Multiphoton imaging and Raman spectroscopy of the bovine vertebral endplate

The interface between the intervertebral disc and the vertebral body is important to the discs' biomechanics and physiology, and is widely implicated in its pathology. This study aimed to explore biochemically and structurally the bony endplate, cartilage endplate and intervertebral disc, below the nucleus and below the annulus in healthy bovine tails. Multiphoton imaging and spontaneous Raman spectroscopy were employed. Raman spectroscopy provided relative quantification of mineral and matrix components across the vertebral endplate and its adjacent areas with microscopic spatial resolution. Microscopy utilising second-harmonic generation (SHG) and two-photon fluorescence (TPF) allowed for the structural identification of distinct endplate regions. The cartilage endplate was revealed as structurally distinct from both the bone and disc, supporting its biomechanical function as a transition zone between the soft and hard tissue components. The collagen fibres were continuous across the tidemark which defines the interface between the mineralised and non-mineralised regions of the endplate. Raman spectroscopy revealed gradients in phosphate and carbonate content through the depth of the endplate and also differences beneath the nucleus and annulus consistent with a higher rate of remodelling under the annulus.