type V and IV collagen metabolisms in patients with inguinal...... elective cholecystectomy served as controls (n = 10). Whole venous blood was collected 35-55 months after operation. Biomarkers for type V collagen synthesis (Pro-C5) and degradation (C5M) and those for type IV collagen synthesis (P4NP) and degradation (C4M2) were measured by a solid-phase competitive...... assay. RESULTS: The turnover of type V collagen (Pro-C5/C5M) was slightly higher postoperatively when compared to preoperatively in the inguinal hernia group (P = 0.034). In addition, the results revealed a postoperatively lower type V collagen turnover level in the inguinal hernia group compared...

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

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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.

Membrane culture and reduced oxygen tension enhances cartilage matrix formation from equine cord blood mesenchymal stromal cells in vitro.

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Co, C; Vickaryous, M K; Koch, T G

2014-03-01

Ongoing research is aimed at increasing cartilage tissue yield and quality from multipotent mesenchymal stromal cells (MSC) for the purpose of treating cartilage damage in horses. Low oxygen culture has been shown to enhance chondrogenesis, and novel membrane culture has been proposed to increase tissue yield and homogeneity. The objective of this study was to evaluate and compare the effect of reduced oxygen and membrane culture during in vitro chondrogenesis of equine cord blood (CB) MSC. CB-MSC (n = 5 foals) were expanded at 21% oxygen prior to 3-week differentiation in membrane or pellet culture at 5% and 21% oxygen. Assessment included histological examination (H&E, toluidine Blue, immunohistochemistry (IHC) for collagen type I and II), protein quantification by hydroxyproline assay and dimethylmethylene assay, and mRNA analysis for collagen IA1, collagen IIA1, collagen XA1, HIF1α and Sox9. Among treatment groups, 5% membrane culture produced neocartilage most closely resembling hyaline cartilage. Membrane culture resulted in increased wet mass, homogenous matrix morphology and an increase in total collagen content, while 5% oxygen culture resulted in higher GAG and type II collagen content. No significant differences were observed for mRNA analysis. Membrane culture at 5% oxygen produces a comparatively larger amount of higher quality neocartilage. Matrix homogeneity is attributed to a uniform diffusion gradient and reduced surface tension. Membrane culture holds promise for scale-up for therapeutic purposes, for cellular preconditioning prior to cytotherapeutic applications, and for modeling system for gas-dependent chondrogenic differentiation studies. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

μ-PIXE and SAXS studies at the bone-cartilage interface

International Nuclear Information System (INIS)

Kaabar, W.; Gundogdu, O.; Laklouk, A.; Bunk, O.; Pfeiffer, F.; Farquharson, M.J.; Bradley, D.A.

2010-01-01

Micro Proton Induced X-ray Emission (μ-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.

{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.

Changes in the expression of collagen genes show two stages in chondrocyte differentiation in vitro

OpenAIRE

1988-01-01

This report deals with the quantitation of both mRNA and transcription activity of type I collagen gene and of three cartilage-specific collagens (types II, IX, and X) during in vitro differentiation of chick chondrocytes. Differentiation was obtained by transferal to suspension culture of dedifferentiated cells passaged for 3 wk as adherent cells. The type I collagen mRNA, highly represented in the dedifferentiated cells, rapidly decreased during chondrocyte differentiation. On the contrary,...

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

Science.gov (United States)

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

2010-04-01

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

Production and characterization of a monoclonal antibody to chicken type I collagen.

Science.gov (United States)

Linsenmayer, T F; Hendrix, M J; Little, C D

1979-01-01

We have shown that lymphocyte-myeloma cell hybridization can be used to produce large amounts of extremely high-titer specific antibodies against type I collagen, a macromolecule normally of low immunogenicity. In a passive hemagglutination assay the antibody had a high titer against chicken type I collagen but showed no activity against chicken type II or rat type I collagen. By using a two-step fluorescence histochemical procedure on sections of embryonic chicken tibia, strong fluorescence was observed in the perichondrium and surrounding connective tissue (known to contain type I collagen) but not over the cartilage (characterized by type II collagen). When used in conjunction with Staphylococcus aureus as a solid phase immunoadsorbant, the antibody was shown to bind to labeled collagen synthesized in vitro by embryonic chicken calvaria. Images PMID:291035

IGF-1 and PDGF-bb Suppress IL-1β-Induced Cartilage Degradation through Down-Regulation of NF-κB Signaling: Involvement of Src/PI-3K/AKT Pathway

Science.gov (United States)

Mobasheri, Ali; Buhrmann, Constanze; Aldinger, Constance; Rad, Jafar Soleimani; Shakibaei, Mehdi

2011-01-01

Objective Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that plays a key role in the pathogenesis of osteoarthritis (OA). Growth factors (GFs) capable of antagonizing the catabolic actions of cytokines may have therapeutic potential in the treatment of OA. Herein, we investigated the potential synergistic effects of insulin-like growth factor (IGF-1) and platelet-derived growth factor (PDGF-bb) on different mechanisms participating in IL-1β-induced activation of nuclear transcription factor-κB (NF-κB) and apoptosis in chondrocytes. Methods Primary chondrocytes were treated with IL-1β to induce dedifferentiation and co-treated with either IGF-1 or/and PDGF-bb and evaluated by immunoblotting and electron microscopy. Results Pretreatment of chondrocytes with IGF-1 or/and PDGF-bb suppressed IL-1β-induced NF-κB activation via inhibition of IκB-α kinase. Inhibition of IκB-α kinase by GFs led to the suppression of IκB-α phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene products involved in inflammation and cartilage degradation (COX-2, MMPs) and apoptosis (caspase-3). GFs or BMS-345541 (specific inhibitor of the IKK) reversed the IL-1β-induced down-regulation of collagen type II, cartilage specific proteoglycans, β1-integrin, Shc, activated MAPKinase, Sox-9 and up-regulation of active caspase-3. Furthermore, the inhibitory effects of IGF-1 or/and PDGF-bb on IL-1β-induced NF-κB activation were sensitive to inhibitors of Src (PP1), PI-3K (wortmannin) and Akt (SH-5), suggesting that the pathway consisting of non-receptor tyrosine kinase (Src), phosphatidylinositol 3-kinase and protein kinase B must be involved in IL-1β signaling. Conclusion The results presented suggest that IGF-1 and PDGF-bb are potent inhibitors of IL-1β-mediated activation of NF-κB and apoptosis in chondrocytes, may be mediated in part through suppression of Src/PI-3K/AKT pathway, which may contribute to their anti-inflammatory effects. PMID

Deciphering chondrocyte behaviour in matrix-induced autologous chondrocyte implantation to undergo accurate cartilage repair with hyaline matrix.

Science.gov (United States)

Demoor, M; Maneix, L; Ollitrault, D; Legendre, F; Duval, E; Claus, S; Mallein-Gerin, F; Moslemi, S; Boumediene, K; Galera, P

2012-06-01

Since the emergence in the 1990s of the autologous chondrocytes transplantation (ACT) in the treatment of cartilage defects, the technique, corresponding initially to implantation of chondrocytes, previously isolated and amplified in vitro, under a periosteal membrane, has greatly evolved. Indeed, the first generations of ACT showed their limits, with in particular the dedifferentiation of chondrocytes during the monolayer culture, inducing the synthesis of fibroblastic collagens, notably type I collagen to the detriment of type II collagen. Beyond the clinical aspect with its encouraging results, new biological substitutes must be tested to obtain a hyaline neocartilage. Therefore, the use of differentiated chondrocytes phenotypically stabilized is essential for the success of ACT at medium and long-term. That is why researchers try now to develop more reliable culture techniques, using among others, new types of biomaterials and molecules known for their chondrogenic activity, giving rise to the 4th generation of ACT. Other sources of cells, being able to follow chondrogenesis program, are also studied. The success of the cartilage regenerative medicine is based on the phenotypic status of the chondrocyte and on one of its essential component of the cartilage, type II collagen, the expression of which should be supported without induction of type I collagen. The knowledge accumulated by the scientific community and the experience of the clinicians will certainly allow to relief this technological challenge, which influence besides, the validation of such biological substitutes by the sanitary authorities. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Is the repair of articular cartilage lesion by costal chondrocyte transplantation donor age-dependent? An experimental study in rabbits.

Directory of Open Access Journals (Sweden)

Janusz Popko

2006-09-01

Full Text Available The repair of chondral injuries is a very important problem and a subject of many experimental and clinical studies. Different techniques to induce articular cartilage repair are under investigation. In the present study, we have investigated whether the repair of articular cartilage folowing costal chondrocyte transplantation is donor age-dependent. Transplantation of costal chondrocytes from 4- and 24-week old donors, with artificially induced femoral cartilage lesion, was performed on fourteen 20-week-old New Zealand White male rabbits. In the control group, the lesion was left without chondrocyte transplantation. The evaluation of the cartilage repair was performed after 12 weeks of transplantation. We analyzed the macroscopic and histological appearance of the newly formed tissue. Immunohistochemistry was also performed using monoclonal antibodies against rabbit collagen type II. The newly formed tissue had a hyaline-like appearance in most of the lesions after chondrocyte transplantation. Positive immunohistochemical reaction for collagen II was also observed in both groups with transplanted chondrocytes. Cartilage from adult donors required longer isolation time and induced slightly poorer repair. However, hyaline-like cartilage was observed in most specimens from this group, in contrast to the control group, where fibrous connective tissue filled the lesions. Rabbit costal chondrocytes seem to be a potentially useful material for inducing articular cartilage repair and, even more important, they can also be derived from adult, sexually mature animals.

Do sarcomere length, collagen content, pH, intramuscular fat and desmin degradation explain variation in the tenderness of three ovine muscles?

Science.gov (United States)

Starkey, Colin P; Geesink, Geert H; Collins, Damian; Hutton Oddy, V; Hopkins, David L

2016-03-01

The longissimus (n=118) (LL), semimembranosus (n=104) (SM) and biceps femoris (n=134) (BF) muscles were collected from lamb and sheep carcases and aged for 5days (LL and SM) and 14days (BF) to study the impact of muscle characteristics on tenderness as assessed by shear force (SF) and sensory evaluation. The impact of gender, animal age, collagen content, sarcomere length (SL), desmin degradation, ultimate pH and intramuscular fat (IMF) on tenderness was examined. The main factors which influenced SF of the LL were IMF, SL and desmin degradation, but for sensory tenderness, IMF, ultimate pH and gender were the main factors. The SF and sensory tenderness of the SM was best predicted by the degree of desmin degradation. For the BF soluble collagen and animal age both influenced SF. Different factors affect tenderness across muscles and not one prediction model applied across all muscles equally well. Copyright © 2015 Elsevier Ltd. All rights reserved.

The effect of estrogen on the expression of cartilage-specific genes in the chondrogenesis process of adipose-derived stem cells

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Farzaneh Sadeghi

2015-01-01

Full Text Available Background: During adolescence, sex hormones play an important role in regulating proliferation, differentiation, maturation, and the scheduled death of chondrocytes. Although some studies have reported the regulatory role of estrogen in the development and progression of cartilage, some of the mechanisms still remain unclear, including the role of estrogen in the expression of cartilage-specific genes in chondrogenesis process, which we cover in this study. Materials and Methods: In the present study, we used adipose-derived stem cells (ADSCs to differentiate into cartilage. Differentiated cartilage cells were used in the control (without estrogen E2 in the culture medium and experimental (with estrogen in the culture medium groups to evaluate the expression of type II collagen and aggrecan as chondrogenic genes markers, with -real-time polymerase chain reaction technique. Results: Our results indicated that estrogen leads to inhibition of type II collagen gene expression and reduction of aggrecan gene expression. Conclusion: Therefore, estrogen probably has negative effects on chondrogenesis process of ADSCs.

Repair of full-thickness articular cartilage defect using stem cell-encapsulated thermogel.

Science.gov (United States)

Zhang, Yanbo; Zhang, Jin; Chang, Fei; Xu, Weiguo; Ding, Jianxun

2018-07-01

Cartilage defect repair by hydrogel-based tissue engineering is becoming one of the most potential treatment strategies. In this work, a thermogel of triblock copolymer poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PLGA-PEG-PLGA) was prepared as scaffold of bone marrow mesenchymal stem cells (BMMSCs) for repair of full-thickness articular cartilage defect. At first, the copolymer solution showed a reversible sol-gel transition at physiological temperature range, and the mechanical properties of such thermogel were high enough to support the repair of cartilage. Additionally, excellent biodegradability and biocompatibility of the thermogel were demonstrated. By implanting the BMMSC-encapsulated thermogel into the full-thickness articular cartilage defect (5.0 mm in diameter and 4.0 mm in depth) in the rabbit, it was found that the regenerated cartilage integrated well with the surrounding normal cartilage and subchondral bone at 12 weeks post-surgery. The upregulated expression of glycosaminoglycan and type II collagen in the repaired cartilage, and the comparable biomechanical properties with normal cartilage suggested that the cell-encapsulated PLGA-PEG-PLGA thermogel had great potential in serving as the promising scaffold for cartilage regeneration. Copyright © 2018 Elsevier B.V. All rights reserved.

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

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

Science.gov (United States)

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

2017-08-01

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

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

DEFF Research Database (Denmark)

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

2003-01-01

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

In Vivo Evaluation of Biocompatibility and Chondrogenic Potential of a Cell-Free Collagen-Based Scaffold

Directory of Open Access Journals (Sweden)

Giovanna Calabrese

2017-11-01

Full Text Available Injured articular cartilage has a limited innate regenerative capacity, due to the avascular nature and low cellularity of the tissue itself. Although several approaches have been proposed to repair the joint cartilage, none of them has proven to be effective. The absence of suitable therapeutic options has encouraged tissue-engineering approaches combining specific cell types and biomaterials. In the present work, we have evaluated the potential of a cell-free Collagen I-based scaffold to promote the augmentation of cartilage-like phenotype after subcutaneous implantation in the mouse. Forty female mice were grafted subcutaneously with scaffolds, while four additional mice without scaffold were used as negative controls. The effects of scaffold were evaluated at 1, 2, 4, 8, or 16 weeks after implantation. Immunohistochemical analysis shows the expression of typical cartilage markers, including type-II Collagen, Aggrecan, Matrilin-1 and Sox 9. These data are also confirmed by qRT-PCR that further show that both COL2A1 and COL1A1 increase over time, but the first one increases more rapidly, thus suggesting a typical cartilage-like address. Histological analysis shows the presence of some pericellular lacunae, after 8 and 16 weeks. Results suggest that this scaffold (i is biocompatible in vivo, (ii is able to recruit host cells (iii induce chondrogenic differentiation of host cells. Such evidences suggest that this cell-free scaffold is promising and represents a potential approach for cartilage regeneration.

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

NARCIS (Netherlands)

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

1995-01-01

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

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

NARCIS (Netherlands)

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

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

Early Changes of Articular Cartilage and Subchondral Bone in The DMM Mouse Model of Osteoarthritis

OpenAIRE

Fang, Hang; Huang, Lisi; Welch, Ian; Norley, Chris; Holdsworth, David W.; Beier, Frank; Cai, Daozhang

2018-01-01

To examine the early changes of articular cartilage and subchondral bone in the DMM mouse model of osteoarthritis, mice were subjected to DMM or SHAM surgery and sacrificed at 2-, 5- and 10-week post-surgery. Catwalk gait analyses, Micro-Computed Tomography, Toluidine Blue, Picrosirius Red and Tartrate-Resistant Acid Phosphatase (TRAP) staining were used to investigate gait patterns, joint morphology, subchondral bone, cartilage, collagen organization and osteoclasts activity, respectively. R...

Tuning the differentiation of periosteum-derived cartilage using biochemical and mechanical stimulation

NARCIS (Netherlands)

Kock, L.M.; Ravetto, A.; Donkelaar, van C.C.; Foolen, J.; Emans, P.J.; Ito, K.

2010-01-01

OBJECTIVE: In this study, we aim at tuning the differentiation of periosteum in an organ culture model towards cartilage, rich in collagen type II, using combinations of biochemical and mechanical stimuli. We hypothesize that addition of TGF-ß will stimulate chondrogenesis, whereas sliding

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

Science.gov (United States)

Moussa, Mayssam; Lajeunesse, Daniel; Hilal, George; El Atat, Oula; Haykal, Gaby; Serhal, Rim; Chalhoub, Antonio; Khalil, Charbel; Alaaeddine, Nada

2017-03-01

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. 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. 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. These results suggest that PRP could be a potential therapeutic tool for the treatment of OA. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

The effects of different doses of IGF-1 on cartilage and subchondral bone during the repair of full-thickness articular cartilage defects in rabbits.

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Zhang, Z; Li, L; Yang, W; Cao, Y; Shi, Y; Li, X; Zhang, Q

2017-02-01

To investigate the effects of different doses of insulin-like growth factor 1 (IGF-1) on the cartilage layer and subchondral bone (SB) during repair of full-thickness articular cartilage (AC) defects. IGF-1-loaded collagen membrane was implanted into full-thickness AC defects in rabbits. The effects of two different doses of IGF-1 on cartilage layer and SB adjacent to the defect, the cartilage structure, formation and integration, and the new SB formation were evaluated at the 1st, 4th and 8th week postoperation. Meanwhile, after 1 week treatment, the relative mRNA expressions in tissues adjacent to the defect, including cartilage and SB were determined by quantitative real-time RT-PCR (qRT-PCR), respectively. Different doses of IGF-1 induced different gene expression profiles in tissues adjacent to the defect and resulted in different repair outcomes. Particularly, at high dose IGF-1 aided cell survival, regulated the gene expressions in cartilage layer adjacent defect and altered ECM composition more effectively, improved the formation and integrity of neo-cartilage. While, at low dose IGF-1 regulated the gene expressions in SB more efficaciously and subsequently promoted the SB remodeling and reconstruction. Different doses of IGF-1 induced different responses of cartilage or SB during the repair of full-thickness AC defects. Particularly, high dose of IGF-1 was more beneficial to the neo-cartilage formation and integration, while low dose of it was more effective for the SB formation. Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

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

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

1999-08-01

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

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

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

2002-11-01

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

Hydroxychloroquine induces inhibition of collagen type II and oligomeric matrix protein COMP expression in chondrocytes

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Tao Li

2016-06-01

Full Text Available The aim of this study was to investigate the effect of hydroxychloroquine on the level of collagen type II and oligomeric matrix protein COMP expression in chondrocytes of knee osteoarthritis. The rate of growth in cartilage cells was analyzed using MTT assay whereas the Col-2 and COMP expression levels were detected by RT-PCR and Western blotting analyses. For the determination of MMP-13 expression, ELISA test was used. The results revealed no significant change in the rate of cartilage cell proliferation in hydroxychloroquine-treated compared to untreated cells. Hydroxychloro-quine treatment exhibited concentration- and time-dependent effect on the inhibition of collagen type II and COMP expression in chondrocytes. However, its treatment caused a significant enhancement in the expression levels of MMP-13 compared to the untreated cells. Therefore, hydroxychloro-quine promotes expression of MMP-13 and reduces collagen type II and COMP expression levels in chondrocytes without any significant change in the growth of cells.

MMP Mediated Degradation of Type VI Collagen Is Highly Associated with Liver Fibrosis - Identification and Validation of a Novel Biochemical Marker Assay

DEFF Research Database (Denmark)

Veidal, Sanne Skovgard; Karsdal, Morten Asser; Vassiliadis, Efstathios

2011-01-01

Background and Aims: During fibrogenesis, in which excessive remodeling of the extracellular matrix occurs, both the quantity of type VI collagen and levels of matrix metalloproteinases, including MMP-2 and MMP-9, increase significantly. Proteolytic degradation of type VI collagen into small...... fragments, so-called neo-epitopes, may be specific biochemical marker of liver fibrosis. The aim of this study was to develop an ELISA detecting a fragment of type VI collagen generated by MMP-2 and MMP-9, and evaluate this assay in two preclinical models of liver fibrosis. Methods: Mass spectrometric...... analysis of cleaved type VI collagen revealed a large number of protease-generated neo-epitopes. A fragment unique to type VI collagen generated by MMP-2 and MMP-9 was selected for ELISA development. The CO6-MMP assay was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon...

Effects of solid acellular type-I/III collagen biomaterials on in vitro and in vivo chondrogenesis of mesenchymal stem cells.

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Gao, Liang; Orth, Patrick; Cucchiarini, Magali; Madry, Henning

2017-09-01

Type-I/III collagen membranes are advocated for clinical use in articular cartilage repair as being able of inducing chondrogenesis, a technique termed autologous matrix-induced chondrogenesis (AMIC). Area covered: The current in vitro and translational in vivo evidence for chondrogenic effects of solid acellular type-I/III collagen biomaterials. Expert commentary: In vitro, mesenchymal stem cells (MSCs) adhere to the fibers of the type-I/III collagen membrane. No in vitro study provides evidence that a type-I/III collagen matrix alone may induce chondrogenesis. Few in vitro studies compare the effects of type-I and type-II collagen scaffolds on chondrogenesis. Recent investigations suggest better chondrogenesis with type-II collagen scaffolds. A systematic review of the translational in vivo data identified one long-term study showing that covering of cartilage defects treated by microfracture with a type-I/III collagen membrane significantly enhanced the repair tissue volume compared with microfracture alone. Other in vivo evidence is lacking to suggest either improved histological structure or biomechanical function of the repair tissue. Taken together, there is a paucity of in vitro and preclinical in vivo evidence supporting the concept that solid acellular type-I/III collagen scaffolds may be superior to classical approaches to induce in vitro or in vivo chondrogenesis of MSCs.

Complex Determinants in Specific Members of the Mannose Receptor Family Govern Collagen Endocytosis

DEFF Research Database (Denmark)

Jürgensen, Henrik J; Johansson, Kristina; Madsen, Daniel H

2014-01-01

Members of the well-conserved mannose receptor (MR) protein family have been functionally implicated in diverse biological and pathological processes. Importantly, a proposed common function is the internalization of collagen for intracellular degradation occurring during bone development, cancer...... invasion, and fibrosis protection. This functional relationship is suggested by a common endocytic capability and a candidate collagen-binding domain. Here we conducted a comparative investigation of each member's ability to facilitate intracellular collagen degradation. As expected, the family members u......PARAP/Endo180 and MR bound collagens in a purified system and internalized collagens for degradation in cellular settings. In contrast, the remaining family members, PLA2R and DEC-205, showed no collagen binding activity and were unable to mediate collagen internalization. To pinpoint the structural elements...

Mechanical stiffness of TMJ condylar cartilage increases after artificial aging by ribose

NARCIS (Netherlands)

Mirahmadi, F.; Koolstra, J.H.; Lobbezoo, F.; van Lenthe, G.H.; Ghazanfari, S.; Snabel, J.; Stoop, R.; Everts, V.

2018-01-01

Objective: Aging is accompanied by a series of changes in mature tissues that influence their properties and functions. Collagen, as one of the main extracellular components of cartilage, becomes highly crosslinked during aging. In this study, the aim was to examine whether a correlation exists

Biostable scaffolds of polyacrylate polymers implanted in the articular cartilage induce hyaline-like cartilage regeneration in rabbits.

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Sancho-Tello, María; Forriol, Francisco; Martín de Llano, José J; Antolinos-Turpin, Carmen; Gómez-Tejedor, José A; Gómez Ribelles, José L; Carda, Carmen

2017-07-05

To study the influence of scaffold properties on the organization of in vivo cartilage regeneration. Our hypothesis was that stress transmission to the cells seeded inside the pores of the scaffold or surrounding it, which is highly dependent on the scaffold properties, determines the differentiation of both mesenchymal cells and dedifferentiated autologous chondrocytes. 4 series of porous scaffolds made of different polyacrylate polymers, previously seeded with cultured rabbit chondrocytes or without cells, were implanted in cartilage defects in rabbits. Subchondral bone was injured during the surgery to allow blood to reach the implantation site and fill the scaffold pores. At 3 months after implantation, excellent tissue regeneration was obtained, with a well-organized layer of hyaline-like cartilage at the condylar surface in most cases of the hydrophobic or slightly hydrophilic series. The most hydrophilic material induced the poorest regeneration. However, no statistically significant difference was observed between preseeded and non-preseeded scaffolds. All of the materials used were biocompatible, biostable polymers, so, in contrast to some other studies, our results were not perturbed by possible effects attributable to material degradation products or to the loss of scaffold mechanical properties over time due to degradation. Cartilage regeneration depends mainly on the properties of the scaffold, such as stiffness and hydrophilicity, whereas little difference was observed between preseeded and non-preseeded scaffolds.

In vitro chondrogenesis with lysozyme susceptible bacterial cellulose as a scaffold.

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Yadav, Vikas; Sun, Lin; Panilaitis, Bruce; Kaplan, David L

2015-12-01

A current focus of tissue engineering is the use of adult human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes for cartilage repair. Several natural and synthetic polymers (including cellulose) have been explored as a biomaterial scaffold for cartilage tissue engineering. While bacterial cellulose (BC) has been used in tissue engineering, its lack of degradability in vivo and high crystallinity restricts widespread applications in the field. Recently we reported the formation of a novel bacterial cellulose that is lysozyme-susceptible and -degradable in vivo from metabolically engineered Gluconacetobacter xylinus. Here we report the use of this modified bacterial cellulose (MBC) for cartilage tissue engineering using hMSCs. MBC's glucosaminoglycan-like chemistry, combined with in vivo degradability, suggested opportunities to exploit this novel polymer in cartilage tissue engineering. We have observed that, like BC, MBC scaffolds support cell attachment and proliferation. Chondrogenesis of hMSCs in the MBC scaffolds was demonstrated by real-time RT-PCR analysis for cartilage-specific extracellular matrix (ECM) markers (collagen type II, aggrecan and SOX9) as well as histological and immunohistochemical evaluations of cartilage-specific ECM markers. Further, the attachment, proliferation, and differentiation of hMSCs in MBC showed unique characteristics. For example, after 4 weeks of cultivation, the spatial cell arrangement and collagen type-II and ACAN distribution resembled those in native articular cartilage tissue, suggesting promise for these novel in vivo degradable scaffolds for chondrogenesis. Copyright © 2013 John Wiley & Sons, Ltd.

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

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

2010-02-11

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

Postnatal development of depth-dependent collagen density in ovine articular cartilage

NARCIS (Netherlands)

Turnhout, van M.C.; Schipper, H.; Lagen, van B.; Zuilhof, H.; Kranenbarg, S.; Leeuwen, van J.L.

2010-01-01

Articular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Adult AC is characterised by a depth-dependent composition and structure of the extracellular matrix that results in depth-dependent mechanical properties, important for the

Postnatal development of depth-dependent collagen density in ovine articular cartilage

NARCIS (Netherlands)

Turnhout, van M.C.; Schipper, H.; Lagen, van B.; Zuilhof, H.; Kranenbarg, S.; Leeuwen, van J.L.

2010-01-01

Background Articular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Adult AC is characterised by a depth-dependent composition and structure of the extracellular matrix that results in depth-dependent mechanical properties, important for

The effect og aging and mechanical loading on the metabolism og articular cartilage

DEFF Research Database (Denmark)

Jørgensen, Adam El Mongy; Kjær, Michael; Heinemeier, Katja Maria

2017-01-01

Objective. The morphology of articular cartilage (AC) enables painless movement. Aging and mechanical loading are believed to influence development of osteoarthritis (OA), yet the connection remains unclear. Methods. This narrative review describes the current knowledge regarding this area......, with the literature search made on PubMed using appropriate keywords regarding AC, age, and mechanical loading. Results. Following skeletal maturation, chondrocyte numbers decline while increasing senescence occurs. Lower cartilage turnover causes diminished maintenance capacity, which produces accumulation...... collagen network damage and proteoglycan loss, leading to irreversible cartilage destruction because of lack of regenerative capacity. Catabolic pathways involve inflammation and the transcription factor nuclear factor-κB. Thus, age seems to be a predisposing factor for OA, with mechanical overload being...

The Effect of Aging and Mechanical Loading on the Metabolism of Articular Cartilage

DEFF Research Database (Denmark)

Jørgensen, Adam El Mongy; Kjaer, Michael; Heinemeier, Katja Maria

2017-01-01

Objective. The morphology of articular cartilage (AC) enables painless movement. Aging and mechanical loading are believed to influence development of osteoarthritis (OA), yet the connection remains unclear. Methods. This narrative review describes the current knowledge regarding this area......, with the literature search made on PubMed using appropriate keywords regarding AC, age, and mechanical loading. Results. Following skeletal maturation, chondrocyte numbers decline while increasing senescence occurs. Lower cartilage turnover causes diminished maintenance capacity, which produces accumulation...... collagen network damage and proteoglycan loss, leading to irreversible cartilage destruction because of lack of regenerative capacity. Catabolic pathways involve inflammation and the transcription factor nuclear factor-κB. Thus, age seems to be a predisposing factor for OA, with mechanical overload being...

Mechanical confinement regulates cartilage matrix formation by chondrocytes

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Lee, Hong-Pyo; Gu, Luo; Mooney, David J.; Levenston, Marc E.; Chaudhuri, Ovijit

2017-12-01

Cartilage tissue equivalents formed from hydrogels containing chondrocytes could provide a solution for replacing damaged cartilage. Previous approaches have often utilized elastic hydrogels. However, elastic stresses may restrict cartilage matrix formation and alter the chondrocyte phenotype. Here we investigated the use of viscoelastic hydrogels, in which stresses are relaxed over time and which exhibit creep, for three-dimensional (3D) culture of chondrocytes. We found that faster relaxation promoted a striking increase in the volume of interconnected cartilage matrix formed by chondrocytes. In slower relaxing gels, restriction of cell volume expansion by elastic stresses led to increased secretion of IL-1β, which in turn drove strong up-regulation of genes associated with cartilage degradation and cell death. As no cell-adhesion ligands are presented by the hydrogels, these results reveal cell sensing of cell volume confinement as an adhesion-independent mechanism of mechanotransduction in 3D culture, and highlight stress relaxation as a key design parameter for cartilage tissue engineering.

Computer-aided cartilage tissue-engineering : a numerical evaluation of the influence of inhomogeneities, collagen architecture and temporal culture effects

NARCIS (Netherlands)

Khoshgoftar, M.

2012-01-01

Hyaline articular cartilage has a crucial role in the distribution of joint mechanical loads and smooth movement of bones. Because of its poor healing capacity, cartilage damage is progressive and may lead to osteoarthritis (OA). Replacing damaged cartilage with tissue engineered (TE) cartilage is

Tissue engineering applications: cartilage lesions repair by the use of autologous chondrocytes

Directory of Open Access Journals (Sweden)

L. De Franceschi

2011-09-01

Full Text Available Promising new therapies based on tissue engineering have been recently developed for cartilage repair. The association of biomaterials with autologous chondrocytes expanded in vitro can represent a useful tool to regenerate this tissue. The scaffolds utilised in such therapeutical applications should provide a pre-formed three-dimensional shape, prevent cells from floating out of the defect, have sufficient mechanical strength, facilitate uniform spread of cells and stimulate the phenotype of transplanted cells. Hyaff®-11 is a hyaluronic-acid based biodegradable polymer, that has been shown to provide successful cell carrier for tissue-engineered repair. From our findings we can state that human chondrocytes seeded on Hyaff®-11 are able to maintain in vitro the characteristic of differentiated cells, expressing and producing collagen type II and aggrecan which are the main markers of cartilage phenotype, down-regulating collagen type I. Moreover, it seems to be a useful scaffold for cartilage repair both in animal models and clinical trials in humans, favouring the formation of a hyaline-like tissue. In the light of these data, we can hypothesise, for the future, the use of autologous chondrocyte transplantation together with gene therapy as a treatment for rheumatic diseases such as osteoarthritis.

The stimulation of mononuclear cells from patients with rheumatoid arthritis to degrade articular cartilage is not modulated by cartilage itself

NARCIS (Netherlands)

van Roon, J. A.; van Roy, J. L.; Lafeber, F. P.; Bijlsma, J. W.

1996-01-01

To study the modulation of mononuclear cell (MNC) activity in patients with rheumatoid arthritis (RA) by constituents released from human articular cartilage, which may be present in vivo during early events of the disease, when articular cartilage is not only mildly damaged. In an attempt to

Imaging Prostate Cancer Microenvironment by Collagen Hybridization

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2016-12-01

of collagen II remodeling in Rheumatoid arthritis and other cartilage-related diseases or wound repair. We did observe trends in the CMP...proteins in vitro and in vivo has been prepared and submitted to Molecular Pharmaceutics . What do you plan to do during the next reporting period to...or care of human subjects, vertebrate animals, biohazards, and/or select agents Nothing to report. PRODUCTS Journal publications: Lucas L