Cruciate Ligament Matrix Metabolism and Development of Laxity 69cathepsin K, when compared with CrCLs from
healthy, young, or aged dogs (Muir et al.
2002). Therefore, cell-signaling pathways that
regulate expression of these proteinases may
form part of the mechanism that leads to up-
regulation of collagen remodeling within the
CrCL (Muiret al. 2002). Levels of cathepsin
K and tartrate-resistant protein (TRAP) expres-
sion in the canine synovium and CrCLwere also
found to be significantly correlated in dogs with
CR (Muiret al. 2005a).
The relationship of cruciate ligament
metabolism and stifle laxity
Detailed comparisons of joint laxity and other
mechanical parameters in stifle joints with nor-
mal CrCLs with other ligament parameters
(e.g., collagen and ECM biochemistry) have
been reported infrequently (Vasseuret al. 1985).
The latter authors compared the biomechan-
ical and histological properties in dogs, and
found a positive relationship with age, increas-
ing weight, and degenerative changes in the
canine CrCL(Vasseuret al. 1985). However, only
a mixed population of dogs of different breeds,
ages, gender, and weight was examined in this
study.
The present authors’ group hypothesized
that increased cranio-caudal stifle joint laxity
would be present in normal stifle joints from a
breed at high risk of developing CR (Labrador
Retriever), compared to a low-risk breed (Grey-
hound). It was also hypothesized that the stifle
joints with increased cranio-caudal laxity (high-
risk breeds) would demonstrate increased ECM
metabolism of their CrCLs. This increased ECM
metabolism could result in reduced ligament
strength and increased joint laxity contributing
to eventual ligament rupture.
Mechanical, biochemical and thermal anal-
yses were used to test the above hypothe-
sis (Comerford 2002; Comerfordet al. 2005).
Mechanical analyses involved the measure-
ment of cranio-caudal laxity and tensile test-
ing of the CrCL.Ex vivomechanical testing
of the stifles from both high- and low-risk
breeds was performed as previously described
(Amis & Dawkins 1991). The stifle joints were
mounted in two different test positions (30◦
and 90◦of flexion) (Figure 8.4), after which the
Figure 8.4 Photograph of a canine stifle joint positioned
in a materials testing machine (Instron 1122, Instron Ltd,
High Wycombe, UK) for cranio-caudal mechanical
testing. The tibia is positioned in the uppermost stainless
steel pot and the load cell is positioned to the top of the
picture.CrCL dimensions were measured and the lig-
aments tested to failure (Wooet al. 1991). Bio-
chemical and thermal analyses to assess ECM
metabolism, such as collagen crosslink anal-
ysis, gelatin and reverse gelatin zymography
(to measure MMPs and TIMPs, respectively),
total and sulfated GAGs, and differential scan-
ning calorimetry, were performed on the CrCLs
from the stifles which had undergone mechani-
cal testing. Cranio-caudal laxity measurements
showed the mean tibial displacements of the
Labrador Retriever stifle joints to be signifi-
cantly greater than those of the Greyhound sti-
flejointsat30◦and 90◦of flexion, indicating
increased laxity within the high-risk stifle joints
(Figure 8.5) (Comerfordet al. 2005). There was a
significantly higher expression of pro-MMP-2 in
the high-risk CrCLs (Labrador Retrievers) com-
pared to the low-risk (Greyhound) CrCLs (Fig-
ure 8.6). Higher pro-MMP-2 levels can be asso-
ciated with increased collagen turnover, though
this was not confirmed by changes in immature
collagen crosslinks in this study. The concen-
trations of pro-MMP-2 had a positive correla-
tion (r=0.5,P=0.02) with cranio-caudal lax-
ity at 90◦, once both dog groups were combined.
The enthalpy of collagen denaturation (indicat-
ing the amount of heat required to denature the
collagen triple helices) was significantly lower
in the Labrador CrCLs, compared to that of