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Myron Spector - One of the best experts on this subject based on the ideXlab platform.
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effects of fgf 2 and igf 1 on adult canine articular chondrocytes in Type II Collagen glycosaminoglycan scaffolds in vitro
Osteoarthritis and Cartilage, 2005Co-Authors: N Veilleux, Myron SpectorAbstract:Summary Objective Chondrocyte-seeded tissue engineering scaffolds hold the promise of enhancing certain cartilage repair procedures. The objective of this study was to evaluate the effects of selected growth factors [fibroblast growth factor (FGF)-2 and insulin-like growth factor (IGF)-1] individually and in combination on adult canine articular chondrocyte-seeded Type II Collagen–glycosaminoglycan (GAG) scaffolds grown in serum-free (SF) medium. Design Approximately 500,000 second passage chondrocytes were seeded into discs of the scaffold, 4mm diameter×2mm thick. The constructs were grown in the following media: serum-containing medium; a basal SF medium; SF with 5ng/ml FGF-2; SF with 25ng/ml FGF-2; SF with 100ng/ml IGF-1; and SF with 5ng/ml FGF-2 plus 100ng/ml IGF-1. The DNA and GAG contents of the scaffolds were determined after 1 day and 2 weeks and the protein and GAG synthesis rates determined at 2 weeks using radiolabels. Histology and Type II Collagen immunohistochemistry were also performed. Results FGF-2 at 5ng/ml was found to substantially increase the biosynthetic activity of the cells and the accumulation of GAG. The histology demonstrated chondrocytes uniformly distributed through a matrix that stained intensely for GAG and Type II Collagen after only 2 weeks. Of interest were the rapid degradation of the Collagen scaffold, despite the fact that the scaffold was carbodIImide cross-linked, and the contraction of the constructs. There were less pronounced effects using the higher dose of FGF-2 and the combination with IGF-1. Conclusions Chondrocyte-seeded Type II Collagen scaffolds cultured in SF medium supplemented with 5ng/ml FGF-2 undergo contraction, demonstrate an increase in construct incorporation of radiolabeled sulfate, and display qualitative signs of chondrogenesis.
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biosynthetic response of passaged chondrocytes in a Type II Collagen scaffold to mechanical compression
Journal of Biomedical Materials Research Part A, 2003Co-Authors: Cynthia R Lee, Alan J Grodzinsky, Myron SpectorAbstract:To investigate the potential utility of mechanical loading in articular cartilage tissue engineering, porous Type II Collagen scaffolds seeded with adult canine passaged chondrocytes were subjected to static and dynamic compressions of varying magnitudes (0-50% static strain) and durations (1-24 h), and at different times during culture (2-30 days postseeding). The effects of mechanical compression on the biosynthetic activity of the chondrocytes were evaluated by measuring the amount of (3)H-proline-labeled proteins and (35)S-sulfate-labeled proteoglycans that accumulated in the cell-scaffold construct and was released to the medium during the loading period. Similar to published results on loading of articular cartilage explants, static compression decreased protein and proteoglycan biosynthesis in a time- and dose-dependent manner (each p < 0.005), and selected dynamic compression protocols were able to increase rates of biosynthesis (p < 0.05). The main difference between the results seen for this tissue engineering system and cartilage explants was in the amount of newly synthesized matrix molecules that accumulated within the construct under dynamic loading, with less accumulating in the Type II Collagen scaffold. In summary, the general biosynthetic response of passaged chondrocytes in the porous Type II Collagen scaffolds is similar to that seen for chondrocytes in their native environment. Future work needs to be directed to modifications of the cell-seeded construct to allow for the capture of the newly synthesized matrix molecules by the scaffold.
Robin A Poole - One of the best experts on this subject based on the ideXlab platform.
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increased Type II Collagen cleavage by cathepsin k and Collagenase activities with aging and osteoarthritis in human articular cartilage
Arthritis Research & Therapy, 2012Co-Authors: Valeria M Dejica, Sheila Laverty, John S Mort, John Antoniou, David J Zukor, Michael Tanzer, Robin A PooleAbstract:The intra-helical cleavage of Type II Collagen by proteases, including Collagenases and cathepsin K, is increased with aging and osteoarthritis (OA) in cartilage as determined by immunochemical assays. The distinct sites of Collagen cleavage generated by Collagenases and cathepsin K in healthy and OA human femoral condylar cartilages were identified and compared. Fixed frozen cartilage sections were examined immunohistochemically, using antibodies that react with the Collagenase-generated cleavage neoepitopes, C2C and C1,2C, and the primary cleavage neoepitope (C2K) generated in Type II Collagen by the action of cathepsin K and possibly by other proteases, but not by any Collagenases studied to date. In most cases, the staining patterns for Collagen cleavage were similar for all three epitopes: weak to moderate mainly pericellular staining in non-OA cartilage from younger individuals and stronger, more widespread staining in aging and OA cartilages that often extended from the superficial to the mid/deep zone of the tissue. In very degenerate OA specimens, with significant disruption of the articular surface, staining was distributed throughout most of the cartilage matrix. Cleavage of Collagen by proteases usually arises pericellularly around chondrocytes at and near the articular surface, subsequently becoming more intense and extending progressively deeper into the cartilage with aging and OA. The close correspondence between the distributions of these products suggests that both Collagenases and cathepsin K, and other proteases that may generate this distinct cathepsin K cleavage site, are usually active in the same sites in the degradation of Type II Collagen.
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cleavage of Type II Collagen by cathepsin k in human osteoarthritic cartilage
American Journal of Pathology, 2008Co-Authors: Valeria M Dejica, Sheila Laverty, John S Mort, David M Percival, John Antoniou, David J Zukor, Robin A PooleAbstract:Cathepsin K is a cysteine protease of the papain family that cleaves triple-helical Type II Collagen, the major structural component of the extracellular matrix of articular cartilage. In osteoarthritis (OA), the anabolic/catabolic balance of articular cartilage is disrupted with the excessive cleavage of Collagen II by Collagenases or matrix metalloproteinases. A polyclonal antibody against a C-terminal neoepitope (C2K) generated in triple-helical Type II Collagen by the proteolytic action of cathepsin K was prepared and used to develop an enzyme-linked immunosorbent assay to study the generation of this epitope and the effects of its presence in normal adult and osteoarthritic femoral condylar articular cartilage. The generation of the C2K epitope in explant culture and the effect of a specific cathepsin K inhibitor were studied. The neoepitope, which is not generated by the Collagenase matrix metalloproteinase-13, increased with age in articular cartilage and was significantly elevated in osteoarthritic cartilage compared with adult nonarthritic cartilage. Moreover, in explants from three of eight OA patients, the generation of the neoepitope in culture was significantly reduced by a specific, nontoxic inhibitor of cathepsin K. These data suggest that cathepsin K is involved in the cleavage of Type II Collagen in human articular cartilage in certain OA patients and that it may play a role in both OA pathophysiology and the aging process.
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differences in Type II Collagen degradation between peripheral and central cartilage of rat stifle joints after cranial cruciate ligament transection
Arthritis & Rheumatism, 2000Co-Authors: Reinout Stoop, Peter M Van Der Kraan, Anthony Peter Hollander, Clark R Billinghurst, Robin A Poole, Pieter Buma, Wim B Van Den BergAbstract:Objective. Type II Collagen degradation is thought to be the key process in cartilage degradation during the development of osteoarthritis (OA). In this study, we investigated the kinetics of Type II Collagen degradation during surgically induced OA. Methods. Experimental OA was induced in male Wistar rats by transecting the cranial (anterior) cruciate ligament (CCL). Hematoxylin and eosin staining was used to study overall cartilage degradation, while immunostained sections were used to demonstrate denatured Type II Collagen (Col2-3/4m antibody) and the Collagenase cleavage site in Type II Collagen (Col2-3/ 4Cshort antibody). Results. During the first 3‐4 weeks, cartilage destruction, associated with chondrocyte death, proteoglycan depletion, and a marked increase in the Collagenase cleavage neoepitope, was mainly located at the margins of the cartilage. From weeks 3‐4, the central part of the cartilage showed increased surface fibrillation and apparent chondrocyte death. In these areas, increased denatured Type II Collagen staining but little cleavage-site staining was present. Conclusion. These results indicate that cartilage degradation after CCL transection in the rat consists of 2 phases. An early phase located at the cartilage margins and a late phase located at the central part of the cartilage. In the early phase, Collagenase-dependent cartilage damage occurred. During the late phase, the level of Type II Collagen denaturation increased.
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comparison of the degradation of Type II Collagen and proteoglycan in nasal and articular cartilages induced by interleukin 1 and the selective inhibition of Type II Collagen cleavage by Collagenase
Arthritis & Rheumatism, 2000Co-Authors: Clark R Billinghurst, William Wu, Mirela Ionescu, Leif Dahlberg, Harold E Van Wart, Jeffrey Chen, A Reiner, Robin A PooleAbstract:OBJECTIVE: To compare interleukin-1alpha (IL-1alpha)-induced degradation of nasal and articular cartilages in terms of proteoglycan loss and Type II Collagen cleavage, denaturation, and release; to examine the temporal relationship of these changes; and to investigate the effects of an inhibitor of Collagenase 2 and Collagenase 3 on these catabolic processes. METHODS: Discs of mature bovine nasal and articular cartilages were cultured with or without human IL-1alpha (5 ng/ml) with or without RS102,481, a selective synthetic inhibitor of Collagenase 2 and Collagenase 3 (matrix metalloproteinase 8 [MMP-8] and MMP-13, respectively) but not of Collagenase 1 (MMP-1). Immunoassays were used to measure Collagenase-generated Type II Collagen cleavage neoepitope (antibody COL2-3/4C(short)) and denaturation (antibody COL2-3/4m), as well as total Type II Collagen content (antibody COL2-3/4m) in articular cartilage and culture media. A colorimetric assay was used to measure total proteoglycan concentration (principally of aggrecan) as sulfated glycosaminoglycans (sGAG). RESULTS: IL-1alpha initially induced a decrease in tissue proteoglycan content in nasal cartilage. A progressive loss of proteoglycan was noted during culture in articular cartilages, irrespective of the presence of IL-1alpha. In both cartilages, proteoglycan loss was followed by IL-1alpha-induced cleavage of Type II Collagen by Collagenase, which was often reflected by increased denaturation. The inhibitor RS102,481 had no clear effect on the reduction in proteoglycan content (measured by sGAG) and Collagen denaturation in either cartilage, but at 10 nM it inhibited the enhanced cleavage of Type II Collagen, partially in nasal cartilage and completely in articular cartilage. CONCLUSION: IL-1alpha-induced cleavage and denaturation of Type II Collagen is observed in both hyaline cartilages and is secondary to proteoglycan loss. It probably involves different Collagenases, since there is no evidence of a rate-limiting role for Collagenase 1 in articular cartilage, unlike the case for nasal cartilage. Inhibitors of this kind may be of value in the treatment of cartilage damage in arthritis. Also, the ability to detect the release of Type II Collagen Collagenase-generated fragments from degraded cartilage offers the potential to monitor cartilage Collagen damage and its control in vivo.
Linda J Sandell - One of the best experts on this subject based on the ideXlab platform.
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cartilage derived retinoic acid sensitive protein and Type II Collagen expression during fracture healing are potential targets for sox9 regulation
Journal of Bone and Mineral Research, 1999Co-Authors: Shinji Sakano, Yong Zhu, Linda J SandellAbstract:Cartilage-derived retinoic acid-sensitive protein (CD-RAP) and mRNA were examined in the mouse fracture model by immunohistochemistry and Northern blot analysis and compared with the expression of Type II Collagen. We also studied the expression of the transcription factor Sox9, reported to enhance Type II Collagen and CD-RAP gene expression in vitro. CD-RAP was first detected in immature chondrocytes on day 5. Intense signals for CD-RAP were found in fracture cartilage on days 7 and 9. CD-RAP decreased at the phase of endochondral ossification. Throughout fracture healing, CD-RAP was detected in cartilage and not in bone or fibrous tissue, thus CD-RAP may be a molecular marker of cartilage formation during fracture healing. Northern blot analysis revealed similar changes in CD-RAP and Type II Collagen mRNA levels. However, with respect to protein levels, CD-RAP decreased faster than Type II Collagen implying the stability is lower than Type II Collagen. Increased levels of Sox9 mRNA and protein were detected on day 5 and coincided with the initial increase of CD-RAP and Type II Collagen mRNAs. Sox9 mRNA levels declined with the progress of chondrocyte hypertrophy, followed by a concomitant decrease in CD-RAP and Type II Collagen mRNA levels. These changes in Sox9 expression compared with the cartilage-specific genes (CD-RAP and Type II Collagen) suggest that cell differentiation during fracture healing may be controlled by specific transcriptional factors which regulate phenotypic changes of the cells.
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transcriptional suppression by interleukin 1 and interferon γ of Type II Collagen gene expression in human chondrocytes
Journal of Cellular Biochemistry, 1994Co-Authors: Mary B Goldring, Keisuke Fukuo, James R Birkhead, Edward Dudek, Linda J SandellAbstract:Type II Collagen is one of the predominant extracellular matrix macromolecules in cartilage responsible for maintenance of integrity of this specialized tissue. We showed previously that interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) are capable of decreasing the levels of alpha 1(II) proCollagen mRNA and suppressing the synthesis of Type II Collagen in cultured human chondrocytes. Data reported here show that these effects of IL-1 and IFN-gamma on the expression of the human Type II Collagen gene (COL2A1) are mediated primarily at the transcriptional level. This conclusion is based on three Types of experimental evidence: (1) in nuclear run-off assays, preincubation of chondrocytes with either IL-1 or IFN-gamma decreased COL2A1 transcription; (2) experiments with the protein synthesis inhibitor cycloheximide and the transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) indicated that the suppression of alpha 1(II) proCollagen mRNA by IL-1 could not be ascribed to decreased mRNA stability; and (3) a plasmid (pCAT-B/4.0) containing 4.0 kb of 5'-flanking sequences of COL2A1 (-577/+3428), encompassing the promoter, exon 1 and the putative enhancer sequence in the first intron, linked to the chloramphenicol acetyltransferase (CAT) reporter gene, was transfected in human chondrocytes. A high level of expression of pCAT-B/4.0 was observed in human chondrocytes incubated with an insulin-containing serum substitute that is permissive for expression of the COL2A1 gene. Expression of pCAT-B/4.0 in these cells was inhibited by either IL-1 or IFN-gamma. Furthermore, expression of pCAT-B/4.0 was not detected in human dermal fibroblasts. When the putative enhancer fragment in the first intron was removed, the expression in chondrocytes was greatly reduced. These studies demonstrate that expression of COL2A1 is tissue specific and that suppression by either IL-1 or IFN-gamma is mediated primarily at the transcriptional level.
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transcriptional suppression by interleukin 1 and interferon γ of Type II Collagen gene expression in human chondrocytes
Journal of Cellular Biochemistry, 1994Co-Authors: Mary B Goldring, Keisuke Fukuo, James R Birkhead, Edward Dudek, Linda J SandellAbstract:Type II Collagen is one of the predominant extracellular matrix macromolecules in cartilage responsible for maintenance of integrity of this specialized tissue. We showed previously that interleukin-1 (IL-1) and interferon-γ (IFN-γ) are capable of decreasing the levels of α1 (II) proCollagen mRNA and suppressing the synthesis of Type II Collagen in cultured human chondrocytes. Data reported here show that these effects of IL-1 and IFN-γ on the expression of the human Type II Collagen gene (COL2A1) are mediated primarily at the transcriptional level. This conclusion is based on three Types of experimental evidence: (1) in nuclear run-off assays, preincubation of chondrocytes with either IL-1 or IFN-γ decreased COL2A1 transcription; (2) experiments with the protein synthesis inhibitor cycloheximide and the transcriptional inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) indicated that the suppression of α1 (II) proCollagen mRNA by IL-1 could not be ascribed to decreased mRNA stability; and (3) a plasmid (pCAT-B/4.0) containing 4.0 kb of 5′-flanking sequences of COL2A1 (−577/+3428), encompassing the promoter, exon 1 and the putative enhancer sequence in the first intron, linked to the chloramphenicol acetyltransferase (CAT) reporter gene, was transfected in human chondrocytes. A high level of expression of pCAT-B/4.0 was observed in human chondrocytes incubated with an insulin-containing serum substitute that is permissive for expression of the COL2A1 gene. Expression of pCAT-B/4.0 in these cells was inhibited by either IL-1 or IFN-γ. Furthermore, expression of pCAT-B/4.0 was not detected in human dermal fibroblasts. When the putative enhancer fragment in the first intron was removed, the expression in chondrocytes was greatly reduced. These studies demonstrate that expression of COL2A1 is tissue specific and that suppression by either IL-1 or IFN-γ is mediated primarily at the transcriptional level.
Pieter Buma - One of the best experts on this subject based on the ideXlab platform.
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Crosslinked Type II Collagen matrices: preparation, characterization, and potential for cartilage engineering.
Biomaterials, 2002Co-Authors: Jeroen Pieper, Joris Van Der Kamp, Theo Hafmans, W.b. Van Den Berg, Peter M Van Der Kraan, Job L. C. Van Susante, Jacques H. Veerkamp, Pieter Buma, A.h.m.s.m. Van KuppeveltAbstract:The limited intrinsic repair capacity of articular cartilage has stimulated continuing efforts to develop tissue engineered analogues. Matrices composed of Type II Collagen and chondroitin sulfate (CS), the major constituents of hyaline cartilage, may create an appropriate environment for the generation of cartilage-like tissue. In this study, we prepared, characterized, and evaluated Type II Collagen matrices with and without CS. Type II Collagen matrices were prepared using purified, pepsin-treated, Type II Collagen. Techniques applied to prepare Type I Collagen matrices were found unsuitable for Type II Collagen. Crosslinking of Collagen and covalent attachment of CS was performed using 1-ethyl-3-(3-dimethyl aminopropyl)carbodIImide. Porous matrices were prepared by freezing and lyophilization, and their physico-chemical characteristics (degree of crosslinking, denaturing temperature, Collagenase-resistance, amount of CS incorporated) established. Matrices were evaluated for their capacity to sustain chondrocyte proliferation and differentiation in vitro. After 7 d of culture, chondrocytes were mainly located at the periphery of the matrices. In contrast to Type I Collagen, Type II Collagen supported the distribution of cells throughout the matrix. After 14 d of culture, matrices were surfaced with a cartilagenous-like layer, and occasionally clusters of chondrocytes were present inside the matrix. Chondrocytes proliferated and differentiated as indicated by biochemical analyses, ultrastructural observations, and reverse transcriptase PCR for Collagen Types I, II and X. No major differences were observed with respect to the presence or absence of CS in the matrices.
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Type II Collagen degradation in articular cartilage fibrillation after anterior cruciate ligament transection in rats
Osteoarthritis and Cartilage, 2001Co-Authors: Reinout Stoop, Clark R Billinghurst, A P Hollander, A R Poole, Pieter Buma, T.h.m. Meijers, P M Van Der Kraan, W.b. Van Den BergAbstract:OBJECTIVE: To investigate the kinetics of early cartilage changes in mechanically induced osteoarthritis (OA) and the association of these changes with damage to the Type II Collagen network. METHODS: Experimental OA was induced by anterior cruciate ligament transsection in the rat knee joint (ACLT-OA). Animals were sacrificed after 2, 7, 14, 28 and 70 days. Knee joints were evaluated using routine histology and immunohistochemistry for denatured (unwound) Type II Collagen to detect Collagen damage. An antibody recognizing the Collagenase cleavage site in Type II Collagen was used to study the role of Collagenase in this process. RESULTS: The first changes of the articular cartilage after anterior cruciate ligament transection occurred in the superficial zone. These changes included loss of superficial chondrocytes, swelling of the remaining chondrocytes and superficial fibrillation. The swelling of the chondrocytes did not result from a change towards the hypertrophic phenoType, since these cells did not stain for Type X Collagen. A marked increase in denatured Type II Collagen staining was present in the fibrillated areas. Staining of the Collagenase cleavage site showed the same distribution as denatured Collagen but was clearly less intense. Collagen damage could never be detected before fibrillation occurred and was not present in non-fibrillated areas. CONCLUSIONS: These results indicate that in this model cartilage degeneration starts at the articular surface and that this degeneration is associated with a localized expression of Type II Collagen degradation products.
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differences in Type II Collagen degradation between peripheral and central cartilage of rat stifle joints after cranial cruciate ligament transection
Arthritis & Rheumatism, 2000Co-Authors: Reinout Stoop, Peter M Van Der Kraan, Anthony Peter Hollander, Clark R Billinghurst, Robin A Poole, Pieter Buma, Wim B Van Den BergAbstract:Objective. Type II Collagen degradation is thought to be the key process in cartilage degradation during the development of osteoarthritis (OA). In this study, we investigated the kinetics of Type II Collagen degradation during surgically induced OA. Methods. Experimental OA was induced in male Wistar rats by transecting the cranial (anterior) cruciate ligament (CCL). Hematoxylin and eosin staining was used to study overall cartilage degradation, while immunostained sections were used to demonstrate denatured Type II Collagen (Col2-3/4m antibody) and the Collagenase cleavage site in Type II Collagen (Col2-3/ 4Cshort antibody). Results. During the first 3‐4 weeks, cartilage destruction, associated with chondrocyte death, proteoglycan depletion, and a marked increase in the Collagenase cleavage neoepitope, was mainly located at the margins of the cartilage. From weeks 3‐4, the central part of the cartilage showed increased surface fibrillation and apparent chondrocyte death. In these areas, increased denatured Type II Collagen staining but little cleavage-site staining was present. Conclusion. These results indicate that cartilage degradation after CCL transection in the rat consists of 2 phases. An early phase located at the cartilage margins and a late phase located at the central part of the cartilage. In the early phase, Collagenase-dependent cartilage damage occurred. During the late phase, the level of Type II Collagen denaturation increased.
Reinout Stoop - One of the best experts on this subject based on the ideXlab platform.
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bone morphogenetic protein bmp 2 enhances the expression of Type II Collagen and aggrecan in chondrocytes embedded in alginate beads
Osteoarthritis and Cartilage, 2004Co-Authors: Tatiana Grunder, Reinout Stoop, Christoph Gaissmaier, Jurgen Fritz, Peter Hortschansky, Jurgen Mollenhauer, Wilhelm K AicherAbstract:Abstract Objective For autologous chondrocyte transplantation (ACT) chondrocytes are expanded in vitro . During expansion these cells may dedifferentiate. This change in phenoType is characterized by a raised expression of Type I Collagen and a decrease in Type II Collagen expression. Since high expression of Type II Collagen is of central importance for the properties of hyaline cartilage, we investigated if the growth factor bone morphogenetic protein-2 (BMP-2) may modulate the chondrogenic phenoType in monolayer cell cultures and in three-dimensional culture systems. Design Chondrocytes from articular knee cartilage of 11 individuals (average age: 39.8 years) with no history of joint disease were isolated and seeded either in monolayer cultures or embedded in alginate beads in presence or absence of human recombinant BMP-2 (hr-BMP-2). Then, cells were harvested and analysis of the chondrogenic phenoType was performed using quantitative RT-PCR, immunocytochemistry and ELISA. Results Addition of BMP-2 to chondrocytes expanded in two-dimensional (2D) cultures during the first subculture (P1) had no effect on mRNA amounts encoding Type II Collagen and interleukin-1β (IL-1β). In contrast, seeding chondrocytes in three-dimensional (3D) alginate cultures raised Type II Collagen expression significantly and addition of BMP-2 enhanced this effect. Conclusions We conclude that chondrocytes during expansion for ACT may benefit from BMP-2 activation only when seeded in an appropriate 3D culture system.
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Type II Collagen degradation in articular cartilage fibrillation after anterior cruciate ligament transection in rats
Osteoarthritis and Cartilage, 2001Co-Authors: Reinout Stoop, Clark R Billinghurst, A P Hollander, A R Poole, Pieter Buma, T.h.m. Meijers, P M Van Der Kraan, W.b. Van Den BergAbstract:OBJECTIVE: To investigate the kinetics of early cartilage changes in mechanically induced osteoarthritis (OA) and the association of these changes with damage to the Type II Collagen network. METHODS: Experimental OA was induced by anterior cruciate ligament transsection in the rat knee joint (ACLT-OA). Animals were sacrificed after 2, 7, 14, 28 and 70 days. Knee joints were evaluated using routine histology and immunohistochemistry for denatured (unwound) Type II Collagen to detect Collagen damage. An antibody recognizing the Collagenase cleavage site in Type II Collagen was used to study the role of Collagenase in this process. RESULTS: The first changes of the articular cartilage after anterior cruciate ligament transection occurred in the superficial zone. These changes included loss of superficial chondrocytes, swelling of the remaining chondrocytes and superficial fibrillation. The swelling of the chondrocytes did not result from a change towards the hypertrophic phenoType, since these cells did not stain for Type X Collagen. A marked increase in denatured Type II Collagen staining was present in the fibrillated areas. Staining of the Collagenase cleavage site showed the same distribution as denatured Collagen but was clearly less intense. Collagen damage could never be detected before fibrillation occurred and was not present in non-fibrillated areas. CONCLUSIONS: These results indicate that in this model cartilage degeneration starts at the articular surface and that this degeneration is associated with a localized expression of Type II Collagen degradation products.
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differences in Type II Collagen degradation between peripheral and central cartilage of rat stifle joints after cranial cruciate ligament transection
Arthritis & Rheumatism, 2000Co-Authors: Reinout Stoop, Peter M Van Der Kraan, Anthony Peter Hollander, Clark R Billinghurst, Robin A Poole, Pieter Buma, Wim B Van Den BergAbstract:Objective. Type II Collagen degradation is thought to be the key process in cartilage degradation during the development of osteoarthritis (OA). In this study, we investigated the kinetics of Type II Collagen degradation during surgically induced OA. Methods. Experimental OA was induced in male Wistar rats by transecting the cranial (anterior) cruciate ligament (CCL). Hematoxylin and eosin staining was used to study overall cartilage degradation, while immunostained sections were used to demonstrate denatured Type II Collagen (Col2-3/4m antibody) and the Collagenase cleavage site in Type II Collagen (Col2-3/ 4Cshort antibody). Results. During the first 3‐4 weeks, cartilage destruction, associated with chondrocyte death, proteoglycan depletion, and a marked increase in the Collagenase cleavage neoepitope, was mainly located at the margins of the cartilage. From weeks 3‐4, the central part of the cartilage showed increased surface fibrillation and apparent chondrocyte death. In these areas, increased denatured Type II Collagen staining but little cleavage-site staining was present. Conclusion. These results indicate that cartilage degradation after CCL transection in the rat consists of 2 phases. An early phase located at the cartilage margins and a late phase located at the central part of the cartilage. In the early phase, Collagenase-dependent cartilage damage occurred. During the late phase, the level of Type II Collagen denaturation increased.