The compressive creep properties of normal and degenerated murine intervertebral discs

Abstract

Identifying mechanisms by which degeneration alters intervertebral disc material properties and biomechanical behavior is important for clarifying back pain risk factors as well as for evaluating the efficacy of novel interventions. Our goal was to quantify and characterize degeneration-dependent changes in the disc’s response to compression using a previously established murine model of disc degeneration. We performed compressive creep tests on normal and degenerated murine intervertebral discs and parameterized the biomechanical response using a previously established fluid-transport model. Using a series of biochemical and histological assays, we sought to determine how biomechanical alterations were attributable to degeneration-related changes in tissue morphology. We observed that with moderate degeneration, discs lost height (mean ± std. dev. of 0.44 ± 0.01 vs. 0.36 ± 0.01 mm, p <0.0001), increased in proteoglycan content (31 ± 4 vs. 43 ± 2 μg/ml of extract, p <0.0002), became less stiff (2.17 ± 0.66 vs. 1.56 ± 0.44 MPa, p <0.053), and crept more. Model results suggested that the increased creep response was mainly due to a diminished strain-dependent nuclear swelling pressure. We also noted that the model-derived tissue properties varied with the applied load magnitude for both normal and degenerated discs. Overall, our data demonstrate that architectural remodeling stimulated by excessive loading diminishes the disc’s ability to resist compression. These results are similar to degeneration-dependent changes reported for human discs. Keywords Intervertebral disc Degeneration Creep Biomechanics Mouse Introduction The intervertebral disc is a viscoelastic structure consisting of a proteoglycan-rich nucleus pulposus surrounded by a collagenous annulus fibrosus. A by-product of its unique composition and architecture is that significant, time-dependent compressive deformation occurs in response to persistent daily loads (referred to as creep). Quantification of disc creep can be used to assess tissue properties that have important clinical implications, as excessive creep deformation has been linked to spinal instability and low back pain [8,16,22] . Historical data demonstrate that the disc’s creep response varies with age, since degeneration alters tissue architecture and biomechanical properties [11,12] . The functional and clinical consequences of degeneration have been described by Kirkaldy–Willis as consisting of three stages: (1) capsular––associated with grade II disc degeneration; (2) abnormal motions––associated with grade III degeneration; and (3) fixed deformity––associated with grade IV degeneration [13] . Importantly, aberrant vertebral motion due to diminished intervertebral constraint has been linked with increased patient symptoms [6,8] . However, the degeneration-related tissue changes responsible for alterations in mechanical response remain unclear. Understanding the mechanisms by which degeneration influences disc tissue, and consequently, creep response may ultimately be used useful for clarifying back pain risk factors as well as for evaluating the efficacy of biomechanical interventions for disc degeneration. Our goal was to quantify and characterize the degeneration-dependent changes in disc biomechanics using an in vivo murine model of disc degeneration. We have previously established that static compression on the murine tail disc leads to morphologic and cellular changes pathognomonic of human disc degeneration [15] . In the current study, we hypothesized that the disc’s ability to resist creep is significantly affected by load-induced degeneration. Also, we questioned whether degeneration-dependent changes in disc biomechanics observed in this murine model are consistent with those reported for humans. To test these hypotheses, we performed mechanical creep tests on normal and degenerated mouse-tail discs and parameterized the creep response using a previously established fluid-transport model. Using a series of biochemical and histological assays, we sought to determine how biomechanical alterations were attributable to degeneration-related changes in tissue morphology. Methods A previously established model of disc degeneration, which established cellular and architectural changes reflective of changes in Thompson grade, was used in this study (approved by the University Committee on Animal Research) [15] . Fifty-eight Swiss Webster mice (male, 12 weeks old) were anesthetized and four 0.4 mm diameter stainless steel pins were used to percutaneously transfix the 9th and 10th caudal vertebrae. In 29 of the animals, the pins were left in place, and these discs served as the unloaded controls. For the remaining animals, the pins were instrumented with calibrated elastics so as to apply a 1.3 MPa compressive stress ( Fig. 1 ). After one week of loading, the elastics were removed, and the animals were allowed to recover for three weeks. This stress level and time were selected because they consistently produce a degenerated disc [15] . The unloaded mice were kept for a comparable time (28 days) and served as age-matched, unloaded controls. Each disc segment was used in only one of the following described experiments. At the specified time, the animals were killed by CO 2 -induced asphyxiation. The study disc and the two adjacent vertebrae were harvested for mechanical testing, measurement of the proteoglycan content, and histological analysis. The specimens used for mechanical testing were radiographed in the dorsal–ventral plane immediately prior to creep testing. The X-rays were digitized at a resolution of 0.03 mm/pixel (ScanMaker IIxe, Microtek, Taiwan, ROC), and disc height was measured using public domain software (NIH Image 1.61, National Institutes of Health, Bethesda, MD). Since the caudal discs are approximately...