Differential Expression of Proteoglycan Epitopes and Growth Characteristics of Intervertebral Disc Cells Grown in Alginate Bead Culture

Abstract

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.