Introduction: In a previous study (Hayes et al., 2007)we reported that novel chondroitin sulphate (CS) sulphation motifs on cell-associated proteoglycans (PGs) may be putative biomarkers of progenitor/stem cell sub-populations resident within the superficial zone of articular cartilage (Dowthwaite et al., 2005). In this study, using the same panel of antibodies, we examine the distribution of novel CS sulphation epitopes in a more clinically relevant model – the developing human knee joint.

Methods: Twelve-14 week human foetal knee joint rudiments were processed into paraffin wax then de-waxed and immunoperoxidase-stained with mAbs 3B3(−), 7D4 and 4C3 using the Vector Universal Elite kit with Nova Red, Mayers Haematoxylin, mounted under coverslips and then photographed.

Results: All three CS sulphation motif epitopes localised prominently at sites of incipient articular cartilage formation at a stage before there was any histological evidence of secondary ossification at the epiphysis. Interestingly, these CS epitopes were also detectable in very defined regions within the perichondrium; growth plate; the fibrocartilage of both meniscus and enthesis; vasculature; and at sites of capillary invasion, with subtle differences in their distribution; for example, 3B3(−) identified the cellular lining of cartilage canals within the epiphyses, whereas 7D4 labelled more their cellular contents.

Discussion: The results of this study show that novel CS sulphation motifs on cell and matrix PGs play important and diverse roles in the development of a wide range of musculoskeletal connective tissues, including articular cartilage. We hypothesize that the unique sulphation sequences on CS-containing PGs are involved in regulating cell proliferation and differentiation events, through interaction with soluble signalling molecules (e.g. growth factors) in the extracellular milieu. These antibodies show considerable promise for uses in tissue engineering applications for identifying and sorting stem/progenitor cells for regeneration of musculoskeletal tissues.

Introduction: The aetiology of dystrophic disc calcification in adult humans is unknown but a well-described clinical disorder with hydroxyapatite as the single mineral phase. Comparable but age-related pathology in the sheep could serve as a model for the human disorder. The objective of this study was to investigate the mineral phase, its mechanisms of formation/association with degeneration in a naturally-occurring animal model of disc calcification.

Methods: Adult sheep lumbar intervertebral discs (n=134) from animals aged 6 (n=4), 8 (n=12) and 11 years (n=2) were evaluated using radiography, morphology, scanning and transmission electron microscopy, energy dispersive X-ray spectroscopy, X-ray powder diffraction, histology, immunohistology and proteoglycan analysis.

Results: Half of the 6 yr, 84% of the 8 yr and 86% of the 11 yr old discs had calcific deposits. These were not well delineated by plain radiography. They were either:

punctate deposits in the outer annulus,

Their maximal incidence was in the lower lumbar discs (L4/5-L6/7) with no calcification seen in the lumbosacral or lower thoracic discs. All deposits were hydroxyapatite with large crystallite sizes (800–1300 angstrom) compared to cortical bone (300–600 angstrom). No type X-collagen, osteopontin or osteonectin, were detected in calcific deposits although positive staining for bone sialoprotein was evident. Calcified discs had less proteoglycan of smaller hydrodynamic size than non-calcified discs.

Discussion: Disc calcification in ageing sheep is due to hydroxyapatite deposition. The variable but large crystal size, lack of protein markers indicate that this does not occur by an ordered endochondral ossification-like process. The decrease in disc proteoglycan content and size suggests an association between calcification and disc degeneration in ageing sheep. There are notable dissimilarities between hydroxyapatite deposition disorder in humans and sheep. No mechanistic explanation can be offered for the different spinal distributions, thoracic and upper lumbar in the former and lumbar in the latter; hydroxyapatite deposition disorder has occasionally been seen in the lumbar spines of four year old sheep during the course of other studies but not at an earlier age. Diffferences in spinal biomechanics may be implicated but hydroxyapatite deposition does not primarily affect the most or least mobile discs in either species. Neither can an explanation be offered for the apparent immunity of the ovine lumbosacral disc to calcification. However, it is known that proteoglycan turnover is faster at this spinal level than at more proximal lumbar discs. While we have been unable to elucidate the mechanism of hydroxyapatite deposition disorder in sheep, clearly it is different from that in normal osteogenesis. We contend this animal provides a useful, naturally-occurring model for investigation of the aetiology and pathogenesis of human hydroxyapatite deposition disorder, notwithstanding obvious differences between sheep and man.

Introduction: The Merino sheep breed has been used extensively for intervertebral disc research but it has not previously been documented that the breed displays a mild form of chondrodystrophy with disproportionate dwarfism. The ovine Merino intervertebral disc is similar to human and chondrodystrophic canine discs in structure, absence of notochordal cells in the adult structure, response to trauma, display of an age-dependant loss of proteoglycans and degenerative spinal pathology including Schmorl’s nodes. In contrast, non-chondrodystrophic breeds have a gelatinous nucleus pulposus, notochordal cells which may persist into adulthood and a low incidence of spinal disorders of discal origin thus are unsuitable as models of the human intervertebral disc.

Methods: Haematoxylin and Eosin, Toluidine blue stained and aggrecan and versican immunolocalised sections were examined by bright field and Nomarsky differential interference contrast microscopy.

Results: The ovine merino intervertebral disc undergoes an age-dependant chondroid transformation of the central nucleus pulposus with the appearance of cell nests of chondrocytic morphology within a hyaline cartilage-like matrix rich in aggrecan and type II collagen but deficient in versican and type I collagen. In contrast, the adjacent nucleus pulposus is a fibrocartilage rich in types I and II collagen, versican and aggrecan; the constituent cells are readily distinguished from the aforementioned cell clusters. The ovine femoral epiphyseal growth plate displays dysplastic changes with relatively short columns of flattened, columnar chondrocytes in the pre-hypertrophic region and a disorganised integration of the hypertrophic cells into metaphyseal endochondral bone in the distal growth plate.

Conclusions: These observations warrant the classification of the Australian merino as a chondrodystrophic breed. The Merino is a useful comparative animal model for the human intervertebral disc.

Calcification of a thoracic intervertebral disc (IVD) with prolapse and root syndromes/spinal cord compression in humans are well-documented entities. The mineral phases have been identified. Similar pathology occurs very rarely in children. It is also seen in dogs, especially the short-legged, chondrodystrophoid (CD) breeds, which are prone to disc degeneration, and in older sheep. The latter exhibit some morphological CD features.

This study is based on radiological/histological/electron microscopic/x-ray diffraction studies of human operative specimens and post-mortem adult animal tissues

The transitional zone (TZ), the interface between the nucleus pulposus and the annulus fibrosus, is the area of the IVD most sensitive in children and adults to the events which lead to dystrophic calcification. The TZ is the “growth plate” of the IVD and the site of maximal proteoglycan and protein synthesis. Giant hydroxyapatite crystallites are the dominant mineral phase in the human (children and adults) and canine pathology. Nucleation occurs in degraded matrix.

The new observation of the type and distribution of calcification in the elderly ovine IVD suggests this animal is a suitable model for further research into the enigmatic phenomenon of so-called dystrophic IVD calcification.

Background: The healthy, adult human disc is innervated but the nerves are restricted to the outer few millimetres of the annulus fibrosus. In degenerate discs with associated back pain, however, the nerves are more numerous and penetrate further in.

We have used a sheep model of intervertebral disc degeneration to monitor the presence and organisation of nerves in the disc as degeneration progresses. This model has been used to study morphological and bio-chemical changes of the disc as it degenerates, in addition to associated alterations in end-plate vascularity and vertebral bone remodelling. One aspect of this model which has not been studied to date is how the innervation of the disc may change with the onset of degeneration. This is the object of the present study.

Materials and Methods: Four-year old, skeletally mature Merino wether sheep (n=64) were divided randomly into lesion and control groups. A surgical incision was created in the anterolateral annulus in the L1–L2 and L3–L4 discs of the lesion group. The control group received the same retroperitoneal surgical approach but the annulus was not incised. Intact lumbar discs encompassed by adjacent vertebral bodies were removed at 3,6,12 and 26 months post operation. Specimens were fixed, decalcified and paraffin embedded before sectioning (7μ thick, vertical sagittal sections) and stained immunohistochemically with the neuronal marker, PGP9.5, together with standard histological stains.

Results: The incised region of the outer annulus underwent collagenous re-organisation, consistent with an active repair process as early as three months post-operatively. However, the inner annular lesion had a poor repair response and propagated with time, sometimes through to the nucleus. In contrast, remodelling of the outer annular lamellae occurred across the cut region. For example, in one sample at two years post injury there were up to six lamellae “bridging the gap”. Nerves were present in all samples but in the sham animals they were very few and confined to the very outer annulus or longitudinal ligament. In the operated animals, nerves were more extensive, occurring in the matrix adjacent to the fissure where there was often blood vessel ingrowth. The maximum number of nerves was seen at 12 months post-operatively, before diminishing in number at 24 months post-op. This paralleled the presence and extent of blood vessel penetration in this experimental model.

Conclusions: We have used an animal model to follow longitudinally the penetration of nerves into the ovine intervertebral disc in association with disc degeneration. Whilst we obviously cannot assess back pain in these animals, and not all nerves are nociceptive, nerves nevertheless are a pre-requisite for the perception of pain. Hence the greater numbers, size and penetration of nerves into degenerate discs demonstrated here has important implications not only for the aetiopathogenesis of degenerative disc disease but also for the treatment of its associated symptoms. Further characterisation of this innervation, i.e. whether autonomic or sensory, may provide an indication as to its nociceptive potential.