Biomechanical overloading initiates intervertebral disc degeneration. We hypothesized that this is due to mechanosensitivity of the cells, which break down the extracellular matrix. Previously, we found that overloading in a loaded disc culture system causes upregulation of remodeling- and inflammatory gene expressions. Fourier Transform Infrared Spectroscopy is a novel technique to identify, visualize and quantify ECM. In this research, we first identified novel spectroscopic markers for disc degeneration, and then applied these markers to investigate the first steps into disc degeneration by overloading.

In dataset 1, 18 discs of 9 goats were injected with chondroitinase ABC (degenerated) or not (control), and obducted 3 months after injection. This was used to find new spectroscopic markers for degeneration. In dataset 2, 42 goat discs were loaded with a physiological loading regime (50–150N) or overloading (50–400N) in a loaded disc culture system. In 18 of these discs, the cell activity was diminished in advance by freeze-thaw cycles and culturing on saline alone (non-vital group)). 24 additional discs were cultured in culture medium immediately post-mortem (vital group). Thereby, we are able to control whether the effect of the overloading is due to cell activity. The discs were fixed in formaldehyde, and 4 μm mid-sagittal were mounted to steel reflectance slides. Infrared spectroscopic mosaic images (23 × 57 images) were collected in transflectance mode at a spectral region of 1025–1150 cm⁻¹. Data was pre-processed by second derivative transformation and MCR-MALS with two factors.

The two factors were transferable between datasets, confirming the reliability. The first factor represents proteoglycans, as confirmed by Saffrin-O staining. In dataset 1, the degenerated group had less proteoglycan factor overall, especially in the nucleus (p<0.05). The second factor was found to have a lower entropy (p<0.01), showing a disorganization in the matrix. In dataset 2, no significant reduction in proteoglycan was found due to overloading in any group. However, the entropy was lower in the overloaded vital group (p<0.05), but not in the overloaded non-vital group (p>0.5).

Therefore, we conclude that infrared spectroscopy is a promising tool to investigate early disc degeneration. Overloading can cause changes in the extracellular matrix, but only due to cell activity. Entropy is an early marker for early disc degeneration, implying that cutting of the extracellular matrix by cell activity is the first step into intervertebral disc degeneration.

The intervertebral disc faces high compressive forces during daily activities. Axial compression induces creeping fluid loss and reduction in disc height. With degeneration, disc fluids and height are progressively lost, altering biomechanics. It is assumed that this loss of fluids is caused by a drop in osmolality in the disc due to proteoglycan depletion. Here we investigate the isolated effect of a reduction in osmosis on the biomechanical properties of the intervertebral disc. Continuous diurnal loading was applied to healthy caprine intervertebral discs in a loaded disc culture system for a total of 6 days. We increased testing bath osmolality with two doses of polyethylene-glycol (PEG), thereby reducing the osmotic gradient between the disc and the surrounding fluid. This way we could study the isolated effect of reduced osmosis on axial creep, without damaging the disc. We evaluated: daily creep and recovery, recovery time-constants and compressive stiffness. Additionally, we investigated water content. There was a strong dose-dependent effect of PEG concentration on water content and axial creep behaviour: disc height, amplitude and rate of creep and recovery were all significantly reduced. Axial compressive stiffness of the disc was not affected. Reduction of water content and amplitude of creep and recovery showed similarity to degenerative disc biomechanics. However, the time-constants increased, indicating that the hydraulic permeability was reduced, in contrast to what happens with degeneration. This suggests that besides the osmotic gradient, the permeability of the tissues determines healthy intervertebral disc biomechanics.

Intervertebral disc degeneration is a common cause of low-back pain, the musculoskeletal disorder with the largest impact world-wide. The complex disease is however not yet well understood, and no treatment is available. This is somewhat in contrast with osteoarthritis, a subject of more extensive research. Intervertebral disc degeneration may though be a type of osteoarthritis, as other vertebrates have a diarthrodial joint instead of an intervertebral disc. We describe the parallel in view of the anatomy, composition and degeneration of the intervertebral disc and articular joint. Not only different embryonic origin and anatomy suggest significant differences between the intervertebral disc and the synovial joint, but their biomechanical properties also partly differ, as articulation is one of the key properties of a synovial joint and does not occur in the intervertebral disc. However, both tissues provide flexibility and are able to endure compressive loads, and both cell behavior and extracellular matrix appear much the same, mainly existing of chondrocytes, proteoglycans and collagen type II, suggesting that the environment of the cell is more important to its behavior than embryonic origin. Moreover, great similarities are found in the inflammatory cytokines, which are mainly IL-1β and TNF-α, and matrix-degrading factors (i.e. MMPs and ADAMTSs) involved in the cascade of degeneration, resulting in overlapping clinical and radiological features such as loss of joint space, subchondral sclerosis, and the formation of osteophytes, causing pain and morning stiffness. Therefore, we state that disc degeneration can result in the osteoarthritic intervertebral disc. This point of view may enhance the synergy between both fields of research, and potentially provide new regenerative strategies for intervertebral disc degeneration.

Daytime spinal loading is twice as long as night time rest, but diurnal disc height changes due to fluid flow are balanced. A direction-dependent permeability of the endplates, favouring inflow over outflow, has been proposed to explain this; however, fluid also flows through the annulus fibrosus. This study investigates the poro-elastic behaviour of entire intervertebral discs in the context of diurnal fluid flow.

Caprine discs were preloaded in saline for 24 hours under different levels of static load. Under sustained load, we modulated the disc's swelling pressure by replacing saline for demi-water and back again to saline, both for 24h intervals. We measured the disc height creep and used stretched exponential models to determine the respective time constants.

Reduction of culture medium osmolality induced an increase in disc height, and the subsequent restoration induced a decrease in disc height. Creep varied with the mechanical load applied. No direction-dependent resistance to fluid flow was observed. In addition, time constants for mechanical preloading were much shorter than for osmotic loading, suggesting that outflow is faster than inflow. However, a time constant does not describe the actual rate of fluid flow: close to equilibrium fluid flow is slower than far from equilibrium. As time constants for mechanical loading are shorter and daytime loading twice as long, the system is closer to the loading equilibrium than to the unloading equilibrium. Therefore, paradoxically, fluid inflow is faster during the night than fluid outflow during the day.

Summary Statement

Conventional imaging techniques lack the ability to objectively assess early stages of intervertebral disc degeneration, characterised by glycosaminoglycan loss. This study shows that MRI T2∗ mapping correlates positively with GAG content and that it provides continuous measurements for disc degeneration.