Low back pain (LBP) is the leading reason for people to miss work and chief complaint of 5% people who visit doctors. Although it has a high impact in society, the causes of LBP cannot always be clarified. However, intervertebral disc (IVD) degeneration has been reported to be a possible leading source for LBP. Since IVD is the biggest avascular tissue in human body, nutrition supply plays very important role for IVD degeneration. IVDs are under mechanical load all the time in vivo, it is also been revealed by in vitro studies that mechanical loadings can alter cellular activities including metabolism. However, how mechanical loadings affect IVD metabolism has not been elucidated yet. Therefore, the purpose of this dissertation is to study the energy metabolism of IVD under compressive loadings.
A bioreactor system was built to apply static and dynamic mechanical loadings to IVD. A pump was utilized to maintain culture medium circulation during tissue culture and loading experiments; a load cell and a data acquisition device were also included in the system to record loading forces.
Adenosine-5’-triphosphate (ATP) is the “energy currency” of cell activities and extracellular ATP is an important molecule which can mediate various physiological activities by activating series of receptors. Therefore it is essential to detect extracellular ATP contents. However, existing techniques have their drawbacks and are not ideal for in vivo/in situ measurement. We thus developed an optical biosensor for in situ measurement of extracellular ATP measurement in biological tissues. This ATP sensor utilized the technique of sol-gel coating and two layers of coatings were applied to the end of optical fibers; the first layer contained Ruthenium complex which can be excited by blue light with 465 nm of wavelength and emit red light with wavelength at 610 nm; the second layer contains two enzymes: glycerol kinase and glycerol 3-phosphate which can oxidase ATP and consume oxygen. During measurement, ATP molecules diffuse into the second layer and are oxidized thus decrease local oxygen concentration; this decrease of oxygen is detected by the first layer and is recorded by NeoFox® software; the signal detected has a linear relationship with the ATP content. This ATP sensor has a broad range of measurement at 10-3 mM to 1.5 mM. A compensation method was established to enable the measurement of ATP contents at different environmental oxygen concentrations. We also demonstrated that the performance of this sensor was not affected by environmental pH and derivatives of ATP such as adenosine diphosphate (ADP) and adenosine monophosphate (AMP) or adenosine.
From static and dynamic compression experiments of porcine IVDs it was found that under both static and dynamic compressive compressions, pH decreased and the contents of lactate and ATP increased significantly in both annulus fibrosus (AF) and nucleus pulposus (NP) regions, suggesting that compressive loading can promote ATP production via glycolysis and reduce pH by increasing lactate accumulation. We also detected high level of extracellular ATP contents in the NP region and compressive loadings significantly decrease extracellular ATP contents. Since ATP can be utilized as intracellular energy currency and regulate a variety of extracellular activities through the purinergic signaling pathway, the findings of this dissertation suggest that compression mediated ATP metabolism could be a novel mechanobiological pathway for regulating IVD metabolism.
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