The effects of TIMP-1 and -2 on canine chondrocytes cultured in three-dimensional agarose culture system.
OBJECTIVE
To investigate the effects of tissue inhibitor of metalloproteinase (TIMP)-1 and -2 on chondrocytes cultured with or without interleukin (IL)-1 beta.


METHODS
Canine articular chondrocytes were cultured in three-dimensional (3-D) agarose constructs. Cells were distributed into each of the two groups, those without IL-1 beta and those with IL-1 beta added to the liquid media. Each group was subdivided into three groups, based on the presence of TIMP-1 or -2. IL-1 beta and TIMPs were added to liquid media bathing the 3-D constructs beginning on day 3. The liquid media and the 3-D constructs were collected on days 9, 15, and 24, and analyzed histologically, biochemically, and immunohistochemically.


RESULTS
Addition of TIMP-1 or -2 resulted in decreases in matrix metalloproteinase (MMP)-3 concentrations of 37 and 41%, and MMP-1 immunoreactivity of 32 and 36%, respectively, compared with the IL-1 beta group, on day 9. Chondrocytes in groups without IL-1 beta maintained viability and produced abundant extracellular matrix (ECM). Chondrocytes in IL-1 beta groups appeared less viable and produced less ECM compared with those without IL-1 beta. Glycosaminoglycan (GAG) concentrations in 3-D constructs (GAG/weight) were significantly (P<0.001) higher in groups without IL-1 beta than in those with IL-1 beta, on days 15 and 24.


CONCLUSIONS
The addition of TIMP was not detrimental to chondrocytes, as used in this study. Despite evidence of decreased MMP levels, TIMPs did not prevent IL-1 beta-associated changes in cellular or ECM characteristics. Further study is necessary before clinically relevant conclusions can be drawn regarding the use of TIMPs in the treatment of osteoarthritis.
Kuroki
K
K
The Comparative Orthopaedic Laboratory, University of Missouri, Columbia, MO 65211, USA.
Cook
J L
JL
Kreeger
J M
JM
Tomlinson
J L
JL
D000362:Agar; D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects*; D003470:Culture Media; D004285:Dogs; D005109:Extracellular Matrix / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D006909:Hydroxyproline / Q000378:metabolism; D007124:Immunoenzyme Techniques; D007375:Interleukin-1 / Q000494:pharmacology; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000494:pharmacology*; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000494:pharmacology*
D003470:Culture Media; D006025:Glycosaminoglycans; D007375:Interleukin-1; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; D000362:Agar; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1; D006909:Hydroxyproline