Oxygen consumption rate trends in degenerate and non-degenerate human intervertebral disc cell samples and their relationships to nutrient metabolism

Abstract

According to the National Institutes of Health, low back pain is an ailment that affects about 35 million people per year in the United States. The degeneration of intervertebral disc (IVD) tissue within the spine has a strong correlation to low back pain1. It is believed that a disruption in nutrient levels, and therefore, the IVD’s nutrient supply, will contribute to IVD degradation. Previous studies at the MUSC/Clemson Bioengineering Program (with: Sarah Cisewski, Hai Yao, PhD) aimed to determine the glycosaminoglycan (GAG) content in the extracellular matrix (ECM) which constitutes healthy cartilage endplate (CEP), which is a component of the IVD. A quantitative analysis of how much GAG is present in healthy tissue set a baseline for the current study involving degenerate and non-degenerate tissue to better understand the relationship between the biochemistry of the human IVD and tissue health. The current study is the next important step in examining the factors that contribute to disc nutrition levels, and thereby those factors which lead to weakened disc health and eventual low back pain. The balance between the rate of nutrient transport through the ECM of the IVD and the rate of consumption by disc cells determines the local nutrient concentration gradient within the IVD2. This study serves to examine the metabolic mechanisms of nutrients within the IVD with the purpose of statistically predicting oxygen consumption, distribution, and transportation. This will be done by establishing the oxygen consumption rate (OCR) at differing glucose levels (1mM, 5mM, and 25mM) in the three tissue regions (nucleus pulposus, annulus fibrosus, and cartilage endplate) of the IVD to determine regional differences in nutrient metabolism. Both degenerate and non-degenerate samples will be used in order to conceptualize the different mechanistic characteristics of nutrient metabolism of the IVD samples based on their grade of health. IVD cell suspensions were placed in a sealed metabolic chamber equipped with a fiber optic oxygen sensor, which recorded the cells’ real time dissolved oxygen concentration. The practical relationship between oxygen consumption and concentration was determined using the Michaelis-Menten (M-M) equation. Values from this metabolic chamber were then curve fit to M-M enzyme kinetic model to obtain the Vmax and Km variable values. Vmax represents the maximum oxygen consumption rate by the cells, and Km represents the oxygen concentration at ½ Vmax. Together, these variables help explain the functional relationship between oxygen concentration and OCR of human IVD cells. Degenerate IVD cells exhibited a markedly higher Vmax value when cultured at the lowest concentration of glucose (1mM) than at the higher concentrations (5mM and 25 mM) with a statistical significance of [p<0.05]. There were no statistically significant differences in the Vmax value for non-degenerate IVD cells due to glucose concentration [p=0.89]. However, non-degenerate NP cells exhibited a significantly higher absolute oxygen consumption rate than CEP cells [p<0.05]. No statistically significant differences were found in the values of Km for either degenerate or non-degenerate samples based on glucose concentration, and no differences between the NP, AF, or CEP occurred in either. The importance of determining the biochemistry of the IVD will help us to describe, explain, and treat lower back pain associated with IVD degeneration.