Impact of glucose depletion on the nucleus pulposus homeostasis — The International Society for the Study of the Lumbar Spine
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Impact of glucose depletion on the nucleus pulposus homeostasis (#1016)

Mathilde PADRONA 1 2 , Hanane EL-HAFCI 1 2 , Manon MAROQUENNE 1 2 , Hervé PETITE 1 2 , Esther POTIER 1 2
  1. Université de Paris, CNRS, INSERM, B3OA, Paris, France
  2. École Nationale Vétérinaire d'Alfort, B3OA, Maisons-Alfort, France

Introduction
Chronic low back pain is commonly associated with intervertebral disc (IVD) degeneration and new therapies aiming to cure the physiopathology behind it are necessary. The avascular nature of the IVD creates a physiological acidic pH and low concentrations of oxygen and glucose in its core (the nucleus pulposus-NP). The effect of degenerated IVD levels of glucose on isolated NP cell viability is divergent in the literature and the impact on NP cell phenotype isn’t known. In our study, we used a bovine NP explant model (keeping intact the extracellular matrix (ECM) which plays a key role in cells’ behavior) and physiological oxygen, osmolarity, pH and glucose levels found in IVD which makes it a more complete and truthful model than those used before. Comparing two glucose conditions, considered as physiological healthy and degenerated levels, the aim of this study is to determine if glucose depletion is one of the main actor of the loss of NP homeostasis.


Methods
NP bovine explants were cultivated into 2 different conditions representative of healthy (2 mM) and degenerated (0.3 mM) glucose levels. Osmolarity (430 mOsm/kg H2O), pH (7.1) and pO2 (4.4%) were all set at physiological healthy values to only evaluate the effects of the glucose variation. The effects of glucose depletion were assessed at 1, 5, 12 and 21 days of culture (n=5/8) using: viability staining (cell viability-CV), RT-qPCR (senescence, NP cell phenotype, anabolic and catabolic phenotype), immunohistochemistry (apoptosis), biochemical assays (ECM characterization), and ELISA (release of inflammatory mediators).


Results
CV showed a significant decrease for the degenerated glucose condition, starting at D8 (46%), when compared to healthy glucose condition (80%), while there was no significant decrease in the healthy glucose condition up to D21 (Fig1). Histological assessment of apoptosis is still ongoing. No increase in the gene expression of the p21WAF1 senescent marker was observed. For both glucose conditions, glucose was still present in the conditioned cell culture media at every medium change, suggesting that cell death is not caused by a complete lack of glucose. Regarding ECM characterization, no effect on proteoglycan and collagen contents was observed with the biochemical assays and histology. As for gene expression of the catabolic (MMP3 and 13, ADAMTS4 and 5, TIMP1 and 2) and anabolic (ACAN, COL1 and 2) phenotype, there was no difference between both glucose conditions. NP cell phenotype genes (T, KRT8, 18 and 19) showed no difference between both glucose conditions. IL-1β, IL-6 and TNF-α concentrations were below the detection limit for both glucose conditions at each time point of analysis.


Discussion
The results showed that a degenerated IVD glucose level has a significant impact on the NP CV in a 3D bovine NP explant model while it had no significant effect on NP cell phenotype and ECM maintenance. These results reveal that glucose is probably not the only player in phenotype and matrix changes in IVD degeneration on a short term, but prove glucose has a clear impact on one of the disc degeneration trait, that is cell death.

 

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