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- Author or Editor: James L. Cook x
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Objective—To characterize chondrocytes from naturally occurring osteochondrosis (OC) lesions of the humeral head of dogs.
Sample Population—15 cartilage specimens from 13 client-owned dogs with humeral head OC and 10 specimens from the humeral head of healthy dogs (controls).
Procedure—Chondrocytes were isolated and cultured in a 3-dimensional system. On days 7, 10, 15, 20, and 25, glycosaminoglycan and hydroxyproline content and cytologic characteristics were evaluated. Expression of collagen types I, II, and X was assessed by use of immunohistochemistry.
Results—Chondrocytes from OC lesions were less viable, compared with control chondrocytes. Glycosaminoglycan content in the OC group was significantly less than in the control group on all days except day 20. Hydroxyproline content was also significantly less in the OC group on days 10, 20, and 25. Expression of collagen type II was significantly less in the OC group, compared with the control group on all days, whereas expression of collagen type I was significantly greater in the OC group on days 20 and 25. Expression of collagen type X was significantly less in the OC group on all days except day 25.
Conclusions and Clinical Relevance—Chondrocytes from naturally occurring OC lesions of the humeral head of dogs cultured in a 3-dimensional system were less viable and less capable of producing appropriate extracellular matrix molecules than chondrocytes from unaffected dogs. Alterations in the synthetic capabilities of chondrocytes from OC-affected cartilage may be a cause or an effect of the disease process. (Am J Vet Res 2002;63:186–193)
Objective—To determine the effects of interleukin (IL)-1β on matrix synthesis and degradation by chondrocytes cultured in a 3-dimensional (3-D) gel medium.
Sample Population—Chondrocytes from 7 dogs.
Procedure—Articular chondrocytes were harvested and cultured in 3-D gel medium alone or with 10 or 20 ng IL-1βml that was added beginning on day 0, 3, 6, or 9. On days 3, 6, 12, and 20 of 3-D culture, samples of the liquid medium were evaluated for glycosaminoglycan (GAG), prostaglandin E2 (PGE2), and matrix metalloprotease (MMP)-3 content. The 3-D plug in each well was evaluated for histologic characteristics of viability, cell morphology, and proteoglycan staining, immunohistochemically stained for collagen type II, and spectrophotometrically analyzed for GAG content.
Results—Significant differences for all variables were detected between controls and each IL-1β group, among groups with different IL-1β concentrations, and among groups with IL-1β added at various time points. Chondrocytes exposed to IL-1β had loss of GAG, increased PGE2 and MMP-3 concentrations, and lack of collagen type-II synthesis. These IL-1β effects appeared to be time and concentration dependent.
Conclusions—Addition of IL-1β to chondrocytes in 3- D gel medium results in time- and concentrationdependent effects on matrix synthesis and degradation and provides an appropriate in vitro model for many of the pathophysiologic events associated with osteoarthritis. (Am J Vet Res 2000;61:766–770)
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)
Objective—To elucidate tissue inhibitor of metalloproteinase (TIMP)-mediated effects on chondrocytes.
Sample Population—Articular cartilage from humeral heads of 6 dogs.
Procedure—Chondrocytes from harvested specimens were cultured in 3-dimensional (3-D) agarose at 106 cells/mL. We prepared 3-D constructs exposed to only tumor necrosis factor (TNF)-α (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-α. Chondrocytes cultured without TIMP or TNF-α 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-α or TNF-α plus TIMP did not increase. On day 9, GAG release from 3-D constructs cultured with TNF-α 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 and Clinical Relevance—Addition of TIMPs did not counteract suppression of GAG and HP accumulation in 3-D constructs exposed to TNF-α. Apparently, adverse effects on chondrocytes exposed to TNF-α cannot be prevented by addition of TIMP alone. (Am J Vet Res 2004;65:1611–1615)