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)
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
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
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
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 investigate the presence or absence of Toll-like receptor (TLR)-2 and TLR-4 in synovial tissues collected from stifle joints (SJs) of dogs with or without osteoarthritis.
Animals—21 purpose-bred research dogs, 3 client-owned dogs with SJ osteoarthritis, and 3 dogs without SJ osteoarthritis.
Procedures—Research dogs underwent arthroscopic surgery in 1 SJ to induce osteoarthritis via cranial cruciate ligament transection (CrCLt; n = 5), femoral condylar articular cartilage groove creation (6), or release of the caudal horn of the medial meniscus (5); 5 dogs underwent sham surgery. Synovial tissue specimens were obtained from both stifle joints of each dog 12 weeks after surgery, and TLR-2 and TLR-4 gene expression were determined via real-time reverse transcription PCR assays. Expression of TLR-4 protein was determined via an immunofluorescence technique in additional specimens obtained from osteoarthritic SJs of dogs with cranial cruciate ligament insufficiency and from dogs with nonosteoarthritic SJs.
Results—Synovial tissues from CrCLt-treated joints had significantly higher TLR-4 gene expression, compared with the contralateral control SJs or any other joint group. TLR-2 gene expression did not differ significantly among groups. Toll-like receptor-4 protein was detected in synovial tissues of osteoarthritic SJs but was rarely evident in nonosteoarthritic SJs.
Conclusions and Clinical Relevance—Increased TLR-4 gene expression in the synovial tissue of SJs with osteoarthritis secondary to CrCLt suggests that activation of innate immunity may play a role in the pathophysiology of SJ osteoarthritis in at least a subset of dogs.
Objective—To determine glycosaminoglycan (GAG)
concentration and immunohistochemical staining
characteristics of type-I, -II, and -X collagen from cartilage
affected by osteochondritis dissecans (OCD) in
Animals—31 dogs with OCD and 11 clinically normal
Procedure—Cartilage samples were evaluated
microscopically, and GAG content was determined.
Immunohistochemical staining was performed for
type-I, -II, and -X collagen. Sections were subjectively
evaluated for location and intensity of staining.
Results—Cartilage affected by OCD had a variety of
pathologic changes and significantly lower GAG concentrations
than did normal cartilage. Normal cartilage
had no detectable type-I collagen. For dogs < 9
months of age, cartilage affected by OCD had significantly
more type-I collagen but significantly less type-
X collagen than did control cartilage. For dogs > 12
months of age, cartilage affected by OCD contained
significantly more type-I collagen than did control cartilage.
There was a significant negative correlation
between immunoreactivity of type-I collagen and that
of type-II and -X collagen. A significant positive correlation
was found between immunoreactivity of type-II
and -X collagen.
Conclusions and Clinical Relevance—Cartilage
affected by OCD contains less GAG, more type-I collagen,
and less type-X collagen, compared with normal
cartilage. A direct correlation between these
changes and the etiopathogenesis of OCD was not
established. (Am J Vet Res 2001;62:876–881)