The biology of the ovine functional spinal unit: facet cartilage and intervertebral disc


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 (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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This paper was published in White Rose E-theses Online.

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