Cruciate ligaments CLs are dense bands of collagenous tissue which stabilise the knee joint Fig' 1'

1
The Organization of Microfibrils and Elastin
Fibres within the Canine Cruciate Ligament
Complex 1KD Smith, 1A Vaughan-Thomas, 2D Spiller,
1JF Innes, 1PD Clegg, 1EJ Comerford. 1Leahurst
Campus, University of Liverpool, Liverpool, U.
K. 2Centre for Cell Imaging, University of
Liverpool, Liverpool, U. K.
INTRODUCTION Cruciate ligaments (CLs) are dense
bands of collagenous tissue which stabilise the
knee joint (Fig. 1). Failure of the anterior
cruciate ligament (ACL) is a major source of
morbidity in the dog. Histological changes
including loss of collagen architecture, loss of
ligament cells and chondrogenic change, are
considered degenerative. While elastin fibres and
microfibrils have important mechanical,
biochemical and cell-regulatory functions,
neither their distribution nor role in
maintaining integrity of the canine ligament have
been investigated. We report the distribution of
elastin fibres and microfibrils in the cruciate
ligaments of two breeds of dogs with a differing
risk of ligament rupture the Greyhound (GH, low
risk) and the Labrador Retriever (LR, high risk).
Materials and Methods Cruciate ligaments were
obtained from dogs euthanased for reasons other
than musculoskeletal disease. Samples were
prepared for histology and confocal laser
scanning microscopy (CLSM). Elastin was
identified using Millers (M) and Verhoeffs (V)
staining. Fibrillin-1 and -2 were localised using
specific primary antibodies and fluorescent
secondary antibody detection by CLSM. Hydrated
tissue was additionally analysed using Nomarski
differential interference microscopy
Figure 2 Elastin fibres (arrows) parallel to
collagen bundles. GH, PCL, V, x100
Figure 3 Microfibrils (arrows) parallel to
collagen bundles. GH PCL, M, x100
Figure 4 Elastin fibres parallel (block arrow)
and oblique (open arrows) to collagen bundles.
GH, ACL, V, x100
Figure 5 Short, thick elastin fibres
perpendicular to collagen bundles. GH, ACL, V,
x100
RESULTS Microfibrils and elastin fibres were
located predominantly within ligament fascicles,
parallel to collagen bundles and with little
regional variation (Figs.2,3). Interfascicular
fibres were more heterogenous in size and
orientation (Figs. 4,5). Elastin fibre
distribution was retained within areas of
degenerative matrix, but a marked difference in
microfibril staining was noted in the GH but not
the LR (Figs 6,7). Fibrillin 1 and fibrillin 2
were found in microfibrils, pericellularly and
between collagen bundles (Figs 8,9).
Figure 6 Moderate CL degeneration in the LR
associated with slightly increased microfibril
staining (blue-grey), particularly
pericellularly. LR ACL, M, x100
Figure 9 Fibrillin 1 immunofluorescence showing
pericellular and interbundle distribution
(arrows). Cell nuclei stained blue (DAPI). GH,
ACL, x100
Figure 7 Moderate CL degeneration in the GH
associated with markedly increased microfibril
staining. GH ACL, M, x100
Figure 8 Fibrillin 2 immunofluorescence showing
high density of microfibrils parallel to collagen
bundles. GH PCL, x40
DISCUSSION The distribution of elastin fibres is
suggestive of a mechanical role in bundle
reorganization following ligament deformation.
Microfibrils and smaller elastin fibres may
affect low strain stiffness or have additional
roles such as cell adhesion or growth factor
regulation. Variations in lateral connections
between collagen bundles may reflect complex
intraligament mechanics. In regions of matrix
degeneration, no differences in elastin fibre
distribution were noted between the GH and LR
CLs. However, as degenerative matrix change
increased, increased microfibril staining was
noted in the GH, but not the LR. This may
represent a fundamental difference between these
breeds, reflecting the differing incidence of CL
disease.