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.

Calcitonin has been recently shown to have a direct protective effect on articular cartilage against joint degenerative disease. It has been proposed that calcitonin might act through the calcitonin receptor (CTR) to activate the cyclic AMP pathway and protect type II collagen degradation. In this study, we examined the presence of the CTR in human articular cartilage and chondrocytes and investigated the potential pharmacological effects and transduction pathway of salmon calcitonin in human chondrocytes.

Five human articular cartilage samples were examined for the expression of the CTR by polymerase chain reaction (PCR), immunostaining and Western blotting. Cyclic AMP levels in human chondrocyte stimulated with salmon calcitonin were measured by ELISA. The effect of salmon calcitonin on the gene expression profiles, including aggrecan, type II collagen, matrix metalloproteinase (MMP)-1, MMP-3 and MMP-13, of human chondrocytes was also examined by Real-time PCR.

It was shown that CTR was not detectable in human cartilage and chondrocytes. The cAMP level in human chondrocytes in vitro was significantly increased by forskolin (100μM) by > 10 fold (P< 0.001), but was not induced by salmon calcitonin (10^-7M, 10^-8M, 10^-9M). Real-time PCR demonstrated that salmon calcitonin tended to reduce the gene expression of MMPs, yet without statistical significance. In contrast to previous reports, our data showed that human cartilage and chondrocytes do not express calcitonin receptors. There was no direct effect of salmon calcitonin on human chondrocytes.

The result suggests that the chondroprotective effect of calcitonin observed in vivo may be indirect via its effect on subchondral bone resorptive activity.

Introduction and Aims: Assessment of the metabolic state of articular cartilage (AC) is important in understanding the initiation and progression of osteoarthritis (OA). The purpose of this study was to evaluate changes in gene expression of the major AC extracellular matrix (ECM) components, in addition to a number of molecules involved in OA, including the novel glycoprotein lubricin, following lateral meniscectomy in a sheep model of OA.

Method: AC tissue from both medial (MTP) and lateral (LTP) tibial plateaux were collected from six non-operated control (NOC) and six lateral meniscectomised (MEN) pure-bred Merino sheep six months post-surgery for semi-quantitative RT-PCR to assess patterns of mRNA expression (relative to GAPDH). Histological evaluation using a modified Mankin score was undertaken in the same sheep to grade the AC and immunohistochemical localisation of gene products was performed.

Results: Cartilage degeneration was evident both macroscopically and histologically in the LTP following MEN, with less marked changes appearing in the MTP. The mean total tissue RNA increased greater than five-fold in the LTP following MEN (p< 0.01). Expression of aggrecan (p< 0.01) and collagen type II (p< 0.01) were found to be significantly elevated in LTP AC following MEN. Increased expression of biglycan (p< 0.01) was observed in LTP AC following MEN, whereas conversely, there was a decreased expression of decorin (p< 0.01), the other fibril associated small leucine rich proteoglycan. Expression of both lubricin (p< 0.01) and connective tissue growth factor (CTGF) (p< 0.05) were also found to decrease following MEN in LTP AC. TGFβ demonstrated no change in expression following MEN. Significant changes in gene expression were generally not seen in the MTP following MEN; however trends were observed reflecting similar gene profile changes to those occurring in the LTP.

Conclusion: Strong up-regulation in gene expression of the major cartilage ECM components was found, reflecting an anabolic response and attempted tissue repair. Significant changes were also observed for other ECM macromolecules thought to be involved in degenerative joint disease, contributing to alterations in the gene expression profile associated with OA.