REGULATION OF GELATINASE-A (MMP-2) PRODUCTION BY OVINE INTERVERTEBRAL DISC NUCLEUS PULPOSUS CELLS GROWN IN ALGINATE BEAD CULTURE BY TRANSFORMING GROWTH FACTOR-β1AND INSULIN LIKE GROWTH FACTOR-I

The aim of this study was to gain information relevant to disc repair processes. Limited degradation of the collagen matrix by matrix metalloproteases (MMPs) may facilitate the loosening of cell-cell and cell-matrix interactions within the injured intervertebral disc (IVD) to favour the penetration of blood vessels and migration of fibroblasts into the defect to promote repair processes. Gelatinase A (MMP-2) has a particularly important role to play in angiogenesis, in the present study we investigated the in vitro regulation of MMP-2 by Transforming Growth Factor-β1 (TGF-β1) and Insulin-like Growth Factor-1 (βIGF-I) in cells from the nucleus pulposus (NP) of the ovine IVD. Ovine NP cells were grown in alginate bead cultures in complete medium (10% foetal calf serum) for 7 days, established in serum-free conditions for 24h, then stimulated with TGF-β1 (0.1 or 10ng/ml) or IGF-I (2 or 50ng/ml) ±Concanavalin A (20μg/ml) for an additional 48h. Conditioned medium was examined for matrix metalloproteases using gelatin zymography, Tissue Inhibitor of Metalloproteinase 2 (TIMP-2) and Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) were immunolocalised in beads. Pro (72kDa) and active (59kDa) MMP-2 were the major gelatinolytic MMPs detected in control cultures, the TGF-β1 and IGF-I treatments significantly decreased levels of the active MMP-2, inclusion of Concanavalin A resulted in a complete reversal of this trend with IGF-I, and to a lesser extent with TGF-β1. Cell surface levels of TIMP-2 and MT1-MMP were decreased by the TGF-β1 treatment while IGF-I only appeared to decrease TIMP-2 expression. The findings of this study provide some insight as to why dense avascular connective tissues such as the intervertebral disc have such a poor healing potential.