Objectives—To define a portion of the nucleotide
sequences of each of the 6 insulin-like growth factor
(IGF) binding proteins (IGFBPs) in horses and describe
patterns of messenger RNA (mRNA) and protein
expression for IGFBPs in normal equine tendons.
Procedure—Total RNA was extracted from the tensile
region of normal superficial digital flexor tendons
and reverse transcribed into complimentary DNA
(cDNA). The cDNA was amplified via PCR, and products
representing portions of each IGFBP were
cloned and sequenced. Nucleotide sequences were
used to deduce the amino acid sequences, and both
nucleotide and predicted amino acid sequences were
compared with those published for bovine, human,
mouse, and ovine IGFBPs. Gene expression was
quantitated by real-time PCR assay, and protein
expression was evaluated by western ligand blot
Results—Clones ranged in size from 262 to 522 bp
and had high degrees of sequence homology with
other mammalian species. Sequence homology
was highest between bovine and equine IGFBPs
(86% to 95%) and amongst the IGFBP-5
sequences from the various species (92% to 95%).
Message for IGFBP-2 to -6, but not IGFBP-1, was
expressed in normal tendon. Protein expression for
IGFBP-2, -3, and -4 was detected by WLB in normal
tendon and markedly increased in damaged tendons.
Conclusions and Clinical Relevance—Results provide
basic information and tools needed for further
characterization of the role of the IGF system in tendon
healing and may lead to the ability to potentiate
the response of healing tendon to exogenous IGF-I via
concurrent manipulation of IGFBPs. (Am J Vet Res 2005;66:300–306)
Objective—To characterize discrete palmar carpal osteochondral fragmentation in horses and to document the effect of osteoarthritis and surgical removal of these fragments on functional outcome.
Design—Retrospective case series.
Procedures—Medical records and radiographic views were reviewed to identify horses that had radiographic evidence of palmar carpal fragmentation, which was subsequently treated by arthroscopic removal. Information collected included cause of fracture, initial and long-term clinical and radiographic findings, and functional outcome.
Results—Palmar carpal fragmentation of 30 carpal bones was identified in 25 unilaterally affected horses. A known traumatic event was reported to cause the fragmentation in 17 of the 25 (68%) horses. Of the 25 horses, 17 (68%) had fragmentation involving the antebrachiocarpal joint, 7 (28%) had fragmentation involving the middle carpal joint, and 1 (4%) had fragmentation involving the carpometacarpal joint. The proximal aspect of the radial carpal bone was the most commonly affected site (12/30 fragments), followed by the accessory carpal bone (6/30). Of the 25 horses, 19 (76%) were not lame (sound) after surgery and returned to their intended use, 4 (16%) were considered pasture sound, and 2 were euthanized (because of severe postoperative osteoarthritis or long bone fracture during recovery from anesthesia). Eight of the 14 horses with preoperative evidence of osteoarthritis returned to function after surgery. Twelve of 17 horses with antebrachiocarpal joint fragments and 6 of 7 horses with middle carpal joint fragments returned to their previous use.
Conclusions and Clinical Relevance—Results indicated that the prognosis for horses after arthroscopic removal of palmar carpal osteochondral fragments is good. Early intervention, before the development of osteoarthritis, is recommended.
Objective—To assess the effects of supraphysiologic
concentrations of insulin-like growth factor-I (IGF-1) on
morphologic and phenotypic responses of chondrocytes.
Sample Population—Articular cartilage obtained
from 2 young horses.
Procedure—Chondrocytes were suspended in fibrin
cultures and supplemented with 25, 12.5, or 0 mg of
IGF-1/ml of fibrin. Chondrocyte morphology and phenotypic
expression were assessed histologically,
using H&E and Alcian blue stains, immunoreaction to
collagen type I and II, and in situ hybridization.
Proteoglycan content, synthesis, and monomer size
were analyzed. The DNA content was determined by
bisbenzimide-fluorometric assay, and elution of IGF-1
into medium was determined by IGF-1 radioimmunoassay.
Results—Both 12.5 and 25 µg of IGF-1/ml enhanced
phenotypic expression of chondrocytes without
inducing detrimental cellular or metabolic effects.
Highest concentration of IGF-1 (25 µg/ml) significantly
increased total DNA content, glycosaminoglycan
(GAG) content, GAG synthesis, and size of proteoglycan
monomers produced, compared with cultures
supplemented with 12.5 µg of IGF-1/ml or untreated
cultures. Histologic examination confirmed these biochemical
effects. Matrix metachromasia, type-II collagen
in situ hybridization and immunoreaction were
increased in cultures treated with 25 µg of IGF-1/ml,
compared with cultures supplemented with 12.5 µg
of IGF-1/ml or untreated cultures.
Conclusions and Clinical Relevance—Chondrocytes
exposed to high concentrations of IGF-1 maintained
differentiated chondrocyte morphology and had
enhanced synthesis of matrix molecules without
inducing apparent detrimental effects on chondrocyte
metabolism. These results suggest that application of
such composites for in vivo use during cartilage grafting
procedures should provide an anabolic effect on
the grafted cells. (Am J Vet Res 2002;63:301–305)
Objective—To evaluate the effects of recombinant human platelet–derived growth factor-BB (rhPDGF-BB) on the metabolic function and morphologic features of equine superficial digital flexor tendon (SDFT) in explant culture.
Animals—6 euthanized horses (2 to 5 years old).
Methods—Forelimb SDFT explants were cultured for 6 days as untreated control specimens or treated with rhPDGF-BB (1, 10, 50, or 100 ng/mL of medium). Treatment effects on explant gene expression were evaluated via real-time PCR analysis of collagen type I, collagen type III, PDGF-A, and PDGF-B mRNA. Explants were assayed for total collagen, glycosaminoglycan, and DNA content; histologic changes were assessed via H&E staining and immunohistochemical localization of collagen types I and III.
Results—No morphologic or proliferative changes were detected in tendon explant sections. After high-dose rhPDGF-BB treatment, gene expression of collagen types I and III was increased and decreased, respectively. Expression of PDGF-A and PDGF-B mRNA was significantly increased at 24 hours, but later decreased to have few or negative autoinductive effects. Although PDGF gene expression waned after 48 hours of culture, collagen type I gene expression was significantly increased at 48 hours and reached peak value on day 6. Glycosaminoglycan and DNA content of explants were unchanged with rhPDGF-BB treatment.
Conclusions and Clinical Relevance—Results suggest that rhPDGF-BB use may be of benefit in the repair of equine tendon, particularly through induction of collagen type I mRNA. Positive autoinductive effects of PDGF-BB in equine SDFT explants were detected early following culture medium supplementation, but these diminished with time.
Objective—To clarify the diagnosis of avulsion of the lateral palmar intercarpal ligament (LPICL), correlate avulsion of this ligament with lameness, determine the prevalence of avulsion of the LPICL in a hospital population, and evaluate the response to surgical removal of the avulsion fragment in horses.
Animals—37 horses with avulsion of the LPICL.
Procedure—Medical records and radiographs of horses with avulsion of the LPICL were reviewed; follow-up information was obtained from race records and from owners via a telephone survey.
Results–Of 6,418 horses evaluated for forelimb lameness from March 1, 1990, to December 31, 2001, 37 (0.5%) had avulsion of the LPICL; each horse had a discrete fragment associated with avulsion of the ligament origin from the ulnar carpal bone. Twenty-six horses underwent arthroscopic fragment removal; 20 of 22 (91%) horses for which follow-up information was available returned to work. Of 9 horses treated conservatively, 5 returned to work. Odds ratio calculations indicated that horses treated surgically were 8 times as likely to return to work than those not treated surgically. Twelve horses had LPICL avulsion without concurrent osteochondral fragmentation in the same or additional joints; follow-up information was available for 9 of those horses, of which 8 returned to athletic work.
Conclusions and Clinical Relevance—In horses, discrete avulsion of the LPICL can be a cause of lameness and arthroscopic debridement may be the treatment of choice. Prognosis for return to work of horses with avulsion of the LPICL is good. (J Am Vet Med Assoc 2005;226:760–766)
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
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 the mRNA expression of
bone morphogenetic protein (BMP)-6 and -2 and a BMP
antagonist (Noggin) in horses with osteochondrosis.
Sample Population—Samples of articular cartilage
from affected stifle or shoulder joints of 10 immature
horses with naturally acquired osteochondrosis and
corresponding joints of 9 clinically normal horses of
similar age; additionally, samples of distal femoral
growth plate cartilage and distal femoral articular cartilage
were obtained from a normal equine fetus.
Procedure—Cartilage specimens were snap-frozen in
liquid nitrogen, and total RNA was isolated. Adjacent
specimens were fixed in 4% paraformaldehyde for
histologic examination. Expression of BMP-6, BMP-2,
and Noggin mRNA was evaluated by real-time quantitative
polymerase chain reaction (PCR) assays. Spatial
tissue mRNA expression of BMP-6 was determined
by in situ hybridization.
Results—Nucleotide sequences were obtained for
portions of the BMP-6 propeptide and mature peptide
region, as well as the signal and mature peptide region
of Noggin. Expression of BMP-6, BMP-2, and Noggin
mRNA was found to be similar in cartilage from normal
and osteochondrosis-affected horses. Spatial expression
of BMP-6 correlated with the middle and deep layers
of articular cartilage; no differences were observed
in overall expression between cartilage specimens
from the 2 groups of horses. No expression of BMP-6
was found in the superficial layer, subchondral bone, or
osteochondrosis-affected cleft fibrous tissue.
Conclusions and Clinical Relevance—Although these
signaling peptides may play important roles in cartilage
differentiation, results did not provide evidence to suggest
that they are involved in the disease process of
osteochondrosis. (Am J Vet Res 2004;65:110–115)
Case Description—3 horses were referred for treatment of subchondral cystic lesions of 1 or both medial femoral condyles.
Clinical Findings—All horses had clinically apparent lameness confirmed to be due to a radiographically evident subchondral cystic lesion of the medial femoral condyle with a large articular component (> 15 mm) and shallow subchondral depth (< 10 mm). Arthroscopic assessment of affected cartilage revealed undulating cartilage with a relatively smooth surface and extensive residual perimeter attachment.
Treatment and Outcome—Resorbable polydioxanone pins were used arthroscopically to reattach the cartilage overlying the subchondral cystic lesions. A biologic graft (bone marrow aspirate concentrate or allogeneic chondrocytes) was injected into the depths of the cystic cavity following cartilage reattachment. Follow-up examination confirmed radiographic resolution of the lesion and elimination of clinical signs within the treated femorotibial joint.
Clinical Relevance—Lesions with a large area of affected articular cartilage have been associated with a decreased rate of return to athletic function following arthroscopic debridement, likely secondary to the loss of subchondral architecture and the production of imperfect fibrocartilage repair. Salvage of the affected cartilage in a select population of horses with progressively expanding but shallow subchondral cystic lesions of the medial femoral condyle is possible and may improve radiographic and clinical outcome.