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Effects of glucosamine and chondroitin sulfate on bovine cartilage explants under long-term culture conditions

Pooi-See ChanDepartment of Animal Science, Michigan State University, East Lansing, MI 48824

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John P. CaronDepartment of Large Animal Clinical Sciences, Michigan State University, East Lansing, MI 48824

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Michael W. OrthDepartment of Animal Science, Michigan State University, East Lansing, MI 48824

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Abstract

Objective—To determine effects of glucosamine (GLN) and chondroitin sulfate (CS) on expression of genes encoding putative mediators of osteoarthritis in bovine cartilage explants cultured for 2 weeks.

Sample Population—Articular cartilage explants harvested from carpal joints of 4 Holstein steers after slaughter.

Procedures—Cartilage disks were treated as follows: fetal bovine serum only (control treatment), human recombinant interleukin (IL)-1β (50 ng/mL; IL-1 treatment), GLN (5 μg/mL) with addition of CS (20 μg/mL; GLN-CS treatment), and human recombinant IL-1β (50 ng/mL) with addition of GLN and CS (IL-1–GLN-CS treatment). Media were analyzed for nitric oxide and prostaglandin E2 (PGE2) release. Explants were subjected to quantitative real-time PCR analysis; expressions of mRNA for inducible nitric oxide synthase, cyclooxygenase-2, microsomal prostaglandin E synthase 1, matrix metalloproteinase (MMP)-3 and -13, aggrecanase-1 and -2, tissue inhibitor of metalloproteinase (TIMP)-3, type II collagen, and aggrecan were assessed.

Results—IL-1–GLN-CS and GLN-CS treatments decreased nitrite release, compared with IL-1 treatment; IL-1–GLN-CS treatment decreased IL-1–induced PGE2 release. Expressions of inducible nitric oxide synthase, cyclooxygenase-2, and microsomal prostaglandin E synthase 1 mRNA were abrogated by GLN-CS and IL-1–GLN-CS treatments. Interleukin-1–induced mRNA expressions of proteolytic enzymes were diminished by IL-1–GLN-CS treatment. Compared with control treatment, GLN-CS treatment decreased MMP-3 and aggrecanase-2 mRNA expression. Transcripts of TIMP-3 were increased by IL-1–GLN-CS treatment, compared with IL-1 treatment. Genes encoding type II collagen and aggrecan on day 14 were upregulated by GLN-CS and IL-1–GLN-CS treatments, compared with control treatment.

Conclusions and Clinical Relevance—Treatment with GLN and CS consistently downregulated mRNA expression for inflammatory mediators and matrix degrading enzymes while increasing TIMP-3 transcripts.

Abstract

Objective—To determine effects of glucosamine (GLN) and chondroitin sulfate (CS) on expression of genes encoding putative mediators of osteoarthritis in bovine cartilage explants cultured for 2 weeks.

Sample Population—Articular cartilage explants harvested from carpal joints of 4 Holstein steers after slaughter.

Procedures—Cartilage disks were treated as follows: fetal bovine serum only (control treatment), human recombinant interleukin (IL)-1β (50 ng/mL; IL-1 treatment), GLN (5 μg/mL) with addition of CS (20 μg/mL; GLN-CS treatment), and human recombinant IL-1β (50 ng/mL) with addition of GLN and CS (IL-1–GLN-CS treatment). Media were analyzed for nitric oxide and prostaglandin E2 (PGE2) release. Explants were subjected to quantitative real-time PCR analysis; expressions of mRNA for inducible nitric oxide synthase, cyclooxygenase-2, microsomal prostaglandin E synthase 1, matrix metalloproteinase (MMP)-3 and -13, aggrecanase-1 and -2, tissue inhibitor of metalloproteinase (TIMP)-3, type II collagen, and aggrecan were assessed.

Results—IL-1–GLN-CS and GLN-CS treatments decreased nitrite release, compared with IL-1 treatment; IL-1–GLN-CS treatment decreased IL-1–induced PGE2 release. Expressions of inducible nitric oxide synthase, cyclooxygenase-2, and microsomal prostaglandin E synthase 1 mRNA were abrogated by GLN-CS and IL-1–GLN-CS treatments. Interleukin-1–induced mRNA expressions of proteolytic enzymes were diminished by IL-1–GLN-CS treatment. Compared with control treatment, GLN-CS treatment decreased MMP-3 and aggrecanase-2 mRNA expression. Transcripts of TIMP-3 were increased by IL-1–GLN-CS treatment, compared with IL-1 treatment. Genes encoding type II collagen and aggrecan on day 14 were upregulated by GLN-CS and IL-1–GLN-CS treatments, compared with control treatment.

Conclusions and Clinical Relevance—Treatment with GLN and CS consistently downregulated mRNA expression for inflammatory mediators and matrix degrading enzymes while increasing TIMP-3 transcripts.

Contributor Notes

Dr. Chan's present address is Kresge Eye Institute, Wayne State University, Detroit, MI 48201.

Supported by Grayson-Jockey Club Research Foundation, Nutramax Laboratories Incorporated, and the Michigan Agricultural Experiment Station.

Address correspondence to Dr. Orth.