Early markers of mechanical modulation in whole bovine intervertebral discs loaded in a multiaxial bioreactor

Intervertebral disc degeneration (IVDD) is a major contributor to low back pain (LBP), with mechanical overloading recognized as a driver of disc pathology. This study investigated early responses of bovine caudal intervertebral discs (IVDs) to repetitive multiaxial loading and assessed effects on nociceptive sensitization. Whole IVDs were cultured ex vivo for 3 or 7 days either free-swelling or under multiaxial loading (0.2 MPa compression, 0–6° flexion, 0–4° torsion, 0.2 Hz, 4 h/day). Disc height, biomechanics, gene expression, biochemical markers, collagen integrity, and cell viability were evaluated. Conditioned medium (CM) from IVDs was applied to primary dorsal root ganglion (DRG) neurons to assess calcium responses and neurite outgrowth. Multiaxial loading induced rapid, region-specific molecular changes, particularly in the outer annulus fibrosus, with upregulation of inflammatory (IL6) and catabolic (MMP13) genes and mild increases in glycosaminoglycan and nitric oxide release. DRG neurons exposed to CM from loaded IVDs exhibited enhanced capsaicin-evoked calcium responses and increased neurite branching, especially in CGRP-positive nociceptors, indicating early discogenic sensitization. Collagen denaturation showed a trend toward higher levels in loaded IVDs, while cell viability, tissue structure, and biomechanical properties remained preserved. Multiaxial loading at the selected magnitude induced early molecular signs of degeneration and neuronal sensitization without detectable alterations in cell viability, tissue structure, or biomechanics, contributing to our understanding of mechanically induced impact on the IVD. Ex vivo multiaxial loading in bioreactors provides a physiologically relevant platform to study the initiation and progression of IVDD and associated discogenic pain mechanisms.