Objective—To clone the 5' end of type III collagen
and describe its pattern of mRNA and protein expression
in normal and healing tendons in horses.
Animals—14 healthy adult horses.
Procedure—The tensile region of collagenase-injured
superficial digital flexor tendons was harvested at
intervals from 1 to 24 weeks after injury. Total RNA
was reverse-transcribed into cDNA for cloning and
sequencing of type III collagen. Equine-specific nucleic
acid probes were developed and used for northern
blot analysis and in situ hybridization. Type III collagen
protein and cyanogen bromide-cleaved collagen peptides
were assessedby gel electrophresis.
Results—Type III collagen mRNA expression and protein
content increased immediately after injury and
remained increased. Type III collagen was localized to
the endotenon in normal tendon and in injured tendon
at 1 week. At 8 and 24 weeks, expression became
more widely distributed throughout the tendon
parenchyma. Injured tendon contained 6 times more
type I than type III collagen mRNA. Quantities of type
III collagen protein were maximal in the first 4 weeks
after injury (approx 33%) and then began to decrease.
Conclusions and Clinical Relevance—Type III collagen
expression is increased initially in endotenon and
subsequently in parenchyma of healing tendon; however,
type III remains the minor collagen throughout
the healing process. The role of type III collagen in
tendon healing is not fully elucidated. (Am J Vet Res 2005;66:266–270)
Objective—To investigate effects of β-aminopropionitrile
and a combination of insulin-like growth factor
(IGF)-I and β-aminopropionitrile on metabolism of
equine tendon fibroblasts.
Sample Population—Flexor tendon explants from 3
Procedure—Explants received 1 of 4 treatments
(control, IGF-I, β-aminopropionitrile, and IGF-I/β-aminopropionitrile)
for 10 days, and message expression for
collagen types I and III was assessed by use of in situ
hybridization. Histologic findings, new protein production,
and quantitative determinations of glycosaminoglycan,
DNA, and de novo collagen synthesis were
Results—Insulin-like growth factor-I stimulated an
anabolic response in tendon. Collagen synthesis and
glycosaminoglycan and DNA content of explants
were all increased. β-Aminopropionitrile significantly
suppressed collagen synthesis, which was not ameliorated
by concurrent IGF-I treatment. β-Aminopropionitrile
caused alterations in cell morphology
characterized by large round cells with eccentric
nuclei and decreased density of collagen fibers.
Protein production and collagen type-III mRNA
expression were reduced in these cells.
Conclusion and Clinical Relevance—Treatment
with β-aminopropionitrile resulted in decreased production
of protein and collagen synthesis, which
could be expected to suppress tendon healing. The
negative effects of β-aminopropionitrile could not be
abrogated by addition of IGF-I to the medium.
Treatment resulted in alterations in cell morphology
and matrix consistency, which could further delay tendon
healing. β-Aminopropionitrile may impair tendon
healing at a cellular level by decreasing collagen production
or increasing rate of degradation of existing
matrix. Because of reduced crosslinking during β-
aminopropionitrile treatment, in combination with
transiently decreased tensile strength, alterations in
collagen content and structure may weaken the healing
tendon. (Am J Vet Res 2001;62:1557–1562)
Objective—To determine molecular changes in the
expression of insulin-like growth factor-I (IGF-I) and
transforming growth factor-β1 (TGF-β1) in horses with
osteochondrosis, and to characterize expression of
matrix aggrecan and collagen types I, II, and X in articular
cartilage of affected joints.
Sample Population—Articular cartilage from affected
stifle or shoulder joints of 11 horses with naturally
acquired osteochondrosis and corresponding joints of
11 clinically normal horses.
Procedure—Harvested specimens were snap frozen
in liquid nitrogen, and total RNA was isolated.
Specimens were fixed in 4% paraformaldehyde for
histologic examinations. Expression of matrix molecules
was assessed by analysis of northern blots and
in situ hybridization, using equine-specific cDNA
probes and riboprobes, respectively. Expression of
IGF-I and TGF-β1 was assessed by use of noncompetitive
quantitative polymerase chain reaction, in situ
hybridization, and immunohistochemical analysis.
Results—Cartilage obtained from osteochondrosis
lesions had significantly greater expression of IGF-I,
compared with normal cartilage. Expression of TGF-
β1 and collagen type I were higher, but not significantly
so, in affected tissues. Expression of aggrecan
or collagen types II and X did not differ between
affected and clinically normal cartilage.
Conclusions and Clinical Relevance—Increased
expression of growth factors and collagen type I was
found in cartilage from osteochondrosis lesions.
However, this probably reflects a healing response to
injured tissue rather than a primary alteration.
Therefore, methods aimed at altering concentrations
of growth factors in cartilage of growing horses
would be unlikely to alter the incidence or progress of
the disease. (Am J Vet Res 2001;62:1088–1094)
Objective—To determine the morphologic and phenotypic
effects of transforming growth factor β1 (TGF-β1)
on cultured equine mesenchymal stem cells (MSC)
and articular chondrocytes.
Sample Population—Bone marrow aspirates and
articular cartilage samples from a 2-year-old and two 8-
Procedure—After initial isolation and culture, MSC
and chondrocytes were cultured in Ham's F-12 medium
supplemented with TGF-β1 at a concentration of 0,
1, 5, or 10 ng/ml. Medium was exchanged on day 2,
and cells were harvested on day 4. Medium was
assayed for proteoglycan (PG) content. Total RNA was
isolated from cell cultures, and expression of aggrecan,
decrin, collagen type-I, and collagen type-II
mRNA was assessed by means of Northern blot analyses.
Cell cultures were stained with H&E or toluidine
blue and examined histologically. Additional cultures
were examined after immunohistochemical staining
for type-I and -II collagen.
Results—MSC cultures exposed to TGF-β1 had an
increased cellular density with cell layering and nodule
formation that was most pronounced in cultures treated
with 5 ng of TGF-β1/ml. Expression of collagen
type-II mRNA in MSC cultures exposed to 5 ng of TGF-
β1/ml was 1.7 times expression in control cultures,
and expression of collagen type-I mRNA was 2.8
times expression in control cultures. Treatment of
MSC with TGF-β1 led to dose-related increases in area
and intensity of type-II collagen immunoreaction.
Conclusion—Results suggest that TGF-β1 enhances
chondrogenic differentiation of bone marrow-derived
MSC in a dose-dependent manner. (Am J Vet Res