The Metabolism of Inner Annulus Fibrosus Cells Regulates Phenotype

Matadar, Raeesa (1), Kandel, Rita (2), Santerre, Paul (3)

(1) Institute of Biomaterials and Biomedical Engineering, University of Toronto

(2) Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital

(3) Faculty of Dentistry, University of Toronto

Introduction: Lower back pain is one of the largest causes of disability globally. It is associated with the degeneration of the intervertebral disc (IVD), a multi-tissue support structure that links the vertebral bodies. Changes in the extracellular matrix (ECM) reduce the disc’s ability to transfer loads resulting in progressive tissue degradation. As there is little capacity for self-repair, regenerating the IVD in vitro is now being explored. Previous work has shown that the addition of dexamethasone and sodium pyruvate contributes to the production and accumulation of appropriate ECM, and increases mitochondrial membrane potential in vitro. Thus, culture conditions appear to trigger metabolic changes that affect ECM accumulation, however, there have been few investigations into this link in annulus fibrosus (AF) cells. The purpose of this study was to investigate the role of the AMPK/PGC1α/PPAR/sox9 signalling axis in phenotype maintenance and appropriate ECM production without the use of a corticosteroid.

Methods: AF cells were isolated enzymatically from bovine caudal tails and seeded onto multilamellar angle-ply nanofibrous PU scaffolds in spinning bioreactors to generate AF tissues in vitro. AF tissues were cultured for up to 3 weeks in DMEM containing either 1mM (low) or 25mM (high) glucose concentrations. Tissues were evaluated histologically, biochemically, immunohistochemically and for gene expression and protein levels.

Results: IAF tissues grown in low-glucose accumulated collagen type II and aggrecan, as well as collagen type I whereas tissues grown in high glucose only accumulated collagen type I. Over time, gene expression showed an increase in PGC1α and mitochondrial transcription factor A, as well as SOX9, COL2A1 and ACAN which are components of the native IAF. We also observed an increase p-AMPK, p-ACC, PGC1α, and sox9 protein levels. ATP levels in tissue were not significantly lower in low-glucose, suggesting a non-canonical activation of AMPK. The next steps will be to inhibit AMPK to confirm its role in phenotype maintenance.

Conclusions: Here we demonstrated that low-glucose medium restored the IAF cell phenotype and activated the AMPK/PGC1α/PPAR/sox9 signalling axis when compared to high-glucose. These studies provide novel insights into the mechanisms regulating IAF phenotype which will facilitate the identification of an ideal medium formulation to enhance tissue formation appropriate for IVD regeneration and functionality.