A stable isotope method for the simultaneous measurement of matrix synthesis and cell proliferation in articular cartilage in vivo K.W. Li, S.A. Siraj,

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Presentation transcript:

A stable isotope method for the simultaneous measurement of matrix synthesis and cell proliferation in articular cartilage in vivo K.W. Li, S.A. Siraj, E.W. Cheng, M. Awada, M.K. Hellerstein, S.M. Turner Osteoarthritis and Cartilage Volume 17, Issue 7, Pages 923-932 (July 2009) DOI: 10.1016/j.joca.2009.01.006 Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Protocol for isolating several metabolites from a single specimen of articular cartilage and analyzing by GC/MS. Proteinase K-solubilized cartilage is subjected to sequential digestion steps with hyaluronidase and chondroitinase ABC to separate the molecular constituents of HA, CS, and DNA. Further hydrolysis and derivatization steps (not shown) are used to prepare glcNAc, galNAc, and dR for GC/MS analysis, providing indices of the fractional synthesis HA, CS and DNA, respectively. Osteoarthritis and Cartilage 2009 17, 923-932DOI: (10.1016/j.joca.2009.01.006) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Age-dependent effects of IL-1 and BMP-7 on the biosynthesis of CS in bovine cartilage explants. Cartilage explants were maintained in medium containing 0 (■), 0.5 ( ), or 5 (□) ng/ml IL-1α, with or without 100ng/ml BMP-7. Fractional CS synthesis in mature bovine explants after (A) 5 and (B) 10 days in culture, as well as that in immature bovine explants after (C) 5 and (D) 10 days is shown. **P<0.05 for BMP-7 main effect; *P<0.05 compared with IL-1-free controls. n=8 from triplicate experiments. Osteoarthritis and Cartilage 2009 17, 923-932DOI: (10.1016/j.joca.2009.01.006) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Age-dependent effects of IL-1 and BMP-7 on the biosynthesis rate of sGAG in bovine cartilage explants, expressed as sGAG synthesized per day. Cartilage explants were maintained in medium containing 0 (■), 0.5 ( ), or 5 (□) ng/ml IL-1α, with or without 100ng/ml BMP-7. Absolute CS deposited is determined from the product of fractional CS synthesis and total tissue content of sGAG and presented normalized to cell number. CS biosynthesis was determined for mature bovine explants maintained for (A) 5 and (B) 10 days and for immature bovine explants maintained for (C) 5 and (D) 10 days. **P<0.05 for BMP-7 main effect; *P<0.05 compared with IL-1-free controls. n=8 from triplicate experiments. Osteoarthritis and Cartilage 2009 17, 923-932DOI: (10.1016/j.joca.2009.01.006) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Age-dependent effects of IL-1 and BMP-7 on the biosynthesis of HA in bovine cartilage explants. Cartilage explants were maintained in medium containing 0 (■), 0.5 ( ), or 5 (□) ng/ml IL-1α, with or without 100ng/ml BMP-7. Fractional HA synthesis in mature bovine explants after (A) 5 and (B) 10 days in culture, as well as that in immature bovine explants after (C) 5 and (D) 10 days is shown. **P<0.05 for BMP-7 main effect; *P<0.05 compared with IL-1-free controls. n=8 from triplicate experiments. Osteoarthritis and Cartilage 2009 17, 923-932DOI: (10.1016/j.joca.2009.01.006) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Age-dependent effects of IL-1 and BMP-7 on cell proliferation in bovine cartilage explants. Cartilage explants were maintained in medium containing 0 (■), 0.5 ( ), or 5 (□) ng/ml IL-1α, with or without 100ng/ml BMP-7. Fractional DNA synthesis in mature bovine explants after (A) 5 and (B) 10 days in culture, as well as that in immature bovine explants after (C) 5 and (D) 10 days is shown. **P<0.05 for BMP-7 main effect; *P<0.05 compared with IL-1-free controls. n=8 from triplicate experiments. Osteoarthritis and Cartilage 2009 17, 923-932DOI: (10.1016/j.joca.2009.01.006) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Fractional synthesis of CS & DNA (cells) in vivo in rat knee cartilage. Osteoarthritis and Cartilage 2009 17, 923-932DOI: (10.1016/j.joca.2009.01.006) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions