Applying the Techniques of Materials Science towards an Understanding of the Process of Canine Intervertebral Disc Degeneration

Abstract

Intervertebral disc degeneration in dogs occurs in an accelerated way and involves calcification, which is associated with disc herniation or extrusion. The degenerative process is complex and involves the transformation of collagen fibres, loss of proteoglycans and notochord cells and a reduction in water content; however, how these processes are linked to future disc extrusion remains unknown. We have employed techniques including Fourier Transform Infra-red Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Uniaxial Compression Loading and Atomic Force Microscopy (AFM) in an attempt to gain a greater understanding of the degenerative process and its consequences on the physical properties of the disc. FTIR verified by TEM demonstrated that calcium phosphate exists in an amorphous state within the disc and that the formation of crystalline particles of hydroxyapatite occurs prior to disc extrusion. AFM identified crystalline agglomerates consistent with hydroxyapatite as well as individual collagen fibres. SEM enabled the identification of regions rich in calcium, phosphorous and oxygen and allowed the visualization of the topographical landscape of the disc. Compression testing generated stress/strain curves which will facilitate investigation into disc stiffness. Ongoing work is aimed at identifying potential areas of intervention in the degenerative process as well as further characterizing the role of calcification in disc extrusion.

Keywords: Hansen; atomic force microscopy; calcification; disc disease; extrusion; intervertebral disk; spinal cord.

Figure 1

Graph of typical smoothed spectra from an extruded intervertebral disc material, showing the PO₄³⁻ V1 vibration peak (encircled region) characteristic of hydroxyapatite mineral content.

Figure 2

(A) TEM image of gel with many needle particles. (B) Polycrystalline electron diffraction (sharp rings).

Figure 3

(A) TEM image of gel with very few needle particles. (B) Amorphous electron diffraction (no sharp rings).

Figure 4

Structures compatible with collagen fibres (red arrows) in extruded material at a scan size of 1000 nanometres.

Figure 5

“Hexagonal-like” particles aggregated. Topographical approach at a nanoscale of 10 µm.

Figure 6

EDS Map Sum Spectrum from samples containing Crystalline HA Calcium (A) and without Crystalline HA Calcium (B) based on FTIR spectra (see Figure 7).

Figure 7

The images (A,C) represent the electron images from the 2 samples represented in Figure 6. The images (B,D) are the respective EDS-layered images showing the distribution of the Ca, P and O in the surface. The smoothed FTIR graph for each sample is presented below the SEM images; the V1 vibration peak consistent with presence of HA can be clearly identified only in the first of the 2 spectra (red arrow).

Figure 8

Stress/strain curves from uniaxial load compression testing.