Abstract
Low back pain causes distress and suffering to patients. The impact of low back pain creates a major socio-economic burden in industrialized societies as well. As the leading cause of disability, low back pain affects more than 80% of the US population at some point in life. Intervertebral disc (IVD) degeneration has been closely associated with low back pain, stimulating interest in finding the causes that lead to IVD degeneration. Therefore, understanding the mechanisms involved in the maintenance of IVD composition may shed light to development of novel therapies for IVD degeneration and low back pain. IVD cells are responsible for maintaining adequate rates of biosynthesis, breakdown and accumulation of extracellular matrix (ECM) constituents in order to preserve the quality and integrity of the ECM and consequently, the IVD’s functional properties. In order to do that, IVD cells produce energy in the form of adenosine triphosphate (ATP). While mechanical stimulation has shown to promote ECM biosynthesis and ATP release, the effects of direct stimulation of the purinergic pathway by extracellular ATP and its derivative, adenosine, on the ECM biosynthesis in IVD cells have not been elucidated. Therefore, the objective of this dissertation is to study the effects of extracellular ATP and adenosine on the ECM biosynthesis of IVD cells, in order to better understand the role of the purinergic pathway in the maintenance of IVD composition. A noninvasive system for detecting membrane potential variations induced by ATP stimulation using a potentiometric dye and image processing techniques was developed. The results indicate that IVD cells have different responses to exogenous ATP stimulation in monolayer and 3-dimensional cultures. Furthermore, AF and NP cells exhibited distinct patterns and magnitudes of membrane potential changes. The ATP-induced response was found to be the result of activation of P2 purinergic receptors in IVD cells. The effects of extracellular ATP on the ECM biosynthesis of IVD cells were studied. Results show up-regulation of aggrecan and type II collagen gene expressions in NP and AF cells by extracellular ATP. Inhibition of gene expression by ATP receptors antagonist suggests that ATP receptors are involved in the ECM biosynthesis in IVD cells. In addition, NP cells were found to be less sensitive to low concentrations of extracellular ATP than AF cells while NP cells exhibited higher accumulation of PG, collagen, and intracellular ATP compared to AF cells. Extracellular adenosine treatment promoted ECM biosynthesis and intracellular ATP production in IVD cells. Gene expression of aggrecan and type II collagen in IVD cells was also up-regulated by extracellular adenosine. Moreover, inhibition of gene expression by adenosine receptors antagonists suggests that adenosine receptors are involved in biological activities in the IVD. The results also suggest that hydrolysis of extracellular ATP promoted by mechanical loading regulates ECM biosynthesis of IVD cells via adenosine formation. The findings of this dissertation contribute to further understanding the role of ATP and adenosine on the ECM biosynthesis of IVD cells. Given that ATP production is fundamental for ECM biosynthesis, this study can provide insight into the role of the purinergic pathway on the ECM biosynthesis in the IVD.