Effects of torsion on intervertebral disc gene expression and biomechanics, using a rat tail model

Abstract

Study design: In vitro and in vivo rat tail model to assess effects of torsion on intervertebral disc biomechanics and gene expression.

Objective: Investigate effects of torsion on promoting biosynthesis and producing injury in rat caudal intervertebral discs.

Summary of background data: Torsion is an important loading mode in the disc and increased torsional range of motion is associated with clinical symptoms from disc disruption. Altered elastin content is implicated in disc degeneration, but its effects on torsional loading are unknown. Although effects of compression have been studied, the effect of torsion on intervertebral disc gene expression is unknown.

Methods: In vitro biomechanical tests were performed in torsion on rat tail motion segments subjected to 4 treatments: elastase, collagenase, genipin, control. In vivo tests were performed on rats with Ilizarov-type fixators implanted to caudal motion segments with five 90 minute loading groups: 1 Hz cyclic torsion to ± 5 ± 15° and ± 30°, static torsion to + 30°, and sham. Anulus and nucleus tissues were separately analyzed using qRT-PCR for gene expression of anabolic, catabolic, and proinflammatory cytokine markers.

Results: In vitro tests showed decreased torsional stiffness following elastase treatment and no changes in stiffness with frequency. In vivo tests showed no significant changes in dynamic stiffness with time. Cyclic torsion upregulated elastin expression in the anulus fibrosus. Up regulation of TNF-α and IL-1β was measured at ±30°.

Conclusion: We conclude that strong differences in the disc response to cyclic torsion and compression are apparent with torsion increasing elastin expression and compression resulting in a more substantial increase in disc metabolism in the nucleus pulposus. Results highlight the importance of elastin in torsional loading and suggest that elastin remodels in response to shearing. Torsional loading can cause injury to the disc at excessive amplitudes that are detectable biologically before they are biomechanically.

Figure 1

A, In vivo testing involved instrumenting an Ilizarov-type apparatus consisting of carbon fiber rings attached to rat tails, with proximal ring held in torque plate and distal ring rotated in the tail holder. B, Newly designed device to apply torsional loading showing the rat holder, torque plate, and tail holder. The motor housing contains a stepper motor mounted on a linear bearing so that torsional loading could be applied without any axial loading and a motor controller.

Figure 2

In vitro biomechanical test applied to caudal motion segments consisted of a torque controlled ramp to failure. This typical result demonstrated a transition from the neutral zone to linear region which occurred at 6.5° ± 0.2°. These findings motivated choice of in vivo test conditions to include rotation in the neutral zone (5°), early in the linear zone (15°) and later in the linear zone with possible damage (30°).

Figure 3

In vivo mechanical measurements were taken every 15 minutes during the 90 minute test. There were no changes in stiffness with time, but stiffness was greater for 30° than 15°, showing nonlinear torsional stiffness behaviors. Stiffness for 5° rotation magnitudes was negligible (not shown) representing loading in the neutral zone.

Figure 4

Fold changes in mRNA levels relative to internal control levels (mean ± SEM) for 13 genes where (A, B) are anabolic genes, (C, D) are catabolic genes and (E, F) are proinflammatory cytokines, and (A, C, E) are the measurements from the AF and (B, D, F) from the NP. The mRNA from internal controls was isolated from pooled tissue from proximal and distal internal controls to eliminate potential level effects. *indicates significantly different from sham; #, significantly different from internal controls for sham group only; P < 0.05.