Abstract
Back pain is the second most common neurological ailment in the United States and the leading cause of pain and disability. More than 80% of the total US population experiences back-pain during their life time and the annual back pain related healthcare costs exceed 100 billion dollars. While the exact cause of low back pain (LBP) is still unknown, degeneration of the intervertebral disc (IVD) has been suggested as a primary contributor. IVD is the largest avascular tissue in the human body and it is composed of three integrated tissues (annulus fibrosus - AF, nucleus pulposus - NP and cartilaginous end plate - CEP). IVD functions as a shock-absorber during motion and provides flexibility to motion of the spine. Maintaining IVD tissue integrity is an energy demanding process. Studies have shown that mechanical loading affects cellular biosynthesis of IVD tissue and may also promote IVD degeneration. However the path to this effect is still unknown. We propose a link between mechanical loading and cell energy production which contributes to altered cellular biosynthesis. Thus, we investigated the effects of mechanical loading on IVD cell energy metabolism under various mechanical loading regimes. Porcine AF and NP cells were isolated and seeded in 2% agarose at a 5,000,000 cells/mL cell density. A custom made bioreactor was used to conduct compression experiments. The experiment groups were: 15% static compression; 30% static compression; 0.1, 1 and 2 Hz dynamic compression at 15% strain magnitude. Experiment duration was 4 hr. ATP concentration in cell-agarose construct and culture media were measured using Luciferin-luciferase method to evaluate ATP production and ATP release from cells respectively. Lactate concentration in media was measured using lactate dehydrogenase enzymatic assay. Nitrite (stable metabolite of nitric oxide - NO) concentration in media was measured by Greiss Assay. DNA content per sample was measured using fluorometric assay. DNA content per sample was used as an internal control; all compressed samples were then normalized to unstrained control group. ATP production of AF cells was up regulated by static and dynamic mechanical loading. Data suggests that AF cell response to mechanical loading is primarily loading amplitude dependent. NP cells exhibited an increased ATP production at 1 Hz dynamic loading but remained comparable to control samples at other tested conditions. AF cells produced an increase in NO production at 1-, 2 Hz dynamic loading. NO production of NP cells was up regulated by mechanical loading at all tested conditions. ATP release was up regulated at higher frequencies in AF cells. In addition to higher frequencies (1 Hz and 2 Hz) NP cell ATP release was also up regulated by 30% static compression. Thus, this study clearly illustrates that mechanical loading affects IVD cell energy production.