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.
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
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
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
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
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
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
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)
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)
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
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 (10–5M) 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
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)
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
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)
Objective—To determine the clinical characteristics and outcome of foals with septic osteitis of the distal phalanx.
Design—Retrospective case series.
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.