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This study assessed the growth characteristics and proteoglycan (PG) production in vitro of cells isolated from various zones; annulus fibrosus (AF), the transitional zone (TZ, an opalescent region situated between the AF and nucleus pulposus, NP) and the NP of the ovine intervertebral disc. The isolated cells were subsequently cultured in calcium alginate microspheres. Cellular metabolic activity was assessed over the 10 days of culture by the bioreduction of a tetrazolium dye substrate to a coloured formazan chromophore which could be measured colorimetrically at 490 nm. These data were correlated with the DNA content of beads measured using the fluorescent dye Hoechst 33258. Viable and non-viable cells in alginate beads were also assessed histochemically using the fluorescent dyes 5-chloromethylfluorescein diacetate and ethidium homodimer-1. PGs synthesised in culture were examined immunohistologically using monoclonal antibodies to defined glycosaminoglycan side chain PG epitopes. The results obtained clearly showed that AF and TZ cells differed from NP cells in the measured indices of cellular metabolism and in the extent of matrix deposition. In contrast to cells from the NP the cells from the AF and TZ were more metabolically active and programmed more to cellular proliferation than to matrix production.

Intervertebral disc (IVD) proteoglycans (PGs) and trypsin inhibitory protein levels were assessed for groups of beagles and greyhounds aged between 4 and 80 months. A significant age-dependent decline in PGs and in trypsin inhibitory protein levels was evident in extracts of the beagle nucleus pulposus (NP). This was confirmed for the semi-purified trypsin inhibitor, using Western blotting. These age changes, however, were not evident in the greyhound NP. Furthermore, no significant ageing trends in PG or trypsin inhibitory protein levels were evident in extracts of the annulus fibrosus of either animal. The early onset of IVD degeneration in the beagle, a canine breed of the chondrodystrophoid (achondroplastic) classification, may be related to this decline in the anti-catabolic effects of the endogenous serine proteinase inhibitory proteins.

Lysozyme was demonstrated to increase in content in the human intervertebral disc in a significant age-dependent manner. In young discs (less than 20 years) the levels were 440 U/g wet weight, whereas in older discs (greater than 65 years) the concentration was 3,460 U/g wet weight. These findings contrasted with the hexuronate level (as a marker of proteoglycans) and neutral sugar content of the tissues which decreased with age. The level of lysozyme determined in the disc tissue also correlated with the morphological grade of disc degeneration.