The effect of vibration on annulus cell signaling

Abstract

Anthony Russo, MD, Albert Banes, PhD, Michell Elfervig, Mari Tsuzaki, Satoru Yamazaki, MD, Paul Weinhold, PhD, Joe Minchew, MD, Chapel Hill, NC, USABackground context: Low back pain is one of the most common medical problems experienced throughout the world, with an annual incidence of 5% to 10%, and the lifetime prevalence is as high as 80%. Degeneration of the intervertebral disc has been implicated as a major cause of low back pain. Understanding of the mechanisms that initiate and perpetuate disc degeneration is limited. Evidence suggests that both biochemical and biomechanical factors play a role in disc degeneration. Of the mechanical risk factors identified, vibratory loading, particularly exposure to whole body vibration, results in a significant increase in low back degenerative disorders with chronic exposure. The mechanism by which vibration may stimulate disc changes is unknown. We hypothesized that direct vibratory loading of annulus cells would trigger an intracellular signaling cascade that could be detected by monitoring intracellular calcium levels.Methods: The outer annulus was isolated from pooled L1–2 to L7–S1 intervertebral discs of adolescent New Zealand white rabbits. Annulus cells were isolated by means of collagenase digestion and grown in Medium 199 containing 5% fetal bovine serum, 20mM HEPES pH 7.2, and antibiotics. Cells were spot plated, at 5 K cells/10 mL spot, in a three-dimensional collagen matrix and cultured to quiescence. The spot cultures were loaded with FURA-2AM for 1 hour at room temperature and then fixed into a tissue culture plate. The cultures were then submerged in EBSS with Ca2+ and fixed to a custom-built stage-mounted vibration jig that delivered 0.1 g amplitude sinusoidal 6 Hz vibration. An upright fluorescence microscope allowed use of a ratio-dye method to convert fluorescent intensity of labeled annulus cells to intracellular calcium concentrations ([Ca2+]ic). Image 1 analysis software was used to quantitate the observed concentrations. After loading onto the stage, cultures were allowed to equilibrate for 30 minutes. A baseline [Ca2+]ic was determined, and then the culture was vibrated for either 15, 30, 45 or 60 seconds. After vibration, [Ca2+]ic was monitored for 5 minutes. After the observation period, all cultures were exposed to 10 mM ATP in EBSS as a positive control, to verify cell reactivity. Separate annulus cultures from each animal were exposed to vibratory stimulus after being submerged in Ca2+ free EBSS, to determine the need for extracellular calcium. Additional cultures were pretreated with 10 mM ATP in EBSS and exposed to vibratory stimulus once [Ca2+]ic had returned to baseline. Changes in [Ca2+]ic were analyzed with one-way analysis of variance and paired Student t test.Results: Rabbit annulus cells average baseline [Ca2+]ic was 170 nM. Cell cultures demonstrated significant increases in [Ca2+]ic after exposure to a vibratory stimulus for 30 and 45 seconds (p<.01), with [Ca2+]ic reaching levels two to three times baseline. No significant change in [Ca2+]ic was seen after 15 seconds of vibration. After 60 seconds of vibration, [Ca2+]ic was elevated but not significantly (p=.32). Trials performed in [Ca2+]-free EBSS demonstrated no change in [Ca2+]ic from baseline after vibration but continued vigorous response to the agonist ATP. Pretreatment of cell cultures with 10 uM ATP resulted in response to the ATP agonist but blocked response to vibration.Conclusions: There are few studies that investigate the mechanism involved in the deleterious effects of whole body vibration on the tissues of the spinal column. We have demonstrated that rabbit annulus fibrosus cells respond to mechanical vibratory stimuli with increases in intracellular calcium concentration. Furthermore, this response is dependent on the presence of extracellular calcium, whereas exposure to ATP results in the release of intracellular Ca2+ stores. This suggests that vibration exerts a direct cellular effect on annulus cells, activating membrane Ca2+ channels. To our knowledge, this is the first study demonstrating the early cellular effects of vibration. Activation of annulus cell surface purinoceptors by ATP before vibratory exposure results in inhibition of the mechanisms triggered by this particular mechanical stimulus. These early results suggest mechanisms involving intracellular calcium are involved in the response of intervertebral disc tissues to vibration, and extracellular ATP acts to modulate the signaling cascade. As the mechanism of increasing intracellular calcium is different for ATP and vibration, this does not simply represent a depletion of calcium stores. Further work is ongoing to delineate the downstream effects of this signaling mechanism and to determine if purinoceptor modulation is protective or deleterious.