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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.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the effects of glucosamine (GLN) and chondroitin sulfate (CS), at concentrations attainable in vivo, on expression of genes encoding proteolytic enzymes, enzyme inhibitors, and macromolecules of articular cartilage in interleukin-1(IL- 1)–challenged bovine cartilage explants.

Sample Population—Articular cartilage explants harvested from 9 steers.

Procedures—Cartilage explants were exposed to media containing 10% fetal bovine serum (FBS) only, IL- 1 (50 ng/mL), IL-1 with GLN (5 µg/mL), IL-1 with CS (20 µg/mL), or IL-1 with GLN and CS for 24 and 48 hours. Cartilage was frozen, and RNA was extracted. Gene expression of matrix metalloproteinases (MMPs)-2, -3, -9, -13, and -14; aggrecanases (Aggs)-1 and -2; tissue inhibitors of metalloproteinases (TIMPs)-1, -2, and -3; and type II collagen and aggrecan were assessed with quantitative real-time polymerase chain reaction.

Results—Upregulated MMP-3, MMP-13, and Agg-1 transcripts at 24 hours were repressed by the GLN and CS combination by at least approximately 6-fold. Glucosamine was effective in suppressing IL-1–induced mRNA expression of MMP-13, Agg-1, and Agg-2, whereas CS was effective in decreasing IL-1–induced MMP-13 transcript at 24 hours. At 48 hours, GLN and CS added separately and in combination significantly abrogated Agg-1 and Agg-2 gene induction. The combination also decreased IL-1–stimulated MMP-13 transcript.

Conclusions and Clinical Relevance—GLN and CS, at concentrations that are within the range measured in synovial fluid and blood after oral administration, may regulate expression of matrix degrading enzymes and their inhibitors at the transcriptional level, providing a plausible mechanism for their purported chondroprotective properties. (Am J Vet Res 2005;66:1870–1876)

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in American Journal of Veterinary Research

Abstract

Objective—To determine the effects of orally administered glucosamine on concentrations of markers of bone and cartilage metabolism in Standardbred horses during race training.

Animals—Twenty 16- to 20-month-old Standardbreds beginning race training.

Procedure—Horses were randomly assigned to 2 groups. One group received glucosamine hydrochloride (4 g, PO, q 12 h), and the second (control) group received glucose (4 g, PO, q 12 h). Serum samples were obtained prior to onset of the study (baseline) and at regular intervals for 48 weeks for determination of concentrations of keratan sulfate (KS), osteocalcin (OC), and pyridinoline crosslinks (PYD).

Results—Osteocalcin concentrations changed significantly with time; mean serum concentrations were significantly higher than baseline values for samples obtained at 24 to 48 weeks after onset of the study. Although a significant effect of time was observed for mean concentration of KS, concentrations did not differ significantly from baseline values at any time during the study when groups were analyzed separately. However, pooled analysis revealed significant increases of mean serum KS concentration at weeks 24 and 30. Significant changes in serum PYD concentrations were not detected. Oral administration of glucosamine did not significantly affect serum concentrations of any of the markers.

Conclusions and Clinical Relevance—Increased serum OC in clinically normal Standardbreds during race training may reflect bone formation that accompanies adaptive remodeling of the appendicular skeleton. For these experimental conditions, glucosamine did not appear to exert a detectable influence on serum concentrations of these 3 markers of connective tissue metabolism. (Am J Vet Res 2002;63:1106–1110)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To compare the inhibitory effects of glucosamine and mannosamine on articular cartilage degradation and the effects on chondrocyte viability in vitro.

Sample Population—Bovine articular cartilage explants.

Procedures—Explants were cultured in commercial medium for 48 hours. Cartilage was exposed to medium containing 10% fetal bovine serum, 10 µg of lipopolysaccharide/mL, and 0.5, 1.0, 2.5, 5.0, and 10.0 mg of glucosamine or mannosamine/mL for 24 hours. Nitric oxide (NO) production (nitrite concentration) and proteoglycan (PG) release (PG concentration) in media were measured . Cartilage extracts were analyzed via zymography to detect gelatinolytic activity. At the end of the experiment, explants were assessed for chondrocyte viability.

Results—Addition of lipopolysaccharide resulted in increased NO production and PG release, but no increase in gelatinolytic activity, compared with controls. Glucosamine and mannosamine at concentrations as low as 0.5 mg/mL inhibited NO production. Glucosamine inhibited PG release at a minimum concentration of 1.0 mg/mL, whereas mannosamine inhibited PG release at a concentration of 0.5 mg/mL. Concentrations of glucosamine ≤ 5.0 mg/mL did not adversely affect chondrocyte viability; however, at a concentration of 10.0 mg/mL, cell death was evident. Mannosamine had a toxic effect at a concentration of 5.0 mg/mL and was associated with pronounced chondrocyte death at a concentration of 10.0 mg/mL.

Conclusions and Clinical Relevance—Glucosamine and mannosamine inhibit selected indices of bovine articular cartilage degradation at concentrations that do not affect chondrocyte viability. The potential for cytotoxic effects at higher concentrations underscores the importance of establishing appropriate dosage regimens for these aminomonosaccharides. (Am J Vet Res 2004;65:1440–1445)

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in American Journal of Veterinary Research

Abstract

Objective—To determine the effects of recombinant equine interleukin -1β (reIL-1β) and 4 anti-inflammatory compounds on the expression and activity of cyclooxygenase (COX)-2 in cultured equine chondrocytes.

Sample Population—Articular cartilage from 9 young adult horses.

Procedure—Reverse transcriptase-polymerase chain reaction methods were used to amplify a portion of equine COX-2 to prepare a cDNA probe. Northern blot analysis was used to quantify the expression of COX-2 in first-passage cultures of equine articular chondrocytes propagated in media containing dexamethasone (DEX), phenylbutazone (PBZ), polysulfated glycosaminoglycan, and hyaluronan, each at concentrations of 10 and 100 µg/ml and each with or without reIL-1β. A commercial immunoassay was used to determine prostaglandin E2 (PGE2) concentrations in conditioned medium of similarly treated cells to quantify COX-2 activity.

Results—Addition of reIL-1β increased the expression of COX-2 in a dose-dependent manner, which was paralleled by an increased concentration of PGE2 in culture medium. Concentration of PGE2 in spent medium from reIL-1β-treated chondrocytes was significantly reduced by DEX and PBZ; however, only DEX significantly reduced gene expression of COX-2.

Conclusions and Clinical Relevance—Prostaglandin E2 is considered to be an important mediator in the pathophysiologic processes of arthritis, and cultured chondrocytes respond to interleukin-1 with enhanced expression and activity of COX-2. Palliative relief in affected horses is probably attributable, in part, to inhibition of PGE2 synthesis; however, analysis of these data suggests that of the 4 compounds tested, only DEX affects pretranslational regulation of the COX-2 gene in cultured equine chondrocytes. (Am J Vet Res 2002;63:1134–1139)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To characterize potential mechanisms of action of glucosamine inhibition of matrix metalloproteinase (MMP) expression and activity in lipopolysaccharide (LPS)-stimulated equine chondrocytes.

Sample Population—Chondrocytes cultured from samples of metacarpophalangeal articular cartilage collected from cadaveric limbs of horses.

Procedure—The effect of glucosamine on MMP activity in conditioned medium from LPS-stimulated cartilage explants was determined by a colorimetric assay with azocoll substrate. Treatments consisted of negative and positive controls, glucose (50mM), and glucosamine (50, 25, 6.25, 3, and 1.5mM). The influence of glucosamine on MMP synthesis was determined in chondrocytes in pellet culture incubated with LPS (20 µg/mL). Concentration of MMP-13 was quantified in spent medium via ELISA; nonspecific MMP activity was determined via azocoll digestion in organomercurial- activated medium. Effects of glucosamine on MMP mRNA concentration in similarly treated chondrocytes were determined by northern blot hybridization with MMP-1, -3, and -13 probes. Statistical analyses were performed with 2-way ANOVA.

Results—Glucosamine had no effect on activated MMP activity but inhibited MMP protein expression, as determined by azocoll digestion (glucosamine, 3 to 50mM) and MMP-13 ELISA (glucosamine, 1.5 to 50mM). Resting mRNA concentrations for MMP-1, -3, and -13 mRNA were significantly lower in cultures exposed to glucosamine at concentrations of 50 and 25mM than those of positive controls.

Conclusions and Clinical Relevance—Glucosamine appears capable of pretranslational, and possibly also translational, regulation of MMP expression; data suggest a potential mechanism of action for chondroprotective effects of this aminomonosaccharide. ( Am J Vet Res 2003;64:666–671)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the effects of prostaglandin E2 (PGE2) on recombinant equine interleukin (IL)-1β-stimulated expression of matrix metalloproteinases (MMP 1, MMP 3, MMP 13) and tissue inhibitor of matrix metalloproteinase 1 (TIMP 1) in vitro.

Sample Population—Cultured equine chondrocytes.

Procedure—Stationary monolayers of first-passage chondrocytes were exposed to graduated concentrations of PGE2 with or without a subsaturating dose (50 pg/ml) of recombinant equine IL-1β (reIL-1β) to induce expression of MMP 1, MMP 3, MMP 13, and TIMP 1, followed by RNA isolation and northern blotting. In subsequent experiments, gene expression was similarly quantified from mRNA isolated from cultures pretreated with phenylbutazone to quench endogenous PGE2 synthesis, followed by exposure to reIL-1β and exogenous PGE2 (5 mg/ml) with appropriate controls.

Results—Exogenous PGE2 (10 mg/ml) significantly reduced reIL-1β-induced expression of MMP 1, MMP 3, MMP 13, and TIMP 1. Abrogation of cytokine induction with this dose of PGE2 was comparable to that for dexamethasone (10–5 M) control. Similarly, pretreatment with phenylbutazone, followed by exposure to reIL-1β and PGE2 (5 mg/ml), was associated with a reduced expression of the genes of interest, an effect that was significant for MMP 1, MMP 13, and TIMP 1.

Conclusions and Clinical Relevance—The MMP and TIMP 1 are important mediators in the pathophysiologic events in osteoarthritis. The potential for physiologically relevant regulation of expression of these genes by PGE2 is a consideration in the use of drugs that inhibit prostanoid synthesis in the treatment of equine arthropathies. (Am J Vet Res 2002;63:987–993)

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in American Journal of Veterinary Research

Abstract

Objective—To determine whether glucosamine and chondroitin sulfate (CS) at concentrations approximating those achieved in plasma by oral administration would influence gene expression of selected mediators of osteoarthritis in cytokine-stimulated equine articular chondrocytes.

Sample Population—Samples of grossly normal articular cartilage obtained from the metacarpophalangeal joint of 13 horses.

Procedure—Equine chondrocytes in pellet culture were stimulated with a subsaturating dose of recombinant equine interleukin (reIL)-1β. Effects of prior incubation with glucosamine (2.5 to 10.0 µg/mL) and CS (5.0 to 50.0 µg/mL) on gene expression of matrix metalloproteinase (MMP)-1, -2, -3, -9, and -13; aggrecanase 1 and 2; inducible nitric oxide synthase (iNOS); cyclooxygenase (COX)-2; nuclear factor κB; and c-Jun- N-terminal kinase (JNK) were assessed by use of a quantitative real-time polymerase chain reaction assay.

Results—Glucosamine at a concentration of 10 µg/mL significantly reduced reIL-1β–induced mRNA expression of MMP-13, aggrecanase 1, and JNK. Reductions in cytokine-induced expression were also observed for iNOS and COX-2. Chondroitin sulfate had no effect on gene expression at the concentrations tested.

Conclusions and Clinical Relevance—Concentrations of glucosamine similar to those achieved in plasma after oral administration in horses exerted pretranslational regulation of some mediators of osteoarthritis, an effect that may contribute to the cartilage- sparing properties of this aminomonosaccharide. Analysis of results of this study indicated that the influence of CS on pretranslational regulation of these selected genes is limited or lacking. (Am J Vet Res 2005;66:1861–1869)

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in American Journal of Veterinary Research

Abstract

Objective—To determine the clinical characteristics and outcome of foals with septic osteitis of the distal phalanx.

Design—Retrospective case series.

Animals—22 foals.

Procedures—Information obtained from medical records included signalment; clinical, laboratory, and radiographic findings; treatment method; and outcome. Foals included in the study had lameness referable to the foot, radiographic evidence of localized lysis or focal loss of bone density of the distal phalanx, and suppurative discharge or necrosis of the affected bone evident at surgery. Foals with a history or evidence of penetrating wounds or subsolar abscessation were excluded.

Results—Mean age of foals at initial evaluation was 40.8 days (range, 3 to 122 days). Twenty-one (95%) foals had lameness as the primary complaint. Lesions consistent with septic osteitis of the distal phalanx localized to specific areas of the bone on the basis of radiographic and surgical findings were located on the solar margin or toe (14/22 [64%]), extensor process (5/22 [23%]), and palmar or plantar process (3/22 [13%]). Hind limbs (18/26 [69%] affected limbs) were more frequently affected. Two foals had > 1 affected limb, 2 had additional sites of osteomyelitis, and 4 had concurrent septic arthritis. Surgical debridement and regional antimicrobial perfusion were performed during general anesthesia. Extensor process lesions were not debrided. Nineteen of 22 (86%) foals survived to be discharged from hospital, and 16 horses reached racing age. Eleven of 16 had race starts, of which 8 had official race starts and 3 had unofficial race starts.

Conclusions and Clinical Relevance—Septic osteitis of the distal phalanx should be considered as a source of lameness in foals with signs referable to the foot and does not necessarily preclude a career in racing. Although infection may occur secondary to bacterial penetration of the hoof or sole, the distal phalanx should also be considered as a potential site for hematogenous septic arthritis or osteomyelitis in foals.

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in Journal of the American Veterinary Medical Association