Objective—To determine the quantity (concentration) and quality (molecular weight) of synovial fluid hyaluronan with respect to presence and severity of osteoarthritis in stifle joints of dogs.
Animals—21 purpose-bred dogs and 6 clinically affected large-breed dogs (cranial cruciate ligament [CrCL] disease with secondary osteoarthritis).
Procedures—Research dogs underwent arthroscopic surgery in 1 stifle joint to induce osteoarthritis via CrCL transection (CrCLt; n = 5 stifle joints), femoral condylar articular cartilage groove creation (GR; 6), or meniscal release (MR; 5); 5 had sham surgery (SH) performed. Contralateral stifle joints (n = 21) were used as unoperated control joints. Synovial fluid was obtained from research dogs at time 0 and 12 weeks after surgery and from clinically affected dogs prior to surgery. All dogs were assessed for lameness, radiographic signs of osteoarthritis, and pathologic findings on arthroscopy as well as for quantity and quality of hyaluronan.
Results—Clinically affected dogs had significantly greater degrees of pathologic findings, compared with dogs with surgically induced osteoarthritis (ie, those with CrCLt, GR, and MR stifle joints), and with respect to lameness scores, radiographic signs of osteoarthritis, pathologic findings on arthroscopy, and synovial fluid hyaluronan concentration. Synovial fluid from stifle joints of dogs with surgically induced osteoarthritis had hyaluronan bands at 35 kd on western blots that synovial fluid from SH and clinically affected stifle joints did not.
Conclusions and Clinical Relevance—Synovial fluid hyaluronan quantity and quality were altered in stifle joints of dogs with osteoarthritis, compared with control stifle joints. A specific hyaluronan protein fragment may be associated with early pathologic changes in affected joints.
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
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
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 assess the cellular, biochemical, and
histologic effects of bipolar radiofrequency-generated
heat on canine articular cartilage.
Sample Population—Articular cartilage explants (n =
72) from 6 canine cadavers and cultured articular
chondrocytes from 5 canine cadavers.
Procedure—Cartilage explants were randomly
assigned to receive no treatment or treatment with
focal (3 seconds) or diffuse bipolar radiofrequency.
Following treatment, methylene blue permeability
assay was performed (n = 12) and remaining samples
(60) were cultured. Immediately and 5, 10, and 20
days after treatment, cultured explants were
assessed for glycosaminoglycan (GAG) and collagen
contents, type II collagen and matrix metalloproteinase
(MMP)-13 immunoreactivity, and modified
Mankin histologic scores. Liquid culture media were
collected every 4 days and GAG content measured.
Additionally, cultured chondrocytes were exposed for
3 seconds to media preheated to 37°, 45°, or 55°C.
Cell viability was determined via 2 different assays
immediately and 24 hours after treatment.
Results—Radiofrequency-treated cartilage had
reduced permeability and considerable histologic
damage, compared with control samples; most treated
samples had reduced collagen II staining and
increased MMP-13 immunostaining. Compared with
other treatments, less GAGs were released from cartilage
after diffuse radiofrequency treatment throughout
the study period. Cell viability was significantly different
between controls and cells treated at 55°C
immediately and 24 hours after heat treatment.
Conclusions and Clinical Relevance—In this study,
bipolar radiofrequency treatment had detrimental
effects on normal articular cartilage cells and extracellular
matrix with probable long-term clinical consequences.
The usefulness of radiofrequency for treatment
of osteoarthritic articular cartilage requires further
investigation. ( Am J Vet Res 2004;65:604–609)
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)