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Effects of proinflammatory cytokines on canine articular chondrocytes in a three-dimensional culture

Keiichi Kuroki DVM, PhD1, Aaron M. Stoker PhD2, and James L. Cook DVM, PhD3
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  • 1 Comparative Orthopaedic Laboratory, University of Missouri, Columbia, MO 65211.
  • | 2 Comparative Orthopaedic Laboratory, University of Missouri, Columbia, MO 65211.
  • | 3 Comparative Orthopaedic Laboratory, University of Missouri, Columbia, MO 65211.

Abstract

Objective—To determine the effects of interleukin (IL)-1 and tumor necrosis factor (TNF)-α on canine chondrocytes cultured in an agarose-based 3-dimensional (3-D) system.

Sample Population—Humeral head articular cartilage chondrocytes obtained from 6 adult dogs.

Procedure—Chondrocytes were cultured in a 3-D system for ≤ 12 days in serum-free medium with IL-1α, IL-1β, or TNF-α at concentrations of 20, 50, or 100 ng/mL. After 1, 3, 6, and 12 days, glycosaminoglycan (GAG) concentrations in 3-D constructs; nitric oxide and prostaglandin E2 (PGE2) concentrations in media samples; and relative expressions of selected genes, including metalloproteinase (MMP)-13 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, were evaluated. Control specimens were comprised of chondrocytes cultured without proinflammatory cytokines.

Results—In control 3-D constructs, GAG content was significantly higher than for all other constructs. Compared with control values, relative expressions of MMP-13, TIMP-1, and TIMP-2 genes in the IL-1β (50 ng/mL) group were significantly higher at day 1; at all evaluations, media concentrations of nitric oxide were significantly higher in all TNF-α–treated cultures; and concentrations of PGE2 in media samples were significantly higher in the IL-1β (50 ng/mL) and IL-1β (100 ng/mL) groups at days 1 and 3, in the IL-1β (100 ng/mL) group at day 6, and in all TNF-α groups at days 1, 3, and 6.

Conclusions and Clinical Relevance—Results suggested that TNF-α more readily induces production of nitric oxide and PGE2 by canine chondrocytes, compared with IL-1β. In vitro, IL-1α appeared to have a minimal effect on canine chondrocytes. (Am J Vet Res 2005;66:1187–1196)

Abstract

Objective—To determine the effects of interleukin (IL)-1 and tumor necrosis factor (TNF)-α on canine chondrocytes cultured in an agarose-based 3-dimensional (3-D) system.

Sample Population—Humeral head articular cartilage chondrocytes obtained from 6 adult dogs.

Procedure—Chondrocytes were cultured in a 3-D system for ≤ 12 days in serum-free medium with IL-1α, IL-1β, or TNF-α at concentrations of 20, 50, or 100 ng/mL. After 1, 3, 6, and 12 days, glycosaminoglycan (GAG) concentrations in 3-D constructs; nitric oxide and prostaglandin E2 (PGE2) concentrations in media samples; and relative expressions of selected genes, including metalloproteinase (MMP)-13 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, were evaluated. Control specimens were comprised of chondrocytes cultured without proinflammatory cytokines.

Results—In control 3-D constructs, GAG content was significantly higher than for all other constructs. Compared with control values, relative expressions of MMP-13, TIMP-1, and TIMP-2 genes in the IL-1β (50 ng/mL) group were significantly higher at day 1; at all evaluations, media concentrations of nitric oxide were significantly higher in all TNF-α–treated cultures; and concentrations of PGE2 in media samples were significantly higher in the IL-1β (50 ng/mL) and IL-1β (100 ng/mL) groups at days 1 and 3, in the IL-1β (100 ng/mL) group at day 6, and in all TNF-α groups at days 1, 3, and 6.

Conclusions and Clinical Relevance—Results suggested that TNF-α more readily induces production of nitric oxide and PGE2 by canine chondrocytes, compared with IL-1β. In vitro, IL-1α appeared to have a minimal effect on canine chondrocytes. (Am J Vet Res 2005;66:1187–1196)