Back pain will affect up to 84 % of people at some point in their lives. It is a
major public health problem in Western society leading to disability and
economic loss through work absence, physician visits and hospitalisation. A
major cause of back pain is the natural degeneration of the intervertebral discs
(IVDs) and osteoarthritis of the facet joints which increase with age. Spinal
fusion is a common treatment that has been used for decades. The procedure
involves the fusion of two vertebrae restricting motion in the functional spinal
unit (FSU). Currently, tissue engineering methods are being researched to
develop facet cartilage and IVD repair and replacement therapies for early
intervention in the disease process with the aim of restoring tissue function and
motion. There is a need for pre-clinical simulations to test these novel early
intervention therapies.
The aim of this study was to characterise facet cartilage and IVD tissue in an
ovine model in order to develop models of the healthy and degenerated ovine
FSU which could be used in pre-clinical simulations of future therapies.
Ovine spines from animals of different ages were dissected and IVDs and
facets from cervical, thoracic and lumbar regions were studied using
photography, μCT and histology. The results showed that degeneration in the
FSU began at 5-6 years of age. Since tissue from one year old sheep was
skeletally immature, tissue from 3-4 year olds was accepted as healthy and
used for further characterisation in order for comparisons to be made between
ovine and human tissue.
IVDs and facet cartilage from cervical, thoracic and lumbar regions of the spines
of 3-4 year old sheep were characterised using photography, histology,
(haematoxylin and eosin, alcian blue, Sirius red and Miller’s elastin stains),
immunohistochemistry, assay for glycosaminoglycan (GAG) content and
indentation testing to determine the biomechanical properties. The cells were
isolated from FSU`s tissues, cultured and phenotyped using
immunofluorescence. Facet cartilage was rich in GAGs throughout the tissue
structure with normal articular cartilage collagen distribution. The percentage of
GAGs in dry weight facet cartilage was 8.47 %. Elastin was also found within
the lamellae layers of the annulus fibrosus (AF) and cartilage end plate (CEP).
Immunohistochemistry showed high expression of collagen II, chondroitin
sulphate, collagen VI, collagen III and fibronectin in both facet cartilage and IVD
tissue with varying distributions and intensities. The isolated FSU cells showed
high expression of collagen types II, III and VI, fibronectin, CD44, SOX-9 and
chondroitin-6-sulphate.
Facet cartilage showed very similar characteristics to other articular cartilages in
terms of the key macromolecules present. Facet cartilage in the cervical region
appeared thicker and contained more GAGs in comparison to the thoracic and
lumbar regions. This may be due to the greater range of motion experienced in
the cervical region and the need for extra support. A relationship between the
presence of collagen types III and VI and elastin was also observed in facet
cartilage. This may be due to the similar role of these proteins in providing
anchorage of the chondrocyte to the ECM in facet cartilage.
In order to mimic degenerated human facets, ovine facets were treated with
chondroitinase ABC in order to remove GAGs from the cartilage. Eight cervical
facets were treated with 0.05 U.ml-1 (w/v) chondroitinase ABC solution and
analysed using photography, indentation, GAG assay, histology and
immunohistochemistry to determine their effect.
The chondroitinase ABC method was shown to remove GAGs from facet
cartilage, as seen in degenerated cartilage. This approach can be used to treat
ovine FSU’s to create degenerate models for in vitro pre-clinical simulations for
testing future therapies such as those developed through tissue engineering.
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