Abstract
As a leading cause of global disability, low back pain is prevalent in the adult population with the highest occurrence in the third decade of life, creating a significant socioeconomic burden. Low back pain is commonly related to degeneration of intervertebral disc (IVD). While the clinical consequences of the ailment are well-documented, its etiology is poorly defined. The cascade of disc degeneration is believed to be initiated by the disappearance of IVD cells and loss of proteoglycan (PG) in the extracellular matrix (ECM). Since essential nutrients are only supplied to disc cells by diffusion from the periphery of the avascular IVD, nutritional deprivation is identified as a leading cause for disc degeneration.
One neglected area of nutrition-related IVD study is ATP metabolism of IVD cells. ATP provides cells the most fundamental form of energy for their survival and function. It can be particularly important for the biosynthesis of PG by IVD cells in which PG serves an additional role as a building block. It is well-known that IVD cells predominantly utilize glycolysis to generate ATP which relies on the availability of oxygen and glucose. Therefore, cells may not function properly with inadequate ATP supply. Furthermore, ATP is constantly released extracellularly which release by IVD cells was found sensitive to mechanical loading. A high level of extracellular ATP is present in the PG-rich disc center and its hydrolysis by the ubiquitous ecto-nucleotidases on the cell membrane may result in the formation of adenosine. While the beneficial roles of extracellular ATP and adenosine have been appreciated in literature, information regarding ATP metabolism and its association with IVD tissue function is largely unknown. Therefore, the overall objective of this dissertation is to quantitively investigate the production, release and extracellular distribution of ATP in the IVD in relation to the maintenance of ECM using a combined experimental and theoretical approach. The role of adenosine as its major hydrolytic product in promoting ECM production was also explored.
Intracellular ATP and PG production of porcine IVD cells under prolonged exposure to both 21% O2 (normoxia) and 5% O2 (hypoxia) within a range of physiological glucose levels were first investigated. The results showed glucose consumption rates of both nucleus pulposus (NP) and annulus fibrosus (AF) cells decreased with reduced glucose supply under both oxygen levels. Hypoxia reduced glucose consumption, intracellular ATP and PG productions of NP cells in general while AF showed the opposite trend. Furthermore, NP cells exhibited a significantly higher glucose consumption rate as well as ATP and PG production than AF cells at all glucose levels under normoxia. However, the difference in both productions became minimal under hypoxia despite NP cells still consumed glucose faster, notably at higher glucose levels. Under all conditions, a strong correlation between the PG production and ATP content was found for both cell types. Furthermore, PG production is more likely to be governed by energy supply rather than gene expression and a glucose level higher than the previously suggested threshold value for cell survival may be needed for notochordal NP cells to maintain normal PG production.
A novel three-dimensional (3D) finite element model of IVD was developed based on the mechano-electrochemical mixture theory to predict extracellular ATP distribution in the porcine and human IVD. Parameters involved in ATP metabolism of porcine IVD cells were experimentally measured and incorporated into the model. The robustness of the approach was demonstrated by a good agreement between the experiment and simulation results from the porcine model. The human model was used to analyze extracellular ATP distribution in the degenerated and non-degenerated human IVD with altered tissue properties and boundary conditions. It was shown that ATP could uniquely bind with PG in the IVD tissue which modulated the distribution of extracellular ATP. A high extracellular ATP content was predicted in the PG-rich disc center and decreased PG content with degeneration resulted in a significant decline of extracellular ATP. The underlying mechanism of the enhancement of ECM biosynthesis of IVD cells by exogenous adenosine was explored. Extracellular adenosine treatment significantly upregulated gene expression of aggrecan and type II collagen and intracellular ATP production in IVD cells which effects were completely suppressed by an adenosine transport blocker but not by various receptor antagonists, suggesting adenosine uptake rather than receptor activation was involved in the pathway.
The outcomes of this dissertation contribute to the knowledge of ATP metabolism of IVD cells and associated biological impacts in disc function. Given the universality of ATP and adenosine in the biological world, this knowledge can provide additional insight for pathways and mechanisms that are involved in maintaining disc integrity and help identify factors that contribute to the pathophysiology of disc degeneration.