The high-throughput phenotyping of the viscoelastic behavior of whole mouse intervertebral discs using a novel method of dynamic mechanical testing

Abstract

Intervertebral disc (IVD) degeneration is highly correlated with lower back pain, and thus understanding the mechanisms of IVD degeneration is critical for the treatment of this disease. Utilizing mouse models to probe the mechanisms of degeneration is especially attractive due to the ease of manipulating mouse models and the availability of transgenics. Yet characterizing the mechanical behavior of mice IVDs remain challenging due to their minute size (approximately 540 μm in height and 1080 μm2 in cross sectional area). We have thus developed a simple method to dynamically characterize the mechanical properties of intact mouse IVDs. The IVDs were dissected with the endplates intact, and dynamically compressed in the axial direction at 1% and 5% peak strains at 1 Hz. Utilizing this novel approach, we examined the effects of in vitro ribosylation and trypsin digestion for 24 or 72 hours on the viscoelastic behavior of the whole murine IVD. Trypsin treatment resulted in a decrease of proteoglycans and loss of disc height, while ribosylation had no effect on structure or proteoglycan composition. The 72 hour ribosylation group exhibited a stiffening of the disc, and both treatments significantly reduced viscous behavior of the IVDs, with the effects being more pronounced at 5% strain. Here we demonstrate a novel high-throughput method to mechanically characterize murine IVDs and detect strain-dependent differences in the elastic and the viscous behavior of the treated IVDs due to ribose and trypsin treatments.

An <em>In Vitro</em> Organ Culture Model of the Murine Intervertebral Disc

“…Determine the mechanical behavior of the IVDs by using displacement-controlled dynamic compression 9 .…”

Viscoelastic heating of insulated bovine intervertebral disc

“…The IVD consists of 2 distinct parts: the fibrocartilaginous outer annulus fibrosus (AF) and the more viscous inner nucleus pulposus (NP). Collectively, the IVD is a viscoelastic tissue . Viscoelastic materials possess mechanical properties that resemble both viscous fluids and elastic solids .…”

“…Collectively, the IVD is a viscoelastic tissue. [1][2][3] Viscoelastic materials possess mechanical properties that resemble both viscous fluids and elastic solids. 4 When cyclically loaded, viscoelastic materials lose energy in the form of heat as a result of viscous dissipation.…”

“…[9][10][11] Over time IVD can degrade as a result of age 12 or injury, resulting in reduced function and significant pain. 1 Heating in the IVD is of particular interest because recent investigations have shown that degenerative human IVD is capable of sensitizing nociceptive neurons to thermal stimuli. 13,14 As a result, nociceptive neurons begin to fire at 37 C, have a T50 of 37.3 C and see a maximal firing response by 38 C. As a result, relatively small changes in temperature in the disc could drive nociception neuron firing in the degenerative IVD.…”

Homeostasis of the intervertebral disc requires regulation of STAT3 signaling by the adhesion G-protein coupled receptor ADGRG6

“…Using micro-indentation we demonstrated a consistent increase in the stiffness (Newton (N)/mm) of ATC;Adgrg6 f/f mutant IVDs under 5% strain cyclic loading ( Figure 3A; p=0.0114; mean w/95% CI) and under 50% monotonic overloading ( Figure 3B; p=0.0026; mean w/95% CI). Stiffening of the IVD is commonly observed with early-onset degenerative changes, which compromises the damage resistance of the tissue (Liu et al, 2015a). We analyzed proteoglycan quantification in these IVDs by dimethylmethylene blue assay and found no significant alterations comparing mutant and littermate control mice at 1.5 months.…”