INTRODUCTION: In the nucleus pulposus (NP) core, [glucose] is low, and, as anaerobic glycolysis leads to lactate production, the pH is low ¹. It has been hypothesized that cells may be metabolically more active when they are loaded but it has not been determined if these changes in metabolism can affect cell survival by interacting with the nutrient/metabolite homeostasis (Figure 1).

METHODS: Bovine NP explants were cultured in a previously developed volume controllable chamber², which can be dynamically loaded in a novel axial compression bioreactor. Explants were cultured for 7 days in degenerative medium (DM, 1 mM glucose, pH 6.8) or healthy medium (HM, 3 mM glucose, pH 7.1) with either non dynamic loading (NDL) or simulated physiological loading (SPL) and compared to native tissue (n=6 for all groups). To determine changes of the extracellular matrix, the sulphated glycosaminoglycan-, hydroxyproline-, and water-content were measured, and the tissue was stained with Alcian blue. Glucose and lactate were measured in the medium supernatants and tissue. A lactate dehydrogenase staining was performed to determine the viable-cell density (VCD). 

RESULTS: The histological appearance as well as tissue composition of NP explants did not change in any of the groups. There was no gradient formation of glucose or lactate in the tissue. Glucose levels in the tissue reached critical values for cell survival (≤ 0.5 mM¹) in all groups. Generally, lactate in the medium was increased in the SPL compared to the NDL groups (Figure 2). The VCD in the periphery of the NPs was not reduced in any of the groups. However, the VCD in the central region of the NP was reduced in the SPL groups (p≤0.01, Figure 3), which led to a significant gradient formation of VCD in these groups (p≤0.05). Within the HM or DM groups, there were no differences in tissue lactate concentration, however, the concentration was significantly increased in the HM compared to the DM groups (p≤0.001).

DISCUSSION: Physiological loading is believed to be essential for homeostasis of the IVD³. Here, we demonstrated that such loading can affect cell metabolism so much so that it was associated with changes in cell viability leading to a new equilibrium in the tissue, in both DM and HM. Urban et al. (2004) described that an interplay between pH and glucose orchestrates cellular survival and that cellular survival between 0 to 0.5 mM glucose is pH-dependent^1,4. We hypothesize, that the formation of a VCD gradient was caused by critically low glucose levels in combination with increased lactate production, decreasing the pH locally. This might have created a temporal gradient of low nutrition and high pH in the central region, in which cell survival was no longer possible. However, at the timepoint of harvest, there was no gradient in lactate and glucose tissue levels, which we believe arose from fast equilibration once a new cell density was reached. Concluding, physiological levels of loading lead to metabolically more active cells, which is relevant for disc degeneration and regeneration.

ACKNOWLEDGEMENTS: EU Horizon 2020 (#825925, www.ipspine.eu).