Effects of tissue inhibitor of metalloproteinases on canine chondrocytes cultured in vitro with tumor necrosis factor-alpha.
OBJECTIVE
To elucidate tissue inhibitor of metalloproteinase (TIMP)-mediated effects on chondrocytes.


METHODS
Articular cartilage from humeral heads of 6 dogs.


METHODS
Chondrocytes from harvested specimens were cultured in 3-dimensional (3-D) agarose at 10(6) cells/mL. We prepared 3-D constructs exposed to only tumor necrosis factor (TNF)-alpha (50 ng/mL). Recombinant human TIMP-1 (255nM), -2 (285nM), or -3 (250nM) was added to liquid media bathing 3-D constructs cultured with TNF-alpha. Chondrocytes cultured without TIMP or TNF-alpha served as control samples. Samples of liquid media were collected on days 6, 9, 15, and 21 of culture for evaluation of glycosaminoglycan (GAG) and nitric oxide concentrations. The 3-D constructs were collected on days 9, 15, and 21 for evaluation of GAG, hydroxyproline (HP), and DNA contents.


RESULTS
GAG content in control samples increased significantly during the study, whereas GAG content in 3-D constructs cultured with TNF-alpha or TNF-alpha plus TIMP did not increase. On day 9, GAG release from 3-D constructs cultured with TNF-alpha was significantly higher than that in other constructs. The HP content in control samples increased during the study and was significantly higher than that in all other constructs on day 21. Concentrations of nitric oxide were significantly lower in control samples on day 6, compared with concentrations for all other constructs.


CONCLUSIONS
Addition of TIMPs did not counteract suppression of GAG and HP accumulation in 3-D constructs exposed to TNF-alpha. Apparently, adverse effects on chondrocytes exposed to TNF-alpha cannot be prevented by addition of TIMP alone.
Kuroki
Keiichi
K
Comparative Orthopaedic Laboratory, Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211, USA.
Cook
James L
JL
Kreeger
John M
JM
D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D004247:DNA / Q000032:analysis; D004285:Dogs; D004338:Drug Combinations; D006025:Glycosaminoglycans / Q000378:metabolism*; D006909:Hydroxyproline / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000494:pharmacology; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000494:pharmacology; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000494:pharmacology; D019714:Tissue Inhibitor of Metalloproteinases / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D004338:Drug Combinations; D006025:Glycosaminoglycans; D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D019717:Tissue Inhibitor of Metalloproteinase-3; D019714:Tissue Inhibitor of Metalloproteinases; D014409:Tumor Necrosis Factor-alpha; D019716:Tissue Inhibitor of Metalloproteinase-2; D009569:Nitric Oxide; D004247:DNA; D006909:Hydroxyproline