Introduction
The intervertebral disc (IVD) is a frequent cause of low back pain (LBP), and severity of IVD degeneration correlates with LBP prevalence. Dorsal root ganglion (DRG) neurons are the first-order neurons that transduce nociceptive/pain signals. Particularly, the spontaneous activation of these DRG neurons in the absence of noxious stimuli is correlated with spontaneous pain. Aberrant spinal mechanical loading is frequently discussed as a potential risk for LBP, but whether the intensively loaded IVD can influence pain-associated sensory nerve sensitization remains unknown. We hypothesized that intensive loading of IVD induces an increased spontaneous activation of DRG neurons.

Methods
Whole organ cultures of bovine tail IVDs were mechanically loaded in a bioreactor. High static loading of 0.2 MPa for 24h/day was applied to the IVD for 7 days to represent 'long-term sitting and standing' compared with a low static loading maintaining the initial disc height. Furthermore, dynamic loading of high frequency and intensity represented the 'wear and tear' loading, whereas low frequency and intensity loading was applied to represent the 'physiological' loading condition (0.32~0.5 MPa at 5 Hz versus 0.02 ~0.2 MPa at 0.2 Hz, for 3 h/day for 5 days).

To investigate the influence of static loading on IVD biology, degeneration-associated gene expressions of the IVD cells were evaluated using real-time RT-PCR. Viability of the disc cells was analyzed using lactate dehydrogenase (LDH) and ethidium-homodimer-1 staining of IVD cryosections.

Conditioned media (CM) were collected to stimulate primary cultures of bovine DRG neurons. Calcium imaging with Fluo-4 was used to evaluate the spontaneous calcium oscillation in the DRG neurons. The phenotypes of DRG neurons were labelled using immunofluorescence following the calcium imaging. Calcium oscillation in calcitonin gene related peptide (CGRP)-positive and CGRP-negative neurons was evaluated separately.

Results
High static loading increased interleukin 6 (IL-6) and matrix metalloproteinase 13 (MMP-13) gene expression in nucleus pulposus (NP) cells by 43.7 (p=0.07) and 13.2 (p=0.05) fold, respectively. High static loading also induced higher cell death compared with low static loading in inner annulus fibrosus (AF) and NP regions. The proportion of dead cells was increased from 3.9% to 15.7% in inner AF (p=0.03) and from 14.3% to 18.5% in NP region.

CM of IVDs subjected to both forms of intensive loading enhanced the spontaneous calcium oscillation in CGRP(-) neurons compared with their control IVD CM. High static loading induced the spontaneous calcium signals in a larger proportion of CGRP(-) neurons (84.32% versus 69.62%, p<0.01). High frequency and intensity cyclic loading elevated the normalized spontaneous calcium fluorescent peak height (∆F/F₀) by 8.6% (p<0.05).

Discussion
The high static loading induced a degenerative-like change in the inner AF and NP region of the IVD. Likewise, consistent with our former studies, high frequency and intensity loading caused an upregulation of IVD degeneration-associated genes.

Both static and cyclic intensive loadings mediated an altered disc-nerve communication which may suggest a biological mechanism associated with discogenic pain. However, how this disc-nerve communication is associated with LBP should be validated using in vivo models and clinical studies in the future.