Project description:Osteoarthritis (OA) is an aging-associated disease of diarthrodial joints that involves changes in the bone, cartilage and soft tissue. Chondrocytes are the only cell type in cartilage and play a pivotal role in tissue homeostasis and cartilage remodeling in OA. Age- or OA-dependent metabolic changes affecting extracelluar matrix composition as well as variations in the response to cytokines and growth factors have been investigated in human chondrocytes. However, the molecular mechanisms causing age-dependent variation of the chondrocyte phenotype are poorly characterized. The proinflammatory mediator nitric oxide (NO) is produced when chondrocytes are exposed to cytokines such as IL-1. NO affects the energy metabolism of cells through interfering with mitochondrial respiration and glycolysis and has been implicated in extracellular matrix synthesis and degradation and chondrocyte death. NO may contribute to the aging process by compromising the energy balance ! in chondrocytes through the generation of an oxidizing environment and this may in turn affect matrix gene expression. In human chondrocytes NO effects on the expression of extacelluar matrix genes or genes related to matrix structure have not been addressed comprehensively. In chondrocytes NO has profound effects on extracellular matrix: Interleukin-1 induced the production of nitric oxide in human articular chondrocytes in alginate culture and simultaneously inhibited the biosynthesis of proteoglycans. NG-monomethyl-L-arginine (L-NMMA) inhibited nitric oxide formation and the suppression of proteoglycan synthesis. The nitric oxide donor, S-nitrosyl-acetyl-D,L- penicillamine (SNAP) also inhibited proteoglycan biosynthesis suggesting that interleukin-1 suppresses matrix synthesis through mechanisms involving the production of NO. Slices of rabbit articular cartilage synthesized large quantities of nitric oxide (NO) following exposure to IL-1 and strongly suppressed the incorporation of 35SO4 into the glycosaminoglycans (GAGs) of cartilage proteoglycans. Simultaneous treatment of cartilage fragments with IL-1 and L-NMMA inhibited NO synthesis in response to IL-1 and restored proteoglycan synthesis. SNAP reversibly mimicked the effect! of IL-1 on proteoglycan synthesis. These data suggest that endogenously synthesized NO is the mediator which reduces cartilage proteoglycan synthesis in response to cytokines such as IL-1. Aggrecan and type II collagen synthesis are also affected by NO. Our hypothesis entails that in human chondrocytes endogenous NO modulates the expression of matrix genes as well as enzymes involved in extracellular matrix remodelling. Experimental approach: In this experiment cultured chondrocytes from 3 donors was left unstimulated (control) or stimulated with IL-1, L-NMMA or IL-1 + L-NMMA for a period of 8 days in order to achieve sufficiently high levels of intracellular NO and stable expression of a subset of genes. Total RNA was isolated using the Trizol procedure. Gene expression will be probed using the Glycov2 chip. In this study cultured chondrocytes from three donors will either be left unstimulated (control) or stimulated with IL-1, L-NMMA or IL-1 L-NMMA for a period of 8 days in order to achieve sufficiently high levels of intracellular NO and stable expression of a subset of genes. 12 Samples were hybridized and analyzed using the GLYCOv2 array, three from each class.

Project description:Osteoarthritis (OA) is an aging-associated disease of diarthrodial joints that involves changes in the bone, cartilage and soft tissue. Chondrocytes are the only cell type in cartilage and play a pivotal role in tissue homeostasis and cartilage remodeling in OA. Age- or OA-dependent metabolic changes affecting extracelluar matrix composition as well as variations in the response to cytokines and growth factors have been investigated in human chondrocytes. However, the molecular mechanisms causing age-dependent variation of the chondrocyte phenotype are poorly characterized. The proinflammatory mediator nitric oxide (NO) is produced when chondrocytes are exposed to cytokines such as IL-1. NO affects the energy metabolism of cells through interfering with mitochondrial respiration and glycolysis and has been implicated in extracellular matrix synthesis and degradation and chondrocyte death. NO may contribute to the aging process by compromising the energy balance ! in chondrocytes through the generation of an oxidizing environment and this may in turn affect matrix gene expression. In human chondrocytes NO effects on the expression of extacelluar matrix genes or genes related to matrix structure have not been addressed comprehensively. In chondrocytes NO has profound effects on extracellular matrix: Interleukin-1 induced the production of nitric oxide in human articular chondrocytes in alginate culture and simultaneously inhibited the biosynthesis of proteoglycans. NG-monomethyl-L-arginine (L-NMMA) inhibited nitric oxide formation and the suppression of proteoglycan synthesis. The nitric oxide donor, S-nitrosyl-acetyl-D,L- penicillamine (SNAP) also inhibited proteoglycan biosynthesis suggesting that interleukin-1 suppresses matrix synthesis through mechanisms involving the production of NO. Slices of rabbit articular cartilage synthesized large quantities of nitric oxide (NO) following exposure to IL-1 and strongly suppressed the incorporation of 35SO4 into the glycosaminoglycans (GAGs) of cartilage proteoglycans. Simultaneous treatment of cartilage fragments with IL-1 and L-NMMA inhibited NO synthesis in response to IL-1 and restored proteoglycan synthesis. SNAP reversibly mimicked the effect! of IL-1 on proteoglycan synthesis. These data suggest that endogenously synthesized NO is the mediator which reduces cartilage proteoglycan synthesis in response to cytokines such as IL-1. Aggrecan and type II collagen synthesis are also affected by NO. Our hypothesis entails that in human chondrocytes endogenous NO modulates the expression of matrix genes as well as enzymes involved in extracellular matrix remodelling. Experimental approach: In this experiment cultured chondrocytes from 3 donors was left unstimulated (control) or stimulated with IL-1, L-NMMA or IL-1 + L-NMMA for a period of 8 days in order to achieve sufficiently high levels of intracellular NO and stable expression of a subset of genes. Total RNA was isolated using the Trizol procedure. Gene expression will be probed using the Glycov2 chip.

Project description:Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score >3, AhlbÃ¤ck Score >2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using quantitative RT-PCR. Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (COL2A1, COMP, aggrecan, CRTL1, SOX9) and genes involved in matrix synthesis (biglycan, COL9A2, COL11A1, TIMP4, CILP2) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (COL10A1, RUNX2, periostin, ALP, PTHR1, MMP13, COL1A1, COL3A1) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, were differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA and OA chondrocytes fulfill the requirements for matrix-associated ACT. Experiment Overall Design: Gene expression profiles of monolayer cultures (ML; passage 2) and Hyaff-11 scaffold cultures (3D; 14 days in vitro) of chondrocytes from 3 normal donors (ND; underwent ACT treatment) and 3 donors suffering from Osteoarthritis (OA; underwent knee replacement surgery) were determined. Comparative analyses between 3D and ML cultures (3D vs. ML) were performed to assess differentiation capacity of ND and OA chondrocytes. Furthermore, OA-related differences were determined comparing OA and ND monolayers as well as scaffold cultures (each OA vs. ND).

Project description:Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score >3, Ahlbäck Score >2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using quantitative RT-PCR. Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (COL2A1, COMP, aggrecan, CRTL1, SOX9) and genes involved in matrix synthesis (biglycan, COL9A2, COL11A1, TIMP4, CILP2) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (COL10A1, RUNX2, periostin, ALP, PTHR1, MMP13, COL1A1, COL3A1) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, were differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA and OA chondrocytes fulfill the requirements for matrix-associated ACT. Keywords: time course, cell type comparison, tissue engineered cartilage; osteoarthritis; Hyaff-11 scaffold; human chondrocytes; gene expression profiling; regenerative medicine; differentiation potential

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