Hyaluronan Oligosaccharides Stimulate MMP and Anabolic Gene Expression in-vitro by Intervertebral Disc Cells and Annular Repair in-vivo.

The role of hyaluronan (HA) oligosaccharides in disc cell mediated matrix metalloproteinase (MMP) and anabolic gene expression in-vitro and annular repair in-vivo were examined in this study. Monolayer and alginate bead cultures of ovine intervertebral disc cells were stimulated with 10-12 mer hyaluronan oligosaccharides (HA-oligos). Annulus fibrosus (AF) monolayers were poorly responsive to the HA-oligos, proMMP-2 levels were marginally elevated and MMP-9 unaffected. ProMMP-2 displayed a strong dose dependant increase in the nucleus pulposus (NP) monolayers. In AF alginate bead cultures, proMMP-2 and active MMP-9 increased up to day 10, in NP cultures proMMP-2 was progressively converted to active MMP-2 over days 7-10 and active MMP-9 levels were elevated on day 10. A steady decline in MMP-2 and 9 activity was evident over days 2-10 in the non-stimulated NP cultures. Disc cell viabilities were ≥ 92 ± 5% in all cultures indicating that the HA-oligo was not cytotoxic. RT-PCR demonstrated an up-regulation in MMP1,13 and ADAMTS1 and the anabolic matrix repair genes ACAN, COL1A1 and COL2A1 in the NP by HA-oligos, AF MMP13, ADAMTS1, 4 and 5, ACAN and COL2A1 were down-regulated this differential regulation is expected to promote clearance of granulation/scar tissue from AF defects and matrix replenishment. AF defect sites contained enlarged annular lamellae in-vivo in response to the HA oligos consistent with an active repair response. Masson Trichrome and Picrosirius red histology and immunolocalisation of type I collagen supported active remodelling in the outer lesion zone by the HA oligo treatment but not the inner lesion.